Systems and methods for multiple detection of biomarkers

Abstract

Methods and systems for the multiple detection and / or measurement of biomarkers in a sample are provided herein. These methods and systems can be used for rapid disease detection and / or monitoring, monitoring of vaccine efficacy and immune response, monitoring of therapeutic agents, and / or monitoring of the safety and efficacy of treatments.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 480,437, filed on January 18, 2023, and U.S. Provisional Application No. 63 / 481,360, filed on January 24, 2023, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Biomarkers found in the body can be used to detect, predict, or manage diseases, but many are present in amounts too low to be detected using commercially available tests. In addition, interference from substances present in a patient's sample can alter the correct value of a diagnostic test result, for example, by interfering with antibody binding, or can increase or decrease an assay signal through cross-linking, steric hindrance, or autoantibody mechanisms.

[0003] There is a clinical need for a simple, inexpensive, and automatable effective solution for new diagnostic technologies that can improve test accuracy by preparing clinical samples, measure biomarkers that are difficult to discover, reduce costs, and ultimately save lives.

Summary of the Invention

[0004] In some embodiments, assay methods are disclosed herein that include the following, in some embodiments, (i) providing a washed substance subject to washing by removing interference from the washed substance using an interference capture composition, wherein the interference capture composition comprises an interference capture portion, and the washed substance is selected from the group consisting of a sample derived from a subject, biomarker capturing particles, a detection antibody, and any combination thereof; and (ii) capturing a biomarker from the sample by contacting the sample with biomarker capturing particles and optionally detecting an antigen using a detection antibody, wherein at least one of the sample, biomarker capturing particles, or detection antibody comprises the washed substance. In some embodiments, the biomarker capturing particles comprise a biomarker capturing portion. In some embodiments, the biomarker capturing particles comprise an antibody, and the biomarker comprises an antigen that binds to an epitope of the antibody. In some embodiments, the biomarker comprises an antibody, and the biomarker capturing particles comprise an antigen epitope that binds to the antibody. In some embodiments, the method includes the step of detecting an antigen using a detection antibody, and removing interference by contacting the antigen with an interference capture composition, which includes removing interference or conjugate nonspecific binding (NSB) from the detection antibody. In some embodiments, the interference capture composition includes interference capture particles, biomarker capture particles, or a combination thereof. In some embodiments, the method includes the steps of contacting the detection antibody with a sample to conjugate a detection biomarker in the sample with the detection antibody, and detecting or measuring the detection antibody conjugated to the detection biomarker. In some embodiments, the interference capture portion is in a liquid reagent. In some embodiments, the liquid reagent further includes a sample preservation reagent or stabilizer. In some embodiments, the liquid reagent further includes a sample preparation reagent or drug. In some embodiments, the interference capture portion is in a solid reagent. In some embodiments, the method includes the step of adding the capture portion to the sample. In some embodiments, the interference capture portion is present in a sample collection device before contact with the sample.In some embodiments, the interference capture portion is biotinylated. In some embodiments, the biomarker capture particles are coated with streptavidin. In some embodiments, the biomarker capture portion contains an antibody or antibody-binding fragment. In some embodiments, the biomarker comprises one or more biomarkers selected from the group consisting of antigens, antibodies, proteins, small molecules, therapeutic agents, hormones, peptides, signaling peptides, exosomes, cells, or any combination thereof. In some embodiments, the biomarker comprises an antibody, and the biomarker capture portion comprises the antigen of the antibody. In some embodiments, the antibody is selected from the group consisting of autoantibodies, therapeutic antibodies, immunoglobulin classes, immunoglobulin subclasses, circulating antibodies, secreted antibodies, alpaca-derived nanobodies, and animal-derived antibodies. In some embodiments, the biomarker comprises an antigen. In some embodiments, the biomarker capture portion comprises an antibody of the antigen. In some embodiments, the antigen comprises a therapeutic agent, drug, small molecule, peptide, protein, vaccine, or immunogen. In some embodiments, the antigen comprises a repeating epitope, and the antigen binds to one or more biomarker capture particles. In some embodiments, the antigen comprises at least a first type of epitope and a second type of epitope. In some embodiments, the biomarker-capturing particles are coated with at least a first type of antibody specific to the first type of epitope and a second type of antibody specific to the second type of epitope. In some embodiments, the biomarker comprises a disease state-specific biomarker. In some embodiments, the biomarker-capturing particles comprise agglutinated particles, which aggregate upon binding to the biomarker to form aggregated biomarker-capturing particles. In some embodiments, the aggregation is visually detectable or detectable via detection techniques. In some embodiments, the aggregated biomarker-capturing particles exhibit color. In some embodiments, the method further comprises the step of eluting the biomarker from the biomarker-capturing particles. In some embodiments, the method further comprises the step of quantifying the biomarker.In some embodiments, the step of quantifying the biomarker includes determining the mass of the biomarker per sample volume. In some embodiments, the sample includes a biological fluid. In some embodiments, the biological fluid includes blood, plasma, serum, saliva, or saline mouth rinse. In some embodiments, the interference includes lipids, triglycerides, bilirubin, hemolysis products, cholesterol, human anti-mouse antibodies (HAMA), rheumatoid interference (RF), manufacturing assay-specific interference (MASI), human anti-animal antibody (HAAA) interference, free biotin interference, anti-streptavidin interference, anti-biotin interference, human anti-polyethylene glycol (PEG) interference, anti-albumin, nonspecific binding, anti-polyvinylpyrrolidone (PVP), anti-polymers, anti-alkaline phosphatase (ALP), anti-ruthenium, anti-fluorescein, anti-acridinium esters, autoantibodies, anti-horseradine peroxidase (HRP), anti-conjugation linkers, anti-amino acid tags, anti-histidine tags, anti-polyhistidine tags, over-the-counter (OTC) supplements, herbal remedies and / or therapeutic agents, or any combination thereof. In some embodiments, the hemolysis products include hemoglobin, lactate dehydrogenase, potassium, or a combination thereof. In some embodiments, the human anti-animal antibody (HAAA) interference includes mouse immunoglobulin, goat immunoglobulin, sheep immunoglobulin, rabbit immunoglobulin, bovine immunoglobulin, or a combination thereof. In some embodiments, the anti-acridinium ester includes ABEI, luminol, isoluminol, or any combination thereof. In some embodiments, the anti-conjugation linker includes LC, LC-LC, PEOn, chromogen, or any combination thereof. In some embodiments, the method further includes the step of contacting the sample with the conjugate. In some embodiments, the method further includes the step of washing the conjugate with interference-capturing particles or biomarker-capturing particles before contacting the sample with the conjugate. In some embodiments, the sample in question is subjected to one or more adjustments to the chemical or physical properties of the sample in order to eliminate one or more interferences.In some embodiments, chemical or physical properties are selected from temperature, color, pH, salinity, conductivity, density, viscosity, surface tension, and protein content. In some embodiments, one or more adjustments include (i) releasing the analyte from the matrix or removing interfering elements by adding a surfactant, detergent, cell lysant, antiprotease agent, protein-based or polymer-based blocking reagent, substitution agent, drug, or any combination thereof to the sample, diluting the sample, or any combination thereof. In some embodiments, the interference capture portion interacts with the interference. In some embodiments, the subject is suspected of having a disease or has been vaccinated against a disease. In some embodiments, the disease includes infectious diseases. In some embodiments, the infectious disease includes viral infections, bacterial infections, or protozoan infections. In some embodiments, the disease includes tick-borne diseases. In some embodiments, the disease includes Lyme disease. In some embodiments, the disease includes celiac disease. In some embodiments, the disease includes severe acute respiratory syndrome. In some embodiments, the disease includes coronavirus infection. In some embodiments, coronavirus infection includes coronavirus disease 2019 (COVID-19). In some embodiments, washing includes washing a sample by removing interferences. In some embodiments, the vaccine is configured to produce components of a pathogen or disease. In some embodiments, the method further includes the step of determining the effectiveness of the vaccine based on the level of eluted biomarkers. In some embodiments, the method further includes the step of re-administering the vaccine to a subject based on the effectiveness of the vaccine. In some embodiments, the eluted biomarkers include antibodies against a pathogen or disease. In some embodiments, the method further includes the step of identifying the likelihood that a subject has the disease based on the eluted biomarkers. In some embodiments, the method further includes the step of identifying the likelihood that the disease is active or acute. In some embodiments, if the subject is identified as having an active or acute disease, the method further includes the step of administering disease treatment to the subject.In some embodiments, the method further includes the step of not administering or discontinuing disease treatment if the subject is identified as not having an active or acute disease. In some embodiments, the step of administering disease treatment includes adjusting the dose or timing. In some embodiments, the biomarker is secreted. In some embodiments, the biomarker includes IgA, IgG, or IgM, or a combination thereof. In some embodiments, the subject is administered a therapeutic compound. In some embodiments, the therapeutic compound includes a therapeutic agent. In some embodiments, the therapeutic compound or a fragment thereof is contained in an antigen. In some embodiments, the captured biomarker includes an autoantibody against the therapeutic compound. In some embodiments, the method further includes the step of determining the level of safety of the therapeutic compound based on a quantified biomarker. In some embodiments, the method further includes the step of administering the therapeutic compound to the subject or adjusting the dose thereof based on a quantified biomarker. In some embodiments, the therapeutic agent includes a therapeutic antibody. In some embodiments, the therapeutic antibody includes an antibody-conjugated fragment. In some embodiments, the captured biomarker includes a therapeutic antibody. In some embodiments, the antigen includes the antigen or biomarker of the therapeutic antibody. In some embodiments, the method further includes the step of determining the pharmacokinetic profile of a therapeutic antibody based on a quantified biomarker. In some embodiments, the method further includes the step of administering or adjusting the dose of a therapeutic antibody to a subject based on a quantified biomarker. In some embodiments, the subject is a human. In some embodiments, the method further includes the step of conjugating the captured biomarker with a detection antibody. In some embodiments, the method further includes the step of measuring the detection antibody. In some embodiments, the method further includes the step of comparing the detection antibody to a standard curve. In some embodiments, the standard curve is generated from biomarker-capturing particles conjugated to a known amount of biomarker. In some embodiments, the method further includes the step of washing the detection antibody with interfering capture particles before detecting the biomarker with the detection antibody.In some embodiments, the detection antibody includes an anti-human antibody. In some embodiments, the detection antibody includes an antibody against an antigen. In some embodiments, the detection antibody is conjugated to a detection reagent. In some embodiments, the detection reagent includes an enzyme or a label. In some embodiments, the label includes a fluorescent tag. In some embodiments, the method further includes the step of multiplexing biomarker-capturing particles with additional biomarker-capturing particles containing a second antigen, wherein the biomarker includes an antibody that binds to the second antigen. In some embodiments, the step of quantifying a biomarker includes multiplexing a detection antibody with a second detection antibody that recognizes a second antigen. In some embodiments, the method further includes multiplexing a detection antibody with an additional detection antibody that recognizes a biomarker in the sample. In some embodiments, the method further includes monitoring the biomarker in a first sample and a second sample over time to determine an increase or decrease in the amount of the biomarker in the second sample of interest compared to the quantified biomarker in the first sample. In some embodiments, the biomarker-capturing particles include microparticles. In some embodiments, the biomarker-capturing particles include beads. In some embodiments, the biomarker-capturing particles include metal. In some embodiments, the biomarker-capturing particles are magnetic or ferromagnetic. In some embodiments, the biomarker-capturing particles include a plurality of biomarker-capturing particles comprising (i) a plurality of magnetic beads and (ii) a plurality of non-magnetic beads, wherein the plurality of magnetic beads may differ in size from the plurality of non-magnetic beads, and one or more of the plurality of magnetic beads and one or more of the non-magnetic beads form a complex with the biomarker. In some embodiments, the concentration of the plurality of non-magnetic beads decreases upon removal of the complex. In some embodiments, washing includes washing biomarker-capturing particles by removing interference. In some embodiments, washing includes washing detection antibodies by removing interference. In some embodiments, the assay sensitivity is at least 76%. In some embodiments, the assay sensitivity is at least 99%. In some embodiments, the assay sensitivity is 100%. In some embodiments, the assay specificity is at least 76%. In some embodiments, the assay specificity is at least 99%. In some embodiments, the assay specificity is 100%.

[0005] In some embodiments, assay methods are disclosed herein that include the steps of: (i) washing a substance by contacting the substance with an interference-capturing composition to remove interference from the substance, wherein the interference-capturing composition comprises an interference-capturing portion, and the substance is selected from the group consisting of a sample derived from a subject, biomarker-capturing particles, a detection antibody, and any combination thereof; and (ii) capturing a biomarker from the sample by contacting the sample with biomarker-capturing particles and optionally detecting an antigen using a detection antibody, wherein at least one of the sample, biomarker-capturing particles, or detection antibody comprises the washed substance. In some embodiments, the biomarker-capturing particles comprise a biomarker-capturing portion. In some embodiments, the biomarker-capturing particles comprise an antibody, and the biomarker comprises an antigen that binds to an epitope of the antibody. In some embodiments, the biomarker comprises an antibody, and the biomarker-capturing particles comprise an antigen epitope that binds to the antibody. In some embodiments, the method comprises the step of detecting an antigen using a detection antibody, wherein removing interference by contacting the substance with an interference-capturing composition comprises removing interference or conjugate nonspecific binding (NSB) from the detection antibody. In some embodiments, the interference capture composition comprises interference capture particles, biomarker capture particles, or a combination thereof. In some embodiments, the method comprises the steps of: contacting a detection antibody with a sample to bind a detection biomarker in the sample to the detection antibody; and detecting or measuring the detection antibody bound to the detection biomarker. In some embodiments, the interference capture portion is in a liquid reagent. In some embodiments, the liquid reagent further comprises a sample preservation reagent or stabilizer. In some embodiments, the liquid reagent further comprises a sample preparation reagent or drug. In some embodiments, the interference capture portion is in a solid reagent. In some embodiments, the method comprises the step of adding the capture portion to a sample. In some embodiments, the interference capture portion is present in a sample collection device before contact with the sample. In some embodiments, the interference capture portion is biotinylated.In some embodiments, the biomarker-capturing particles are coated with streptavidin. In some embodiments, the biomarker-capturing portion includes an antibody or antibody-binding fragment. In some embodiments, the biomarker includes one or more biomarkers selected from the group consisting of antigens, antibodies, proteins, small molecules, therapeutic agents, hormones, peptides, signaling peptides, exosomes, cells, or any combination thereof. In some embodiments, the biomarker includes an antibody, and the biomarker-capturing portion includes the antigen of the antibody. In some embodiments, the antibody is selected from the group consisting of autoantibodies, therapeutic antibodies, immunoglobulin classes, immunoglobulin subclasses, circulating antibodies, secreted antibodies, alpaca-derived nanobodies, and animal-derived antibodies. In some embodiments, the biomarker includes an antigen. In some embodiments, the biomarker-capturing portion includes an antibody of the antigen. In some embodiments, the antigen includes a therapeutic agent, drug, small molecule, peptide, protein, vaccine, or immunogen. In some embodiments, the antigen includes a repeating epitope, and the antigen binds to one or more biomarker-capturing particles. In some embodiments, the antigen includes at least a first type of epitope and a second type of epitope. In some embodiments, the biomarker-capturing particles are coated with at least a first-type antibody specific to a first-type epitope and a second-type antibody specific to a second-type epitope. In some embodiments, the biomarker includes a disease-state-specific biomarker. In some embodiments, the biomarker-capturing particles include agglutinated particles, which aggregate upon binding to the biomarker to form aggregated biomarker-capturing particles. In some embodiments, the aggregation is visually detectable or detectable via detection techniques. In some embodiments, the aggregated biomarker-capturing particles exhibit color. In some embodiments, the method further includes the step of eluting the biomarker from the biomarker-capturing particles. In some embodiments, the method further includes the step of quantifying the biomarker.In some embodiments, the step of quantifying the biomarker includes determining the mass of the biomarker per sample volume. In some embodiments, the sample includes a biological fluid. In some embodiments, the biological fluid includes blood, plasma, serum, saliva, or saline mouth rinse. In some embodiments, the interference includes lipids, triglycerides, bilirubin, hemolysis products, cholesterol, human anti-mouse antibodies (HAMA), rheumatoid interference (RF), manufacturing assay-specific interference (MASI), human anti-animal antibody (HAAA) interference, free biotin interference, anti-streptavidin interference, anti-biotin interference, human anti-polyethylene glycol (PEG) interference, anti-albumin, nonspecific binding, anti-polyvinylpyrrolidone (PVP), anti-polymers, anti-alkaline phosphatase (ALP), anti-ruthenium, anti-fluorescein, anti-acridinium esters, autoantibodies, anti-horseradine peroxidase (HRP), anti-conjugation linkers, anti-amino acid tags, anti-histidine tags, anti-polyhistidine tags, over-the-counter (OTC) supplements, herbal remedies and / or therapeutic agents, or any combination thereof. In some embodiments, the hemolysis products include hemoglobin, lactate dehydrogenase, potassium, or a combination thereof. In some embodiments, the human anti-animal antibody (HAAA) interference includes mouse immunoglobulin, goat immunoglobulin, sheep immunoglobulin, rabbit immunoglobulin, bovine immunoglobulin, or a combination thereof. In some embodiments, the anti-acridinium ester includes ABEI, luminol, isoluminol, or any combination thereof. In some embodiments, the anti-conjugation linker includes LC, LC-LC, PEOn, chromogen, or any combination thereof. In some embodiments, the method further includes the step of contacting the sample with the conjugate. In some embodiments, the method further includes the step of washing the conjugate with interference-capturing particles or biomarker-capturing particles before contacting the sample with the conjugate. In some embodiments, the sample in question is subjected to one or more adjustments to the chemical or physical properties of the sample in order to eliminate one or more interferences.In some embodiments, chemical or physical properties are selected from temperature, color, pH, salinity, conductivity, density, viscosity, surface tension, and protein content. In some embodiments, one or more adjustments include (i) releasing the analyte from the matrix or removing interfering elements by adding a surfactant, detergent, cell lysant, antiprotease agent, protein-based or polymer-based blocking reagent, substitution agent, drug, or any combination thereof to the sample, diluting the sample, or any combination thereof. In some embodiments, the interference capture portion interacts with the interference. In some embodiments, the subject is suspected of having a disease or has been vaccinated against a disease. In some embodiments, the disease includes infectious diseases. In some embodiments, the infectious disease includes viral infections, bacterial infections, or protozoan infections. In some embodiments, the disease includes tick-borne diseases. In some embodiments, the disease includes Lyme disease. In some embodiments, the disease includes celiac disease. In some embodiments, the disease includes severe acute respiratory syndrome. In some embodiments, the disease includes coronavirus infection. In some embodiments, coronavirus infection includes coronavirus disease 2019 (COVID-19). In some embodiments, washing includes washing a sample by removing interferences. In some embodiments, the vaccine is configured to produce components of a pathogen or disease. In some embodiments, the method further includes the step of determining the effectiveness of the vaccine based on the level of eluted biomarkers. In some embodiments, the method further includes the step of re-administering the vaccine to a subject based on the effectiveness of the vaccine. In some embodiments, the eluted biomarkers include antibodies against a pathogen or disease. In some embodiments, the method further includes the step of identifying the likelihood that a subject has the disease based on the eluted biomarkers. In some embodiments, the method further includes the step of identifying the likelihood that the disease is active or acute. In some embodiments, if the subject is identified as having an active or acute disease, the method further includes the step of administering disease treatment to the subject.In some embodiments, the method further includes the step of not administering or discontinuing disease treatment if the subject is identified as not having an active or acute disease. In some embodiments, the step of administering disease treatment includes adjusting the dose or timing. In some embodiments, the biomarker is secreted. In some embodiments, the biomarker includes IgA, IgG, or IgM, or a combination thereof. In some embodiments, the subject is administered a therapeutic compound. In some embodiments, the therapeutic compound includes a therapeutic agent. In some embodiments, the therapeutic compound or a fragment thereof is contained in an antigen. In some embodiments, the captured biomarker includes an autoantibody against the therapeutic compound. In some embodiments, the method further includes the step of determining the level of safety of the therapeutic compound based on a quantified biomarker. In some embodiments, the method further includes the step of administering the therapeutic compound to the subject or adjusting the dose thereof based on a quantified biomarker. In some embodiments, the therapeutic agent includes a therapeutic antibody. In some embodiments, the therapeutic antibody includes an antibody-conjugated fragment. In some embodiments, the captured biomarker includes a therapeutic antibody. In some embodiments, the antigen includes the antigen or biomarker of the therapeutic antibody. In some embodiments, the method further includes the step of determining the pharmacokinetic profile of a therapeutic antibody based on a quantified biomarker. In some embodiments, the method further includes the step of administering or adjusting the dose of a therapeutic antibody to a subject based on a quantified biomarker. In some embodiments, the subject is a human. In some embodiments, the method further includes the step of conjugating the captured biomarker with a detection antibody. In some embodiments, the method further includes the step of measuring the detection antibody. In some embodiments, the method further includes the step of comparing the detection antibody to a standard curve. In some embodiments, the standard curve is generated from biomarker-capturing particles conjugated to a known amount of biomarker. In some embodiments, the method further includes the step of washing the detection antibody with interfering capture particles before detecting the biomarker with the detection antibody.In some embodiments, the detection antibody includes an anti-human antibody. In some embodiments, the detection antibody includes an antibody against an antigen. In some embodiments, the detection antibody is conjugated to a detection reagent. In some embodiments, the detection reagent includes an enzyme or a label. In some embodiments, the label includes a fluorescent tag. In some embodiments, the method further includes the step of multiplexing biomarker-capturing particles with additional biomarker-capturing particles containing a second antigen, wherein the biomarker includes an antibody that binds to the second antigen. In some embodiments, the step of quantifying the biomarker involves the detection antibody recognizing the second antigen. This includes multiplexing with a second detection antibody. In some embodiments, the method further includes the step of multiplexing the detection antibody with an additional detection antibody that recognizes a biomarker in the sample. In some embodiments, the method further includes the step of monitoring the biomarker in the first and second samples over time to determine whether the amount of biomarker in the second sample of interest has increased or decreased compared to the quantified biomarker in the first sample. In some embodiments, the biomarker-capturing particles include microparticles. In some embodiments, the biomarker-capturing particles include beads. In some embodiments, the biomarker-capturing particles include metal. In some embodiments, the biomarker-capturing particles are magnetic or ferromagnetic. In some embodiments, the biomarker-capturing particles include a plurality of biomarker-capturing particles comprising (i) a plurality of magnetic beads and (ii) a plurality of non-magnetic beads, wherein the plurality of magnetic beads may differ in size from the plurality of non-magnetic beads, and one or more of the plurality of magnetic beads and one or more of the non-magnetic beads form a complex with the biomarker. In some embodiments, the concentration of the plurality of non-magnetic beads decreases upon removal of the complex. In some embodiments, washing includes washing the biomarker-capturing particles by removing interference. In some embodiments, washing includes washing the detection antibody by removing interference. In some embodiments, the assay sensitivity is at least 76%. In some embodiments, the assay sensitivity is at least 99%. In some embodiments, the assay sensitivity is 100%. In some embodiments, the assay specificity is at least 76%. In some embodiments, the assay specificity is at least 99%. In some embodiments, the assay specificity is 100%.

[0006] In some embodiments, assay methods for determining the efficacy of a pharmaceutical agent are disclosed herein, comprising the steps of (i) providing a sample of a subject to be administered the pharmaceutical agent; (ii) capturing a biomarker from the sample by contacting the sample with biomarker-capturing particles comprising a biomarker-capturing portion; (iii) quantifying the eluted biomarker; and (iv) determining the efficacy of the pharmaceutical agent. In some embodiments, the step of quantifying the eluted biomarker includes detecting an antigen using a detection antibody. In some embodiments, the method further comprises the step of washing a substance by removing interference from the substance by contacting the substance with an interference-capturing composition, the interference-capturing composition comprising an interference-capturing portion, and the substance being selected from the group consisting of a sample, biomarker-capturing particles, a detection antibody, and any combination thereof. In some embodiments, the pharmaceutical agent includes a vaccine, a therapeutic agent, or any combination thereof. In some embodiments, the sample, biomarker-capturing particles, or any combination thereof are subjected to washing before capturing a biomarker from the sample, and the washing includes removing interference by contacting with an interference-capturing composition. In some embodiments, the biomarker-capturing particles comprise a biomarker-capturing portion. In some embodiments, the biomarker capturing particles include an antibody, and the biomarker includes an antigen that binds to an epitope of the antibody. In some embodiments, the biomarker includes an antibody, and the biomarker capturing particles include an antigen epitope that binds to the antibody. In some embodiments, the method includes detecting an antigen using a detection antibody, and removing interference by contacting it with an interference capturing composition, which includes removing interference or conjugate nonspecific binding (NSB) from the detection antibody. In some embodiments, the interference capturing composition includes interference capturing particles, biomarker capturing particles, or a combination thereof. In some embodiments, the method includes contacting the detection antibody with a sample to bind a detection biomarker in the sample to the detection antibody, and detecting or measuring the detection antibody bound to the detection biomarker. In some embodiments, the interference capturing portion is in a liquid reagent.In some embodiments, the liquid reagent further comprises a sample preservation reagent or stabilizer. In some embodiments, the liquid reagent further comprises a sample preparation reagent or drug. In some embodiments, the interference capture portion is in a solid reagent. In some embodiments, the method comprises the step of adding the capture portion to a sample. In some embodiments, the interference capture portion is present in a sample collection device before contact with the sample. In some embodiments, the interference capture portion is biotinylated. In some embodiments, the biomarker capture particles are coated with streptavidin. In some embodiments, the biomarker capture portion comprises an antibody or antibody-binding fragment. In some embodiments, the biomarker comprises one or more biomarkers selected from the group consisting of antigens, antibodies, proteins, small molecules, therapeutic agents, hormones, peptides, signaling peptides, exosomes, cells, or any combination thereof. In some embodiments, the biomarker comprises an antibody, and the biomarker capture portion comprises the antigen of the antibody. In some embodiments, the antibody is selected from the group consisting of autoantibodies, therapeutic antibodies, immunoglobulin classes, immunoglobulin subclasses, circulating antibodies, secretory antibodies, alpaca-derived nanobodies, and animal-derived antibodies. In some embodiments, the biomarker comprises an antigen. In some embodiments, the biomarker capture portion includes an antibody of an antigen. In some embodiments, the antigen includes a therapeutic agent, drug, small molecule, peptide, protein, vaccine, or immunogen. In some embodiments, the antigen includes a repeating epitope, and the antigen binds to one or more biomarker capture particles. In some embodiments, the antigen includes at least a first type of epitope and a second type of epitope. In some embodiments, the biomarker capture particles are coated with at least a first type antibody specific to the first type of epitope and a second type antibody specific to the second type of epitope. In some embodiments, the biomarker includes a disease state-specific biomarker. In some embodiments, the biomarker capture particles include agglutinated particles, which aggregate upon binding to the biomarker to form aggregated biomarker capture particles.In some embodiments, aggregation is visually detectable or detectable via detection techniques. In some embodiments, the aggregated biomarker-capturing particles exhibit color. In some embodiments, the method further includes the step of eluting the biomarker from the biomarker-capturing particles. In some embodiments, the method further includes the step of quantifying the biomarker. In some embodiments, the step of quantifying the biomarker includes determining the biomarker mass per sample volume. In some embodiments, the sample includes a biological fluid. In some embodiments, the biological fluid includes blood, plasma, serum, saliva, or saline mouth rinse. In some embodiments, the interference includes lipids, triglycerides, bilirubin, hemolysis products, cholesterol, human anti-mouse antibodies (HAMA), rheumatoid interference (RF), manufacturing assay-specific interference (MASI), human anti-animal antibody (HAAA) interference, free biotin interference, anti-streptavidin interference, anti-biotin interference, human anti-polyethylene glycol (PEG) interference, anti-albumin, nonspecific binding, anti-polyvinylpyrrolidone (PVP), anti-polymers, anti-alkaline phosphatase (ALP), anti-ruthenium, anti-fluorescein, anti-acridinium esters, autoantibodies, anti-horseradine peroxidase (HRP), anti-conjugation linkers, anti-amino acid tags, anti-histidine tags, anti-polyhistidine tags, over-the-counter (OTC) supplements, herbal remedies and / or therapeutic agents, or any combination thereof. In some embodiments, the hemolysis products include hemoglobin, lactate dehydrogenase, potassium, or a combination thereof. In some embodiments, the human anti-animal antibody (HAAA) interference includes mouse immunoglobulin, goat immunoglobulin, sheep immunoglobulin, rabbit immunoglobulin, bovine immunoglobulin, or a combination thereof. In some embodiments, the anti-acridinium ester includes ABEI, luminol, isoluminol, or any combination thereof. In some embodiments, the anti-conjugation linker includes LC, LC-LC, PEOn, chromogen, or any combination thereof. In some embodiments, the method further includes the step of contacting a sample with a conjugate.In some embodiments, the method further includes the step of washing the conjugate with interference-capturing particles or biomarker-capturing particles before contacting the sample with the conjugate. In some embodiments, the sample of interest is subjected to one or more adjustments to the chemical or physical properties of the sample in order to eliminate one or more interferences. In some embodiments, the chemical or physical properties are selected from temperature, color, pH, salinity, conductivity, density, viscosity, surface tension, and protein content. In some embodiments, one or more adjustments include (i) releasing the analyte from the matrix or removing the interfering element by adding a surfactant, detergent, cell lysant, antiprotease agent, protein-based or polymer-based blocking reagent, substitution agent, drug, or any combination thereof to the sample, diluting the sample, or any combination thereof. In some embodiments, the interference-capturing portion interacts with the interference. In some embodiments, the subject is suspected of having a disease or has been vaccinated against a disease. In some embodiments, the disease includes infectious diseases. In some embodiments, the infectious disease includes viral infections, bacterial infections, or protozoan infections. In some embodiments, the disease includes tick-borne diseases. In some embodiments, the disease includes Lyme disease. In some embodiments, the disease includes celiac disease. In some embodiments, the disease includes severe acute respiratory syndrome. In some embodiments, the disease includes coronavirus infection. In some embodiments, coronavirus infection includes coronavirus disease 2019 (COVID-19). In some embodiments, washing includes washing a sample by removing interference. In some embodiments, the vaccine is configured to produce components of a pathogen or disease. In some embodiments, the method further includes the step of determining the efficacy of the vaccine based on the level of eluted biomarkers. In some embodiments, the method further includes the step of re-administering the vaccine to a subject based on the efficacy of the vaccine. In some embodiments, the eluted biomarkers include antibodies against a pathogen or disease.In some embodiments, the method further includes a step of identifying the likelihood that a subject has a disease based on eluted biomarkers. In some embodiments, the method further includes a step of identifying the likelihood that the disease is active or acute. In some embodiments, if the subject is identified as having an active or acute disease, the method further includes a step of administering a disease treatment to the subject. In some embodiments, if the subject is identified as not having an active or acute disease, the method further includes a step of not administering or discontinuing a disease treatment. In some embodiments, the step of administering a disease treatment includes adjusting the dose or timing. In some embodiments, the biomarker is secreted. In some embodiments, the biomarker includes IgA, IgG, or IgM, or a combination thereof. In some embodiments, the subject is administered a therapeutic compound. In some embodiments, the therapeutic compound includes a therapeutic agent. In some embodiments, the therapeutic compound or a fragment thereof is contained in an antigen. In some embodiments, the captured biomarker includes an autoantibody against the therapeutic compound. In some embodiments, the method further includes a step of determining the level of safety of the therapeutic compound based on quantified biomarkers. In some embodiments, the method further includes the step of administering a therapeutic compound to a subject or adjusting the dose thereof based on a quantified biomarker. In some embodiments, the therapeutic agent comprises a therapeutic antibody. In some embodiments, the therapeutic antibody comprises an antibody-conjugated fragment. In some embodiments, the captured biomarker comprises a therapeutic antibody. In some embodiments, the antigen comprises an antigen or biomarker of the therapeutic antibody. In some embodiments, the method further includes the step of determining the pharmacokinetic profile of the therapeutic antibody based on a quantified biomarker. In some embodiments, the method further includes the step of administering a therapeutic antibody to a subject or adjusting the dose thereof based on a quantified biomarker. In some embodiments, the subject is human. In some embodiments, the method further includes the step of conjugating the captured biomarker with a detection antibody. In some embodiments, the method further includes the step of measuring the detection antibody.In some embodiments, the method further includes the step of comparing the detection antibody with a standard curve. In some embodiments, the standard curve is generated from biomarker-capturing particles bound to a known amount of biomarker. In some embodiments, the method further includes the step of washing the detection antibody with interfering capture particles before detecting the biomarker using the detection antibody. In some embodiments, the detection antibody includes an anti-human antibody. In some embodiments, the detection antibody is against an antigen. The method includes an antibody. In some embodiments, the detection antibody is conjugated to a detection reagent. In some embodiments, the detection reagent includes an enzyme or a label. In some embodiments, the label includes a fluorescent tag. In some embodiments, the method further includes the step of multiplexing a biomarker-capturing particle with an additional biomarker-capturing particle containing a second antigen, wherein the biomarker includes an antibody that binds to the second antigen. In some embodiments, the step of quantifying the biomarker includes multiplexing the detection antibody with a second detection antibody that recognizes the second antigen. In some embodiments, the method further includes the step of multiplexing the detection antibody with an additional detection antibody that recognizes a biomarker in the sample. In some embodiments, the method further includes the step of monitoring the biomarker in a first sample and a second sample over time to determine an increase or decrease in the amount of the biomarker in the second sample of interest compared to the quantified biomarker in the first sample. In some embodiments, the biomarker-capturing particle includes microparticles. In some embodiments, the biomarker-capturing particle includes beads. In some embodiments, the biomarker-capturing particle includes a metal. In some embodiments, the biomarker-capturing particle is magnetic or ferromagnetic. In some embodiments, the biomarker capturing particles comprise a plurality of biomarker capturing particles comprising (i) a plurality of magnetic beads and (ii) a plurality of non-magnetic beads, wherein the plurality of magnetic beads may differ in size from the plurality of non-magnetic beads, and one or more of the plurality of magnetic beads and one or more of the non-magnetic beads form a complex with the biomarker. In some embodiments, the concentration of the plurality of non-magnetic beads decreases upon removal of the complex. In some embodiments, washing includes washing the biomarker capturing particles by removing interference. In some embodiments, washing includes washing the detection antibody by removing interference. In some embodiments, the assay sensitivity is at least 76%. In some embodiments, the assay sensitivity is at least 99%. In some embodiments, the assay sensitivity is 100%. In some embodiments, the assay specificity is at least 76%.In some embodiments, the assay specificity is at least 99%. In some embodiments, the assay specificity is 100%.

[0007] In some embodiments, kits are disclosed herein that include (a) interference-capturing particles containing an interference-capturing moiety, and (b) biomarker-capturing particles containing a biomarker-capturing moiety. In some embodiments, the kit further includes a detection antibody. In some embodiments, the interference-capturing moiety includes human immunoglobulins, antibodies, animal antibodies, antibody fragments, polymerized antibodies, mouse antibody fragments, aptamers, proteins, enzymes, small molecules, streptavidin, avidin, neutraavidin, MIPs, polymers, conjugation linkers, or any combination thereof. In some embodiments, the biomarker-capturing moiety includes antibodies, antibody fragments, polypeptide binders, monobodies, non-immunoglobulin binders, DARPin, aphibodies, antikalin, molecularly imprinted polymers (MIPs), aptamers, chimeric antibodies, therapeutic antibodies, antigens, proteins, small molecules, therapeutic agents, hormones, peptides, signaling peptides, exosomes, cells, disease-specific antigens, antibodies, biomarkers, or any combination thereof. In some embodiments, the kit is intended for use in the methods disclosed herein. In some embodiments, the kit further includes a sample container. In some embodiments, the kit further includes instructions for use. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows an exemplary sample preparation method. [Figure 2A] Figure 2A shows an exemplary detection / measurement process. [Figure 2B] Figure 2B shows an exemplary detection / measurement process. [Figure 2C] Figure 2C shows an exemplary detection / measurement process. [Figure 2D] Figure 2D shows an exemplary detection / measurement process. [Figure 2E]Figure 2E shows an exemplary detection / measurement process. [Figure 3A] Figure 3A shows the protocol for detecting Lyme infection. [Figure 3B] Figure 3B shows the protocol for detecting Lyme infection. [Figure 4A] Figure 4A shows an image of the sample well. [Figure 4B] Figure 4B shows an image of the sample well. [Figure 4C] Figure 4C shows an image of the sample well. [Figure 4D] Figure 4D shows an image of the sample well. [Figure 4E] Figure 4E shows an image of the sample well. [Figure 4F] Figure 4F shows an image of the sample well. [Figure 4G] Figure 4G shows an image of the sample well. [Figure 4H] Figure 4H shows an image of the sample well. [Figure 4I] Figure 4I shows an image of the sample well. [Figure 4J] Figure 4J shows an image of the sample well. [Figure 5] Figure 5 shows an exemplary aggregation of biomarker-capturing particles. [Figure 6] Figure 6 shows graph data from a comparison of the capture bead signal with and without BSA beads. [Figure 7] Figure 7 is a graph showing the cleaning effectiveness of the captured beads. [Figure 8] Figure 8 is an image showing an exemplary custom catalyst 96-slot magnet. [Figure 9] Figure 9 is an image showing an exemplary custom catalyst 96-slot magnet. [Figure 10] Figure 10 is an image illustrating an exemplary pipetting technique. [Figure 11] Figure 11 shows an example calibration curve for IgG. [Figure 12A]Figures 12A - 12B show data from the COVID-19 antibody test (lU / mL) versus the GenScript® cPass™ neutralizing antibody detection kit (IU / mL). Figure 12A shows the NIBSC IgG IU / mL versus cPass SemiQuant IU / mL with a fitted line. [Figure 12B] Figures 12A - 12B show data from the COVID-19 antibody test (lU / mL) versus the GenScript® cPass™ neutralizing antibody detection kit (IU / mL). Figure 12B shows a summary of data for different parameters including 95%CI, SE, t, and p-values. [Figure 13A] Figure 13A shows exemplary calibration curves for IgG, IgM, and IgA. [Figure 13B] Figure 13A shows exemplary calibration curves for IgG, IgM, and IgA. [Figure 13C] Figure 13A shows exemplary calibration curves for IgG, IgM, and IgA. [Figure 14A] Figure 14A shows long-term test research data of antibodies. [Figure 14B] Figure 14B shows long-term test research data of antibodies. [Figure 14C] Figure 14C shows long-term test research data of antibodies. [Figure 15] Figure 15 shows the interference signal by washing with clean beads. [Figure 16] Figure 16 shows a gel photograph of SDS PAGE. [Figure 17A] Figures 17A - 17C show dose-response curves for AB40, AB42, and pTaul81, respectively. [Figure 17B] Figures 17A - 17C show dose-response curves for AB40, AB42, and pTaul81, respectively. [Figure 17C] Figures 17A - 17C show dose-response curves for AB40, AB42, and pTaul81, respectively. [Figure 18A]Figures 18A–18C show that levels of amyloid β1-40 (AB40 or AB1-40, Figure 18A), phosphorylated tau (pTau181, Figure 18B), and AB40+pTau181 (Figure 18C) were lower in SOR samples from patients with moderate Alzheimer's disease (N=2) compared to SOR samples from healthy controls (N=6). [Figure 18B] Figures 18A–18C show that levels of amyloid β1-40 (AB40 or AB1-40, Figure 18A), phosphorylated tau (pTau181, Figure 18B), and AB40+pTau181 (Figure 18C) were lower in SOR samples from patients with moderate Alzheimer's disease (N=2) compared to SOR samples from healthy controls (N=6). [Figure 18C] Figures 18A–18C show that levels of amyloid β1-40 (AB40 or AB1-40, Figure 18A), phosphorylated tau (pTau181, Figure 18B), and AB40+pTau181 (Figure 18C) were lower in SOR samples from patients with moderate Alzheimer's disease (N=2) compared to SOR samples from healthy controls (N=6). [Modes for carrying out the invention]

[0009] Methods and systems for the multiple detection and / or measurement of biomarkers in a sample are provided herein. These methods and systems may be used for rapid disease detection and / or monitoring, monitoring of vaccine efficacy and immune response, monitoring of therapeutic agents, and / or monitoring of the safety and efficacy of treatments.

[0010] definition As used herein and in the claims, the following terms have the meanings set forth below.

[0011] In relation to numbers, "about" refers to the number plus or minus 15%. The term "about" refers to a range from which 15% of the lowest value has been subtracted and 15% of the highest value has been added.

[0012] An "affinity assay" generally refers to an assay that determines the presence or absence of an analyte in a sample, and / or an assay that directly or indirectly quantifies the amount of an analyte in a sample based on specific or relatively specific interactions between the analyte and a molecule that preferentially binds to the analyte. Affinity assays include assays that rely in at least in some respects on the specific or relatively specific binding affinity of one entity to another. Affinity assays include, but are not limited to, assays that rely on the binding interaction between a receptor and a ligand, the binding interaction between an enzyme and its substrate, the binding interaction between a polynucleotide and its complement or substantial complement, and the binding interaction between a small molecule and a binding protein that specifically binds to that small molecule. Immunoassays include, for example, assays that rely on the interaction between an antigen and an antibody that recognizes the antigen. Immunoassays also include, for example, assays that use an antibody or a fragment thereof to bind to a target antigen in a sample. Affinity assays also include, for example, competitive assays and sandwich assays. Such assays include those that rely on the interaction of a surface-bound antigen to detect a target antibody in a sample, and those that rely on the interaction of a surface-bound antibody or a fragment thereof to detect a target antigen in a sample. As used herein, antigens are not limited to polypeptides or proteins, but may also include small molecules (e.g., haptens) and antibodies (e.g., antibodies can be used as antigens to produce other antibodies that recognize them). Generally, antigens as used herein include any analytes of interest in a sample immunoassayed with an antibody or a fragment thereof using the compositions or methods of this disclosure.

[0013] When used interchangeably herein, “analyte,” “target,” or “biomarker” generally refers to a molecule or complex that is detected and / or quantified. Non-exclusive examples of biomarkers include, but are not limited to, polypeptides (e.g., proteins, phosphorylated or other post-translational modified forms of proteins, antibodies, etc.), antigens, small molecules, and nucleic acids (e.g., DNA, RNA, mRNA, ribosomal RNA, microRNA, transcription factor binding sites, genomic DNA or RNA, etc.). Biomarkers may include antibodies, autoantibodies, therapeutic antibodies, immunoglobulin classes such as IgG, IgM, IgA, IgE, immunoglobulin subclasses such as IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, circulating antibodies, secreted antibodies, animal-derived antibodies, antigens, proteins, small molecules, therapeutic agents, hormones, peptides, signaling peptides, exosomes, cells, or any combination thereof. In some embodiments, biomarkers may include disease state biomarkers. In some embodiments, disease conditions may include heart disease, Alzheimer's disease, Parkinson's disease, dementia, traumatic brain injury (TBI), neurodegenerative diseases, cancer, kidney disease, autoimmune diseases, infections, sexually transmitted diseases (STDs) or sexually transmitted infections (STIs), infertility, women's health conditions, anemia, bone diseases, endocrine disorders, inflammation, metabolic diseases, therapeutic drug monitoring, sleep apnea, liver disease, respiratory diseases, or any combination thereof. In some embodiments, animal-derived antibodies may be derived from alpacas, mice, pigs, monkeys, rats, sheep, goats, cattle, or any combination thereof.

[0014] When used interchangeably herein, “biomarker capture particle,” “target capture particle,” or “capture particle” generally refers to a particle capable of interacting with a biomarker in a sample. In some embodiments, a biomarker capture particle may include a capture portion capable of interacting with a biomarker in a sample. In some embodiments, a biomarker capture particle may include a coating, such as a streptavidin coating, that is added to or present in the sample and can interact with the capture portion. In some embodiments, a biomarker capture particle may include a capture bead.

[0015] "Blocker" generally refers to proteins, polymers, surfactants, detergents, or combinations thereof. In some embodiments, the binding of the capture portion on the particles described herein (e.g., nanoparticles, microparticles) is blocked by a blocker such as a protein, polymer, surfactant, detergent, or combination thereof. Blockers include proteins such as albumin, bovine serum albumin, human serum albumin, ovalbumin, gelatin, casein, acid hydrolyzed casein, γ-globulin, purified IgG, animal serum, polyclonal antibodies, monoclonal antibodies, polymers such as polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), combinations of proteins and polymers, peptides, pegylation reagents such as (PEO)n-NHS or (PEO)n-maleimide, triblock copolymers such as Pluronic F108, F127, and F68, and Triton The selection is made from the group consisting of nonionic detergents such as X-100, polysorbate 20 (Tween-20), and Tween 80 (nonionic); zwitterionic detergents such as CHAPS; ionic detergents such as sodium dodecyl sulfate (SDS), deoxycholate, cholate, and sarcosyl; surfactants; sugars such as sucrose; and commercially available blockers such as heterophilic blocking reagents (Scantibodies), MAK33 (Roche® Diagnostics), immunoglobulin inhibitor reagent (IIR) (Bioreclamation), Heteroblock (Omega Biologicals), Blockmaster (JSR), TRUblock (Meridian Life Sciences), and StabilCoat® & StabilGuard® (Surmodics). In some embodiments, the blocker is bound to the particles described herein (e.g., covalently, non-covalently). In some embodiments, the blocker does not bind to the particles described herein (e.g., covalently, non-covalently).

[0016] When used interchangeably herein, “capture molecule,” “biomarker capture moiety,” or “target binding element” generally refer to a molecule configured to capture a specific biomarker of interest (by direct or indirect binding to the biomarker) and bound to a solid support such as a particle (e.g., microspheres, microbeads, etc.) (e.g., covalently or noncovalently, directly or via a linker, e.g., streptavidin biotin, etc.). A capture moiety may bind directly to a biomarker and may be specific to that biomarker. A capture moiety may bind to one or more molecules that bind to a biomarker and specifically capture it. Non-limiting examples of capture sites include, but are not limited to, interference, polypeptides (e.g., antibodies, SH2 and other polypeptide-binding domains, short synthetic peptides, and antigens), polynucleotides (e.g., polynucleotide capture probes, transcription factor binding sites, aptamers), antigens, biomarkers, polysaccharides, lipids, small molecules, molecularly imprinted polymers (MIPs), chimeric antibodies, therapeutic antibodies, recombinant antibodies, monoclonal antibodies, polyclonal antibodies, and their antibody fragments such as Fab, F(ab')2, Fc, scFv, alpaca-derived nanobodies, phage display VHH constructs, and engineered variants such as diabodies, triabodies, minibodies, and single-domain antibodies. Captured subparticles may be combined, mixed, or pooled in multiple or pooled different biomarker capturing particles (e.g., capture beads) to form a multiplexed capture biomarker, where each capture bead is coated with a different target binding element or capture subparticle, or with a mixture of different target binding elements or capture subparticles, and all particles are magnetic, or some particles are magnetic and others are non-magnetic.

[0017] As used herein, “specificity” refers to the ability to accurately fail to detect (measure) the level, concentration, or presence of a biomarker for a true negative result that might otherwise be reported as a false positive. In clinical trials, specificity refers to the ability of the trial to accurately identify patients who do not have the disease. Therefore, a trial with 100% specificity accurately identifies all patients who do not have the disease. In a trial, this typically means that negative results do not have a signal that exceeds background noise. For example, pre-analysis washing of samples with interfering particles can mitigate sample interference, which could otherwise cause crosslinking (e.g., HAMA or RF) of human immunoglobulins (e.g., hIgG, hIgA, hIgM, hIgE classes, or hIgA1, hIgA2, hIgG1, hIgG2, hIgG3, hIgH4 subclasses) to the solid-phase biomarker-capturing portion, or heterophilic autoantibodies or NSBs, resulting in false high signals or false positive results (HAMA-like or RF-like crosslinking of the conjugate to the solid phase causes false high signals in sandwich immunoassays, and heterophilic immunoglobulins, autoantibodies, or NSBs cause false high signals in serological, antibody, or autoantibody assays). If such interference is washed away, mitigated, or reduced within the assay blocking threshold or assay design, the biomarker will not be detected by the conjugate for true negative results. In some embodiments, methods are disclosed herein that can provide a specificity of at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0018] As used herein, “sensitivity” refers to the ability to accurately detect (measure) the level, concentration, or presence of a biomarker for a true positive result that might otherwise be overlooked and reported as a false negative. In clinical trials, sensitivity refers to the ability of a test to accurately identify patients with the disease. A test with 100% sensitivity accurately identifies all patients with the disease. In a test, this typically means that a positive result has a signal that exceeds background noise. For example, pre-analysis washing of a sample with interfering capture particles can sterically block the solid-phase biomarker capture portion or compete for binding to the solid-phase capture reagent (e.g., free biotin interference competes with a biotin-labeled antibody or antigen for binding to the streptavidin solid phase or conjugate), mitigating sample interference that could lead to false low-signal or false-negative results. If such interference is washed, mitigated, or reduced within the assay blocking threshold or assay design, the biomarker is accurately captured by the solid phase and subsequently detected by the conjugate for a true positive result. This specification discloses methods that can improve the sensitivity or potential of biomarker detection, particularly in the case of low-abundance biomarkers or biomarkers at concentrations below the detection limit (<Limit of Detection (LoD) or <Limit of Quantification (LoQ)) of the current assay, by capturing and purifying biomarkers from a sample using antibody or antigen-coated biomarker-capturing particles into a matrix-free buffer for subsequent biomarker detection. In some embodiments, the methods disclosed herein can capture, purify, concentrate, or concentrate biomarkers to smaller volumes, resulting in a six-fold or greater increase in the biomarker level, which can then be detected above the LoD or LoQ of the assay. In other words, the interference of washing the sample allows subsequent biomarker-capturing particles to accurately detect and capture targeted biomarkers in the washed sample matrix for subsequent purification and concentration for high-sensitivity detection.In some embodiments, methods are disclosed herein that can provide sensitivity of at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0019] As used herein, "accuracy" may refer to specificity, sensitivity, or a combination thereof.

[0020] The terms "coupled with," "bound with," or their grammatical equivalents generally refer to a covalent bond (either directly or indirectly, via one or more carbon-carbon, carbon-nitrogen, carbon-oxygen bonds, etc.) or non-covalent bond (either directly or indirectly) or interaction between two parts or entities. The term "coupled with" is not intended to imply the orientation or direction of the bond. Entities known to interact specifically with each other can be covalently bonded. One non-limiting example of entities known to interact specifically and that can be covalently bonded is an antigen and its specific antibody, which can be made to be covalently bonded, for example, by bonding chemistry. Non-covalent bonds can include affinity, ionicity, van der Waals (e.g., dipole / dipole forces or London forces), hydrogen bonds (e.g., between polynucleotide double chains), and hydrophobic interactions. When the bond is non-covalent, the association between entities is preferably specific. Non-limiting examples of specific non-covalent associations include binding interactions between biotin and biotin-binding proteins (e.g., avidin, SA, neutraavidin, SA fragments, avidin fragments, neutraavidin fragments, or mixtures thereof); binding of biotinylated Fab, biotinylated immunoglobulins or fragments thereof, biotinylated small molecules (e.g., receptor hormones or ligands), biotinylated polynucleotides, biotinylated macromolecules (e.g., proteins or natural or synthetic polymers) to biotin-binding proteins such as avidin, SA, neutraavidin, SA fragments, avidin fragments, neutraavidin fragments, or mixtures thereof; binding of substrates to enzymes; binding of glycoproteins to glycoprotein-specific lectins; binding of ligands to ligand-specific receptors; binding of antibodies to antigens that produce antibodies; and double-strand formation between polynucleotides and polynucleotides that are complementary or substantially complementary to each other.

[0021] "Determining," "measuring," "evaluating," "assessing," "assaying," or "analyzing" generally refer to forms of measurement, as used interchangeably herein. These terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative, semi-quantitative, qualitative, or quantitative and qualitative determinations. Assessments may be relative or absolute. "Detecting the presence of" may, depending on the context, include determining the quantity of something that is present, in addition to determining whether it is present or not.

[0022] "Diagnostic" or "test" generally refers to chromatographic methods such as immunohistochemical extraction (IE) and solid-phase extraction (SPE), spectrophotometric and mass spectrometry (i.e., HPLC, MS, LC-MS, LC-MS / MS, MALDI-TOF), radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), chemiluminescent immunoassay (CLIA), fluorescence immunoassay (FIA), turbidimetric or particle-enhanced turbidimetric immunoassay (PETIA), and chemical and molecular diagnostics such as flow cytometry (PCR synthesis, RT-PCR, PCR-ELISA, fluorescence immunoassay). This refers to any antibody-based diagnostic test, non-antibody-based diagnostic test, sample preparation methods or devices for subsequent analysis, by any test for monitoring such as diagnosis, prognosis, screening, risk assessment, risk stratification, and therapeutic drug monitoring, including situ hybridization, next-generation sequencing (NGS), lateral flow (LF), microarrays, multiplex testing, point-of-care (PoC), direct-to-consumer (DTC), CLIA and CLIA-neglected tests and devices, research-use only (RUO) tests, in vitro diagnostic (IVD) tests, laboratory-developed (LDT) tests, companion diagnostics, and any test for monitoring such as diagnosis, prognosis, screening, risk assessment, risk stratification, and therapeutic drug monitoring.

[0023] "Interference" generally refers to substances or conditions present in a sample, such as a biological sample, that can alter the correct value of the result by interfering with the capture portion or particles, or increase or decrease the assay signal through cross-linking, steric hindrance, or autoantibody mechanisms. False results can occur unexpectedly in any sample without practical means to conspicuously identify such specimens that may cause problems. The consequences of such interference are that false results may affect patient care, potentially leading to unnecessary invasive, diagnostic, or therapeutic procedures, or the inability to treat patients with false-negative test results. Examples of interference include autoantibodies such as polyclonal and / or monoclonal antibodies of goats, rabbits, sheep, cattle, mice, horses, pigs, and donkeys; dependent affinity or dependent affinity-like interference such as rheumatoid factor (RF), human anti-mouse antibody (HAMA), and human anti-animal antibody (HAAA); as well as chemiluminescent substrates (isoluminol, luminol, ABEI, ruthenium, acridinium esters), fluorescent labels (fluorescein or other fluorophores and dyes), and anti-alkali interference. Phosphatases (ALP), anti-horseradish peroxidase (HRP), antihistidines, capture moieties (streptavidin, neutraavidin, avidin, polyA, polyDT, aptamers, antibodies, Fab, F(ab')2, antibody fragments, recombinant proteins, enzymes, proteins, biomolecules, polymers) and their binding partners (i.e., biotin, fluorescein, PolyDT, PolyA, antigens, etc.), conjugation linkers (LC, LC-LC, PEO, PEO) n), and solid-phase blocking proteins (bovine serum albumin, human serum albumin, ovalbumin, gelatin, purified poly and monoclonal IgG from mouse, goat, sheep and rabbit, etc., polyvinyl alcohol or PAA, polyvinylpyrrolidone or PVP, Tween-20, Tween-80, TritonTriblock copolymers such as X-100, Pluronic, and Tetronic, as well as other commercially available blockers, blocking proteins, and polymer-based blocking reagents such as those from Surmodics and Scantibodies; anti-amino acid tags, recombinant tags, or affinity tags on proteins; peptides, antigens, antibodies, e.g., poly(his) tags (6-his tags, 8-his tags), Strep tags, chitin-binding proteins (CBP), maltose-binding proteins (MBP), glutathione-S-transferase (GST), epitope tags including FLAG tags, ALFA tags, V5 tags, Myc tags, HA tags, Spot tags, T7 tags, and NE tags; green fluorescent protein (GFP); lipids, triglycerides, bilirubin, hemolytic products (e.g., hemoglobin, enzymes, potassium), cholesterol, free biotin interference, anti-biotin interference, human anti-polyethylene glycol (PEG) interference, anti-albumin, nonspecific binding (NSB), anti-polymer interference Examples of manufacturing assay-specific interference include body, anti-polyvinylpyrrolidone (PVP) antibodies, anti-polyvinyl alcohol (PVA), autoantibodies, anti-conjugation linkers such as LC, LC-LC, and PEOn, over-the-counter (OTC) supplements, herbal remedies, or therapeutic agents that may cause problems or erroneous results that adversely affect test performance or accuracy, non-antibody-based diagnostic tests such as molecular diagnostics or mass spectrometry (i.e., HPLC, MS, LCMS, LC-MS / MS), or antibody-based tests such as radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), chemiluminescence immunoassays (CLIA), and fluorescence immunoassays (FIA), chemistry such as turbidimetric or particle-enhanced turbidimetric immunoassays (PETIA), antibody-oligoconjugates in immunoPCR, lateral flow, flow cytometry, point-of-care (PoC), and CLIA and CLIA used in test designs or assay formulations that have been abandoned.

[0024] An “interference capture composition” generally refers to a composition comprising one or more interference capture portions that interact with a sample interference such that the interference interacts with and binds to the interference capture composition when exposed to the interference capture composition. In some embodiments, the interference capture composition may comprise “interference capture particles” or “clean beads,” as used interchangeably herein. In some embodiments, the interference capture particles may comprise particles described herein comprising interference capture portions. In some embodiments, when the interference capture particles are isolated or removed from the sample via magnetic coupling, magnetic centrifugation, centrifugation, or filtration, the sample, essentially free of particles, will have a significantly lower level, concentration, threshold, or titer of interference such that it no longer interferes with the testing, measurement, or characterization of the sample.Examples of interference capture moieties include human immunoglobulins (IgA, IgG, IgM, IgE) targeting autoantibody interference, animal antibodies (mouse, goat, sheep, rabbit, cattle, llama, alpaca) targeting heteroaffinity antibodies such as HAMA, RF, and HAAA, polymerized antibodies or mouse antibody fragments (Fc, Fab, F(ab')2) targeting HAMA and RF interference, small molecules targeting proteins, enzymes, or anti-signaling interference (ALP, HRP, fluorescein / fluorophores, luminol, isoluminol, acridinium esters, ABEI, ruthenium, luciferin), streptavidin, avidin, or neutraavidin (bisnorbiotin, biotin sulfoxide) targeting biotin interference or biotin metabolite interference, and anti Examples of sample interferences include polymers that target specific interference via epitope binding of bodies, aptamers, antibody fragments, MIPs, or bilirubin, albumin, lipids, triglycerides, cholesterol, jaundice (bile pigments), hemoglobin, herbs (e.g., hemolytic products, stipulation, jaundice, tube additives, administration of radioactive or fluorescent compounds, drugs, herbal medicines, and dietary supplements are all exogenous interferences that can adversely affect immunoassays), conjugation linkers such as LC, LC-LC, PEOn, PEG, polyhistidine (His tag), bead polymers or copolymers, iron oxide or exposed iron crystals on the surface, PVP used as a wetting agent for polymerization, functional groups, or the beads themselves as some sample interferences that interact with any NSB interactions with the beads.

[0025] "Magnetic particle" or "magnetic bead" generally refers to a particle or bead that can be attracted to or may be attracted to a magnetic field, respectively. Magnetic particles or magnetic beads may include a magnetic core, such as a magnetic metal oxide core. Magnetic particles or magnetic beads may include paramagnetic particles that are only slightly attracted to a magnetic field and do not retain magnetic properties when the external field is removed.

[0026] "Non-specific binding" generally refers to the binding of different molecules (e.g., different types, different sizes, different nucleotide sequences, etc.) that have roughly the same affinity for a particle.

[0027] "Particles" generally refer to substances in solid form, such as beads, magnetic beads, latex beads, fluorescent beads, streptavidin beads, multiplex immunoassay beads, nanobeads, microbeads, bead surface chemistry, antibody-coated beads, or antigen-coated beads. Particles can have a variety of shapes, which may be regular or irregular. Particles can have sizes ranging from about 10 nanometers to about 100 micrometers. Particles may include adsorbed or covalently bonded silane coatings on their surface, for example, a wide variety of biocompatible adsorbents covalently bonded by a selected coupling chemical reaction, thereby coating the surface of the particles with functional groups. Suitable silanes useful for coating particle surfaces, such as aminosilanes, include p-aminopropyltrimethoxysilane, N-2-aminoethyl-3-aminopropyltrimethoxysilane, triamino-functional silanes, n-dodecyltriethoxysilane, and n-hexyltrimethoxysilane. Alternatively, or in addition, particles may not have any coating on their surface.

[0028] The saliva test generally detects autoantibodies against p53, free PSA, total PSA, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), beta-2-microglobulin (B2M), beta-human chorionic gonadotropin (beta-hCG), bladder tumor antigen (BTA), CA125, CA15-3, CA19-9, CA50, CA72-4, and CA242, as well as Campylobacter infection, hepatitis, hepatitis B, hepatitis C, novel coronavirus (COVID-19), influenza (Flu), measles, meningococcal disease, HPV, HSV, HIV, and R Infections such as SV (infectious disease), sexually transmitted infections or STIs (STDs or STIs) such as syphilis, hepatitis, trichomoniasis, gonorea, chlamydia, and human immunodeficiency virus (HIV), autoimmune diseases such as celiac disease, vector-borne diseases such as Lyme disease, Zika, dengue, and malaria, or amyloid-beta or β-amyloid (1-40, 1-42), total tau, phosphorylated tau 181, 205, 212, 217, or 231, neuronal filament light (NfL), glial acid fibrillary protein (GFAP), NSE (neuron-specific This refers to the use of saliva or saline oral rinse collection at home, or saliva collection at a doctor's office, retailer (Walgreens, CVS, Walmart, etc.), or specialist clinician (neurologist) saliva collection for age-based risk screening by detecting disease-specific biomarkers in saliva, such as neuronal markers that suggest or show symptoms of neurodegenerative diseases (Alzheimer's disease or Parkinson's disease), such as enolase, sTREM2 (Triggering receptor expressed on myeloid cells 2), α-synuclein, neurograin, NPTX2 (Neuronal penttraxin-2), BACE-1 (Beta-secretase 1), which help diagnose disease or detect early onset of disease, or tumor-specific antigens that suggest or show symptoms of cancer onset, cancer stage or progression, or cancer recurrence.Saliva-based testing may also include testing athletes suspected of traumatic brain injury (TBI) or concussion, or rubbing in / out TBI or concussion in the emergency room (ER) or emergency department (ED) for victims of car accidents, falls, explosions or impacts, or Shark'n Baby Syndrome, requiring accurate and highly sensitive detection of neuronal markers in saliva-based samples. When target capture beads are added to a saliva or saline mouth rinse in a collection device, the target capture beads may maximize the recovery of biomarkers during or in the collection device, thereby treating, exposing, or incubating the entire or whole sample with the target capture beads. This is important for any biomarkers that could otherwise interact with the collection device or sample tube surface hydrophobically or via NSBs, and which, in the absence of target capture beads, could be lost in the sample or no longer detectable, thus maximizing their capture and recovery from the sample. Target capture beads may contain different antibodies that bind to one or more different biomarkers or panels of biomarkers during pre-analytical capture in the collection device or sample tube. Furthermore, if these biomarkers are also present inside cells, extracellular particles, exosomes, virions, or bacteria, they can also be lysed in the presence of target capture beads to maximize the capture efficiency and yield of the total biomarkers from the sample. Since the entire sample is exposed to antibody-coated target capture beads with or without lysis buffer or drugs, this can also improve the capture of low-abundance biomarkers for subsequent concentration or enrichment (e.g., instead of the sample volume being limited to fit the sample volume requirements specific to the test system, such as 5–300 μL, an entire 1–5 mL saliva sample is the sample for neuronal marker capture). This novel approach to capturing neuronal markers during or after saliva-based sample collection in collection kits maximizes the sensitivity of their subsequent detection.

[0029] "Sample" or "biofluid" generally refers to any human or animal serum, plasma (i.e., EDTA, lithium heparin, sodium citrate), blood, whole blood, processed blood, semen or seminal plasma, cells, tissues, biopsy material, DNA, RNA, or any fluid, dissolved solid, processed solid material, oral fluid such as oral mucosal exudate (OMT), saliva (passively drawn or swab), oral samples (collected by swab or brush), saline mouth rinse (SOR) or mouth rinse, gingival crevice fluid (GCF), sputum, sweat, tears, mucus, urine, feces (liquid and / or solid), vaginal fluid, milk, cerebrospinal fluid, peritoneal fluid, pleural fluid, or digestive fluids tested for diagnosis, prognosis, screening, risk assessment, risk stratification, and monitoring such as therapeutic drug monitoring.

[0030] The terms “subject,” “individual,” and “patient” are often used interchangeably herein. “Subject” may be a biological entity. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject may be a tissue, cell, or offspring of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed or suspected of being at high risk of disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk of disease.

[0031] The terms "treatment" or "treating" are used in reference to pharmaceutical or other intervention regimens aimed at obtaining beneficial or desired outcomes in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic and / or preventive benefits. A therapeutic benefit may refer to the eradication or improvement of the symptom or underlying condition being treated. A therapeutic benefit may also be achieved by the eradication or improvement of one or more physiological symptoms associated with an underlying disorder, such that improvement is observed in the subject, even though the subject may still be suffering from the underlying disorder. Preventive effects include delaying, preventing, or eliminating the onset of a disease or illness; delaying or eliminating the onset of symptoms of a disease or illness; delaying, halting, or reversing the progression of a disease or illness; or any combination thereof. For a preventive benefit, a subject at risk of developing a particular disease, or who reports one or more physiological symptoms of a disease, may receive treatment, even if a diagnosis of the disease has not been made.

[0032] The section headings used herein are for organizational purposes only and should not be interpreted as limiting the subjects described.

[0033] Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art in the field relating to the claimed subject matter. Where applicable, terms having a commonly understood meaning are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the generally understood meaning in the art.

[0034] Throughout this application, various embodiments may be presented in scope form. It should be understood that the scope form is merely for convenience and brevity and should not be interpreted as an inflexible limitation to the scope of this disclosure. Therefore, a scope description should be considered to have all possible sub-scopes specifically disclosed, as well as the individual numbers within those scopes. For example, a scope description such as 1–6 should be considered to specifically disclose sub-scopes such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, as well as the individual numbers within those scopes, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0035] As used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. For example, the term "sample" includes multiple samples and mixtures thereof. Similarly, the plural forms of terms, such as "biomarker," include both singular and plural forms (i.e., one or more) and mixtures thereof, unless the context clearly indicates otherwise.

[0036] Methods and systems for multiple detection and / or measurement This disclosure provides a method and system for multiple detection and / or measurement of one or more biomarkers in a biological sample with high accuracy and efficiency.

[0037] In one embodiment, the present disclosure provides a method for detecting and / or measuring one or more biomarkers in a biological sample, comprising the steps of (a) providing a sample to be measured and (b) capturing a biomarker from the sample by contacting the sample with biomarker-capturing particles (a “capture” or “capturing” process).

[0038] In some embodiments, the biological sample is washed by removing assay interference from the sample containing interfering particles, or is subjected to washing and preparation before (b).

[0039] sample In some embodiments, the sample may include serum, plasma (i.e., EDTA, lithium heparin, sodium citrate), blood, whole blood, processed blood, semen or seminal plasma, cells, tissues, biopsy material, DNA, RNA, or any fluid, dissolved solid, processed solid material, oral fluid such as oral mucosal exudate (OMT), saliva (passively drawn or swab), oral samples (collected by swab or brush), saline mouth rinse (SOR) or mouth rinse, gingival crevicular fluid (GCF), sputum, sweat, tears, mucus, urine, feces (liquid and / or solid), vaginal fluid, milk, cerebrospinal fluid, peritoneal fluid, pleural fluid, and digestive fluids. In some embodiments, the sample may include human-derived samples. In some embodiments, the sample may include animal-derived samples.

[0040] In some embodiments, the sample contains a volume of approximately 10 μL to approximately 100 mL. In some embodiments, the volume of the sample is approximately 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 550 μL, 600 μL, 650 μL, 700 μL, 750 μL, 800 μL, 850 μL, 900 μL, and 900 μL. The volume may be 50 μL, approximately 1 mL, approximately 1.5 mL, approximately 2 mL, approximately 2.5 mL, approximately 3 mL, approximately 3.5 mL, approximately 4 mL, approximately 4.5 mL, approximately 5 mL, approximately 5.5 mL, approximately 6 mL, approximately 6.5 mL, approximately 7 mL, approximately 7.5 mL, approximately 8 mL, approximately 8.5 mL, approximately 9 mL, approximately 9.5 mL, approximately 10 mL, approximately 20 mL, approximately 40 mL, approximately 50 mL, approximately 75 mL, approximately 100 mL, approximately 150 mL, or approximately 200 mL, or within a range defined by any of the aforementioned amounts. Sample volume must be at least 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 550 μL, 600 μL, 650 μL, 700 μL, 750 μL, 800 μL, 850 μL, or 900 μL. L, minimum 950 μL, minimum 1 mL, minimum 1.5 mL, minimum 2 mL, minimum 2.5 mL, minimum 3 mL, minimum 3.5 mL, minimum 4 mL, minimum 4.5 mL, minimum 5 mL, minimum 5.5 mL, minimum 6 mL, minimum 6.5 mL, minimum 7 mL, maximum May include as low as 7.5 mL, as low as 8 mL, as low as 8.5 mL, as low as 9 mL, as low as 9.5 mL, as low as 10 mL, as low as 20 mL, as low as 40 mL, as low as 50 mL, as low as 75 mL, as low as 100 mL, as low as 150 mL, or as low as 200 mL.In some embodiments, the sample volume is less than 10 μL, less than 20 μL, less than 30 μL, less than 40 μL, less than 50 μL, less than 60 μL, less than 70 μL, less than 80 μL, less than 90 μL, less than 100 μL, less than 150 μL, less than 200 μL, less than 250 μL, less than 300 μL, less than 350 μL, less than 400 μL, less than 450 μL, less than 500 μL, less than 550 μL, less than 600 μL, less than 650 μL, less than 700 μL, less than 750 μL, less than 800 μL, less than 850 μL The sample volume is less than 900 μL, less than 950 μL, less than 1 mL, less than 1.5 mL, less than 2 mL, less than 2.5 mL, less than 3 mL, less than 3.5 mL, less than 4 mL, less than 4.5 mL, less than 5 mL, less than 5.5 mL, less than 6 mL, less than 6.5 mL, less than 7 mL, less than 7.5 mL, less than 8 mL, less than 8.5 mL, less than 9 mL, less than 9.5 mL, less than 10 mL, less than 20 mL, less than 40 mL, less than 50 mL, less than 75 mL, less than 100 mL, less than 150 mL, or less than 200 mL. In some embodiments, the sample may contain a volume of less than 200 μL. In some cases, the sample is a point-of-care (POC) finger puncture sample, a pediatric sample, a geriatric sample, or an IV drug user sample, where blood collection is difficult. In some embodiments, the sample may contain a volume of about 200 μL to about 5 mL. In some embodiments, the sample may contain a volume greater than approximately 5 mL, and a larger volume may be required for biomarker enrichment.

[0041] In some embodiments, the sample includes biological fluids. In some embodiments, biological fluids include whole blood, plasma, serum, oral fluid (saliva, drol, oral mucosal exudate (OMT), or oral rinse, e.g., saline oral rinse), mucus, urine, semen, vaginal fluid, milk, cerebrospinal fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid (CSF), tissue, sweat, tears, or digestive fluids.

[0042] subject In some embodiments, subjects are suspected of having a disease, have been vaccinated against a disease, or have been screened for a disease. In some embodiments, subjects have a disease. In some embodiments, the disease may include infectious diseases, viral infections, bacterial infections, protozoan infections, tick-borne diseases, Lyme disease, severe acute respiratory syndrome, or coronavirus infections, such as coronavirus disease 2019 (COVID-19).

[0043] In some embodiments, the disease includes infectious diseases. In some embodiments, the disease includes viral infections, bacterial infections, or protozoan infections. In some embodiments, the disease includes tick-borne diseases. In some embodiments, the disease includes Lyme disease. In some embodiments, the disease includes severe acute respiratory syndrome, the disease includes coronavirus infection, and coronavirus infection includes coronavirus disease 2019 (COVID-19).

[0044] In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0045] biomarkers In some embodiments, the biomarker may include antibodies, autoantibodies, therapeutic antibodies, immunoglobulin classes such as IgG, IgM, IgA, and IgE, immunoglobulin subclasses such as IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4, circulating antibodies, secretory antibodies, and alpaca-derived nanobodies, as well as animal-derived antibodies such as those from mice, pigs, monkeys, rats, sheep, goats, or cattle.

[0046] In some embodiments, therapeutic antibodies may include FDA-approved monoclonal antibody drugs, such as infliximab for Crohn's disease, rituximab for lymphoma, ustekinumab for psoriasis, and tocilizumab for the treatment of rheumatoid arthritis, as well as FDA-approved antibody drugs for emergency use, such as bebuterobimab, a monoclonal antibody against the SARS-CoV-2 spike protein.

[0047] In some embodiments, the biomarker may include an antigen, pathogen, protein, small molecule, therapeutic agent, hormone, peptide, signaling peptide, exosome, or cell.

[0048] In some embodiments, biomarkers may include disease-specific antigens, antibodies, or biomarkers for conditions such as heart disease, Alzheimer's disease, traumatic brain injury (TBI), neurodegenerative diseases, cancer, kidney disease, autoimmune diseases, infections, bacterial infections, viral infections, fungal infections, sexually transmitted diseases (STDs) or sexually transmitted infections (STIs), fertility issues, anemia, endocrinology, inflammation, metabolic disorders, sleep apnea, liver disease, or respiratory diseases. Detection of biomarkers may be used for the diagnosis or prognosis of diseases.

[0049] In some embodiments, the biomarkers may include neuronal markers for Alzheimer's disease or neurodegenerative diseases. In some embodiments, the neuronal markers may include amyloid-beta or β-amyloid (1-38, 1-40, 1-42), also known as Abeta38, Abeta40, or Abeta42, total tau, aggregates of hyperphosphorylated tau protein, or PHF-Tau(Ser202 / Thr205), PHF-Tau(Thr181), PHF-Ta, also known as phosphorylated tau 181 (pTaul81) or 231 (pTau231). Paired helical filaments (PHF-Tau) such as u(Thr217) and PHF-Tau(Thr231), neuronal filament light (NfL), glial acid fibrillary protein (GFAP), NSE (neuron-specific enolase), sTREM2 (trigger receptor expressed on myeloid cells), α-synuclein, neurogranin, NPTX2 (neuronal pentraxin-2), and BACE-1 (β-secretase 1). Neuronal markers for traumatic brain injury (TBI) or concussion may include S100B, γ-enolase (NSE), α-II spectrin, astroglial protein, NfL, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), and tau.

[0050] In some embodiments, the biomarker may include one or more biomarkers, such as antibodies, antigens, and therapeutic agents, or a combination thereof, in multiple detection and / or measurement. Multiple detection and / or measurement of different biomarkers may be used, in particular, in the diagnosis of disease staging, monitoring of vaccine effectiveness, and monitoring of treatment effectiveness.

[0051] In some embodiments, the biomarker-capturing particles may include beads, for example, biomarker-capturing beads. In some embodiments, the biomarker-capturing particles may include magnetic particles or paramagnetic particles. In some embodiments, the biomarker-capturing particles may include non-magnetic particles. In some embodiments, the biomarker-capturing particles described herein may have an average diameter of about 0.010 micrometers (μm) to about 3.00 micrometers, or preferably 0.05 micrometers to 2.8 micrometers in diameter, or more preferably 0.05 micrometers to 1.6 micrometers in diameter, or preferably about 0.05 micrometers to about 0.55 micrometers in diameter.

[0052] particle In some embodiments, the particles may include fine particles. In some embodiments, the particles may include nanoparticles. In some embodiments, the particles may include beads. In some embodiments, the particles may include metal. In some embodiments, the particles may be magnetic.

[0053] In some embodiments, the particles described herein (e.g., fine particles, nanoparticles) may include a core or a support. In some embodiments, the core or support may be a paramagnetic or superparamagnetic material that can be subjected to forces in a magnetic field gradient but is not permanently magnetized. Non-limiting examples of paramagnetic or superparamagnetic materials may include iron oxide, ferromagnetic iron oxide, Fe2O3 or Fe3O4, maghemite, or combinations thereof.

[0054] In some embodiments, the core or support may include ceramics, glass, latex, silica, metals, alloys, colloidal metals such as gold, silver or alloys, or polymers.

[0055] In some embodiments, the particles may comprise an organic polymer or copolymer. In some embodiments, the organic polymer or copolymer is hydrophobic. In some embodiments, the organic polymer or copolymer may comprise, but is not limited to, materials selected from the group consisting of polystyrene, derivatized polystyrene, poly(divinylbenzene), styrene-acylate copolymer, styrene-butadiene copolymer, styrene-divinylbenzene copolymer, poly(styrene-oxyethylene), polymethyl methacrylate, polymethacrylate, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, N,N'-methylenebisacrylamide, polyolefin, polyethylene, polypropylene, polyvinyl chloride, polyacrylonitrile, polysulfone, poly(ethersulfone), pyrolytic materials, block copolymers, and copolymers of the aforementioned, silicone, or biodegradable polymers such as silica, methylolmelamine, dextran, or poly(ethylene glycol)-dextran (PEG-DEX), or combinations thereof.

[0056] In some embodiments, the surface of the particles may contain one or more functional groups for covalent bonding (coupling, conjugation, or bonding) of binders, bonding partners, capture moieties, or combinations thereof, such as carboxyl, tosyl, epoxy, amine, sulfhydryl, hydroxyl, ester, and maleimide; click chemistry functionalities [copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), strain-enhanced azide-alkyne cycloaddition (SPAAC), strain-enhanced alkyne-nitrone cycloaddition (SPANC), and alkenes and azide[3+2] cycloaddition, alkenes and tetrazine reverse demand Diels-Alder, and alkenes and tetrazole photoclick reactions], hydrazone coupling functional groups such as S-HyNic (succinimidyl-6-hydrazino-nicotinamide) and S-4FB (N-succinimidyl-4-formylbenzamide) heterobifunctional crosslinking agents, as well as photoreactive chemistry functionalities such as one or more functional groups for covalent bonding (coupling, conjugation, or bonding) of binders, bonding partners, capture moieties, or combinations thereof.

[0057] In some embodiments, the biomarker-capturing particles may include any preferred particles described herein or any combination thereof.

[0058] In some embodiments, the biomarker-capturing particles may include a capture portion. In some embodiments, the capture portion may be coated onto the surface of the biomarker-capturing particles. In some embodiments, the capture portion may be covalently or non-covalently bonded to the biomarker-capturing particles. The covalent bond may include a bond via one or more functional groups selected from the group consisting of carboxyl, hydroxyl, tosyl, epoxy, aldehyde, amine, amide, amino, hydrazide, isothiocyanate, maleimide, and sulfhydryl, with any preferred bonding chemistry. In some embodiments, the capture portion may be bonded to the biomarker-capturing particles using an amine-reactive reagent such as sulfo-NHS-LC-biotin.

[0059] In some embodiments, the capture portion (i.e., an antibody or antibody fragment such as SH-Fab) can be bound / coated to the biomarker capture particle by a cleavable bond. In some embodiments, the cleavable bond may be a disulfide bond (RSSR). After washing or isolating the particles from the sample matrix, the particles can then be treated with a solution containing a reducing agent such as TCEP or DTT to cleave the disulfide bond and release the capture portion-biomarker complex into the solution for further processing or measurement.

[0060] In some embodiments, depending on the application for which the affinity assay is designed, the substance may function as one or the other of a binding pair consisting of a capture portion and a biomarker. Such a substance may, for example, be used as a capture portion (analyte binder) or may be used to generate the capture portions used herein (for example, by using them as haptens / antigens to generate specific antibodies). In some embodiments, such a substance may be a biomarker. Affinity assays, including immunoassays, can be designed to detect the presence and / or levels of such biomarkers in a sample.

[0061] In some embodiments, substances can be used to associate with particles and capture molecules or biomarkers that interact with them (e.g., antibodies or fragments thereof that are specific to the listed substances, binding proteins, or enzymes).

[0062] An unrestricted list of substances that can function as one or the other of a binding pair consisting of a capture portion and a biomarker includes: nitric oxide synthase (iNOS), CA19-9, IL-1α, IL-1β, IL-2, IL-3, IL-4, ILt, IL-5, IL-7, IL-10, IL-12, IL-13, sIL-2R, sIL-4R, sIL-6R, SIV core antigen, IL-1RA, TNF-α, IFNγ, GM-CSF; PSA, pPSA in PSA, BPSA, non-α1 antichymotrypsin complexed PSA, α1-antithymotrypsin complexed PSA, prostatic kallikrein such as hK2, hK4, and hK15, isoforms of PSA (prostate-specific antigens) such as ek-rhK2, Ala-rhK2, TWT-rhK2, XarhK2, HWT-rhK2, and other kallikrein; HIV-1 p24; ferritin, L-ferritin, troponin I, BNP, leptin, digoxin, myoglobin, type B natriuretic peptide or brain natriuretic peptide (BNP), NTproBNP, CNP, NT-proCNP(1-50), NT-CNP-53(51-81), CNP-22(82-103), CNP-53(51-103), atrial natriuretic peptide (ANP); human growth hormone, bone alkaline phosphatase, human follicle-stimulating hormone, human luteinizing hormone, prolactin; human chorionic gonadotropins (e.g., CGα, CGβ); soluble ST2, thyroglobulin; anti-thyroglobulin; IgE, IgG, IgG1, IgG2, IgG3, IgG4, B. anthracis protective antigen, B. anthracis lethal factor, B. anthracis spore antigen, F. tularensis LPS, S. aureas enterotoxin B, Y. pestis capsular F1 antigen, insulin, alpha-fetoprotein (e.g., AFP 300), carcinoembryonic antigen (CEA), CA 15.3 antigen, CA 19.9 antigen, CA 125 antigen, HAV Ab, HAV Igm, HBc Ab, HBc Igm, HIV 1 / 2, HBsAg, HBsAb, HCVAb, anti-p53, histamine; neopterin; s-VCAM-1, serotonin, sFas, sFas ligand, sGM-CSFR, s1CAM-1, thymidine kinase, IgE, EPO, intrinsic factor Ab, haptoglobulin, anticardiolipin, anti-dsDNA, anti-Ro, Ro, anti-La, anti-SM, SM, anti-nRNP, anti-histone, anti-Scl-70, Scl-70, antinuclear antibody, anti-centromere antibody, SS-A, SS-B, Sm, U1-RNP, Jo-1, CK, CK-MB, CRP, ischemic albumin, HDL, LDL, oxLDL, VLDL, troponin T, troponin I, troponin C, microalbumin, amylase, ALP, ALT, AST, GGT, IgA, IgG, prealbumin, anti-streptrysin, Chlamydia, CMV IgG, ToxoIgG, ToxoIgM, Apolipoprotein A, Apolipoprotein B, C3, C4, Propargin factor B, Albumin, α1 acid glycoprotein, α1 antitrypsin, α1 microglobulin, α2 macroglobulin, anti-streptrysin O, antithrombin-III, Apolipoprotein A1, Apolipoprotein B, β2 microglobulin, Ceruloplasmin, Complement C3, Complement C4, C-reactive protein, DNase B, ferritin, free κ light chain, free λ light chain, haptoglobin, immunoglobulin A, immunoglobulin A (CSF), immunoglobulin E, immunoglobulin G, immunoglobulin G (CSF), immunoglobulin G (mouse), immunoglobulin G subclass, immunoglobulin M, immunoglobulin M (CSF), κ light chain, λ light chain, lipoprotein (a), microalbumin, prealbumin, propergin factor B, rheumatoid factor, ferritin, transferrin, transferrin (urine), rubella IgG, Thyroglobulin antibody, Toxoplasma IgM, Toxoplasma IgG, IGF-I, IGF-binding protein (IGFBP)-3, hepsin, pim-1 kinase, E-cadherin, EZH2, and α-methylacyl-CoA racemase, TGF-β, IL6SR, GAD, IA-2, CD-64, neutrophil CD-64, CD-20, CD-33, CD-52, cytochrome P450 isoforms, s-VCAM-1, sFas, sICAM, hepatitis B surface antigen, thromboplastin, HIV p24, HIV gp41 / 120, HCVMay contain C22, HCV C33, hemoglobin A1c, and GAD65, IA2, vitamin D, 25-OH vitamin D, 1,25(OH)2 vitamin D, 24,25(OH)2 vitamin D, 25,26(OH)2 vitamin D, 3-epimer of vitamin D, FGF-23, sclerostin, procalcitonin, calcitonin, c. diphysil toxin A & B, h. pylori, HSV-1, and HSV-2.

[0063] In some embodiments, the substance that can function as one or the other of a binding pair consisting of a capture portion and a biomarker may include a portion, such as an antibody or a fragment thereof, that is specific to any of the WHO International Biological Reference Preparations that are maintained, characterized, and / or in circulation by the WHO International Biological Standards (updated June 30, 2005, available at http: / / www.who.int / bloodproducts / re_materials, the list of which is incorporated herein by reference).

[0064] A partial list of such preferred international reference standards, identified by the WHO code in parentheses following the substance, includes: human recombinant thromboplastin (rTF / 95), rabbit thromboplastin (RBT / 90), thyroid-stimulating antibody (90 / 672), recombinant human tissue plasminogen activator (98 / 714), high molecular weight urokinase (87 / 594), prostate-specific antigen (96 / 668), prostate-specific antigen 90:10 (96 / 700); Human plasma protein C (86 / 622), human plasma protein S (93 / 590), rheumatoid arthritis serum (W1066), serum amyloid A protein (92 / 680), streptokinase (00 / 464), human thrombin (01 / 580), bovine complex thromboplastin (OBT / 79), anti-D positive control intravenous immunoglobulin (02 / 228), pancreatic islet cell antibody (97 / 550), lipoprotein a (IFCC) SRM 2B), human parvovirus B19 DNA (99 / 800), human plasmin (97 / 536), human plasminogen activator inhibitor 1 (92 / 654), platelet factor 4 (83 / 505), prekallikrein activator (82 / 530), human brain CJD control and human brain sporadic CJD preparation 1 and human brain sporadic CJD preparation 2 and human brain variant CJD (none; each is from WHO TRS ECBS Report No. 926, 53rd Report, brain (Cited in homogenate), human serum complement components C1q, C4, C5, factor B, and full functional complement CH50 (W1032), human serum immunoglobulin E (75 / 502), human serum immunoglobulins G, A, and M (67 / 86), human serum protein albumin, α-1-antitrypsin, α-2-macroglobulin, ceruloplasmin, complement C3, transferrin (W1031), anti-D negative control intravenous immunoglobulin (02 / 226), hepatitis A RNA (00 / 560), hepatitis B surface antigen subtype adw2 genotype A (03 / 262 and 00 / 588), hepatitis B virus DNA (97 / 746), hepatitis C virus RNA (96 / 798), HIV-1 p24 antigen (90 / 636), HIV-1 RNA (97 / 656), HIV-1 RNA genotype (set of 10 I01 / 466), human fibrinogen concentrate (98 / 614),Human plasma fibrinogen (98 / 612), elevated A2 hemoglobin (89 / 666), elevated F hemoglobin (85 / 616), hemoglobin cyanide (98 / 708), low molecular weight heparin (85 / 600 and 90 / 686), unfractionated heparin (97 / 578), blood coagulation factor VIII and von Willebrand factor (02 / 150), human blood coagulation factor VIII concentrate (99 / 678), human blood coagulation factor XIII plasma (02 / 206), human blood coagulation factors II, VII, IX, X (99 / 826), human blood coagulation factors II and X concentrate (9 8 / 590), human fetal carcinogen antigen (73 / 601), human Creactive protein (85 / 506), recombinant human ferritin (94 / 572), apolipoprotein B (SP3-07), β-2-microglobulin (B2M), human β-thromboglobulin (83 / 501), human blood coagulation factor IX concentrate (96 / 854), human blood coagulation factor IXa concentrate (97 / 562), human blood coagulation factor Leiden, human gDNA samples FV wild type, FVL homozygote, FVL heterozygote (03 / 254, 03 / 260, 03 / 248), human blood coagulation factor Factor VII concentrate (97 / 592), human blood coagulation factor VIIa concentrate (89 / 688), human anti-syphilis serum (HS), human anti-tetanus immunoglobulin (TE-3), human anti-thrombin concentrate (96 / 520), human plasma antithrombin (93 / 768), human anti-thyroglobulin serum (65 / 93), anti-toxoplasma serum (TOXM), human anti-toxoplasma serum (IgG) (01 / 600), human anti-varicella-zoster immunoglobulin (W1044), apolipoprotein A-1 (SP1-01), human anti-interferon-β serum (G038-501-572) ), human anti-measles serum (66 / 202), antinuclear ribonucleoprotein serum (W1063), antinuclear factor (allogeneic) serum (66 / 233), anti-parvovirus B19 (IgG) serum (91 / 602), anti-poliovirus serotype 1, 2, 3 (66 / 202), human anti-rabies immunoglobulin (RAI), human anti-rubella immunoglobulin (RUBI-1-94), anti-smooth muscle serum (W1062), human anti-double-stranded DNA serum (Wo / 80), human anti-E complete blood typing serum (W1005), human anti-echinococcus serum (ECHS), human anti-hepatitis A immunoglobulin (97 / 646),This product contains human anti-hepatitis B immunoglobulin (W1042), human anti-hepatitis E serum (95 / 584), anti-human platelet antigen-1a (93 / 710), anti-human platelet antigen-5b (99 / 666), human anti-interferon alpha serum (B037-501-572), human alpha-fetoprotein (AFP), ancrod (74 / 581), human anti-A blood typing serum (W1001), human anti-B blood typing serum (W1002), human anti-C complete blood typing serum (W1004), anti-D (anti-Rh0) complete blood typing reagent (99 / 836), human anti-D (anti-Rh0) incomplete blood typing serum (W1006), and human anti-D immunoglobulin (01 / 572).

[0065] Depending on the application for which the affinity assay is designed, other examples of substances that can function as one or the other of a binding pair consisting of a capture portion and a biomarker include compounds that can be used as haptens to produce antibodies capable of recognizing the compound, including but not limited to any salts, esters, or ethers of the following: hormones, progestins, corticosteroids, and dehydroepiandrosterone, including but not limited to progesterone, estrogen, and testosterone, as well as any non-protein / non-polypeptide antigen listed by the WHO as an international reference standard. A partial list of such preferred international reference standards, identified by the WHO code in parentheses following the substance, includes vitamin B12 (WHO 81.563), folic acid (WHO 95 / 528), homocysteine, transcobalamin, T4 / T3, and other substances disclosed in the WHO catalog of International Biological Reference Preparations (available on the WHO website, e.g., on the page http: / / www.who.int / bloodproducts / ref_materials / as updated June 30, 2005), which is incorporated herein by reference.

[0066] The methods and systems described herein may include the aforementioned WHO reference standards or mixtures containing the reference standards. Depending on the application for which the affinity assay is designed, other examples of substances that can function as one or the other of a binding pair consisting of an analyte binder (capture portion) and the analyte include drugs of abuse.Abuse drugs include, for example, drugs and their metabolites (e.g., metabolites present in blood, urine, and other biological substances), as well as any salts, esters, or ethers thereof: heroin, morphine, hydromorphone, codeine, oxycodone, hydrocodone, fentanyl, demerol, methadone, dalvon, stadol, talwin, palegoric, buprenex; stimulants such as amphetamine and methamphetamine; methylamphetamine, ethylamphetamine, methylphenidate, ephedrine, pseudoephedrine, ephedra, ma Huang, methylenedioxyamphetamine (MDS), phentermine, phenylpropanolamine; amifenazole, bemiglide, benzfetamine, bromataan, chlorphentermine, clopropamide, crocetamide, diethylpropion, dimethylamphetamine, doxapram, etamiban, fencamfamine, meclofenoxate, methylphenidate, niketamide, pemoline, pentetrazole, fendimethrazine, fenmetrazine, phentermine, phenylpropanolamine, picrotoxin, piperadol, prolintan, stricnin, synephrine, phencyclidine, and analogs such as Angeldust, PCP, ketamine; inhibitors such as barbiturates, glutetimide, methaquaron, and meprobamate, methhexital, thiamil, thiopental, amobal This may include: Vital, pentobarbital, secobarbital, butarbital, butabarbital, tarbutal, and aprobarbital, phenobarbital, mefobarbital; benzodiazapens such as estazolam, flurazepam, temazepam, triazolam, midazolam, alprazolam, chlordiazepoxide, clorazepate, diazepam, harazepam, lorazepam, oxazepam, prazepam, quazepam, clonazepam, flunitrazepam; GBH drugs such as gamma-hydroxybutyrate and gamma-butyrolactone; glutethimide, methaquan, meprobamate, carisoprodol, zolpidem, zaleplon; cannabinoid drugs such as tetrahydracannabinol and its analogues; cocaine, 3-4 methylenedioxymethamphetamine (MDMA); and hallucinogens such as mescaline and LSD.

[0067] In some embodiments, the capture portion includes antibodies, antibody fragments, polypeptide binders, antibody polymers, antibody fragment polymers, antibody and antibody fragment polymers, receptors, receptor ligands, ligand binders, ligands for ligand binders, enzymes, irreversibly inactivated enzymes, alkaline phosphatase, horseradish peroxidase, peptides, proteins, polymers, fluorophores, fluorescent dyes, quantum dots (Qdots), fluorescent protein labels, DNA stains, chemicals, and chemiluminescent chemicals such as luminol, isoluminol, isoluminol derivatives, acridinium esters, ruthenium, and N-(4-aminobutyl)-N-ethyl-isoluminol (ABEI), e.g., sulfo-NHS-biotin, sulfo-NHS-LC-biotin, sulfo-NHS-LC-LC-biotin, sulfo-NHS-SS-biotin, NHS-PEO4-biotin, NHS-biotin, NHS-LC-biotin, NHS- Amine-reactive labeling reagents such as LC-LC-biotin, PFP-biotin, TFP-PEO-biotin, or NHS-iminobiotintrifluoroacetamide, for example, sulfhydryl-reactive biotin labeling reagents such as maleimide-PEO2-biotin, biotin-BMCC, PEO-iodoacetylbiotin, iodoacetyl-LC-biotin, or biotin-HPDP, for example, biotinPEO-amine or biotinPEO-LC-amine The luboxyl-reactive biotin-labeling reagents may include, for example, carbohydrate-reactive biotin-labeling reagents such as biotin hydrazide, biotin-LC-hydrazide, and photoreactive biotin-labeling reagents such as psoralen-PEO-biotin, monobodies, non-immunoglobulin binders, DARPin, afibodies, antikalin, MIP, aptamers, chimeric antibodies, therapeutic antibodies, alpaca-derived nanobodies, phase-represented VHH constructs, or nucleic acids.In some embodiments, the surface of biomarker-capturing particles may be co-coated with animal antibodies or animal serum to block interference, such as human anti-animal antibody (HAAA) or rheumatoid factor (RF) interference, or with manufacturer assay-specific reagents such as streptavidin, ALP, HRP, BSA-fluorescein, BSA-ABEI, BSA-acridinium, or BSA-ruthenium to block manufacturer assay-specific interference (MASI).

[0068] In some embodiments, the antibody fragment may include Fab, F(ab')2, Fc, scFv, or engineered variants such as diabodies, triabodies, minibodies, VHH constructs, and single-domain antibodies. In some embodiments, the antibody fragment may be recombinant, monoclonal, or polyclonal.

[0069] In some embodiments, the antibodies may include autoantibodies, therapeutic antibodies, immunoglobulin classes such as IgG, IgM, IgA, and IgE, immunoglobulin subclasses such as IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4, circulating antibodies, secretory antibodies, alpaca-derived nanobodies, and animal-derived antibodies. In some embodiments, the therapeutic antibodies may include FDA-approved monoclonal antibody drugs, including infliximab for Crohn's disease, rituximab for lymphoma, ustekinumab for psoriasis, and tocilizumab for the treatment of rheumatoid arthritis, as well as FDA-approved antibody drugs for emergency use, including bebuterobimab, a monoclonal antibody against the SARS-CoV-2 spike protein.

[0070] In some embodiments, target capture particles or biomarker capture particles may include single antigen-coated beads, two or more antigen-coated beads, a pool of different antigen-coated beads, beads coated with multiple recombinant antigens, or beads coated with viral or bacterial lysates, in order to improve the possibility and sensitivity of detecting human immunoglobulins, immunoglobulin subclasses, or combinations thereof, such as IgA, IgG, IgM, and IgE, against an antigen. Biomarker capture particles may be used in serological tests.

[0071] In some embodiments, biomarker-capturing particles may include beads coated with a single antibody, beads coated with two or more antibodies, a pool of beads coated with different antibodies, beads coated with multiple antibodies, beads coated with a capture portion (e.g., antibody fragments, aptamers, MIPs, alpaca-derived nanobodies), or a pool or combination of such beads, to improve the likelihood and sensitivity of detecting an antigen or biomarker, or for the multiple detection of multiple antigens or biomarkers. This is an antigen assay, typically performed by a sandwich assay, competitive assay, piggyback assay, delayed addition, or delayed capture assay. Biomarker-capturing particles may be used in antigen assays.

[0072] In some embodiments, the biomarker-capturing particles may comprise a pool of different biomarker-capturing particles coated with antibodies and antigens, by combining different assay formats that may be required to perform combination or "combo" assays, to detect both antibodies and antigens in the same test, or to detect either an antigen or a biomarker. The eluate may be measured by mass spectrometry (LC-MS, LC-MS / MS, MALDI-TOF, etc.), ELISA, CLIA, FIA, chemical, molecular (PCR, NGS), or any other assay method or system, by cleaving or eluting the antibodies, antigens, or biomarkers captured from the biomarker-capturing particles, or by neutralizing the eluate and measuring it.

[0073] In some embodiments, the biomarker-capturing particles may include latex beads. In some embodiments, the latex beads may include polymer beads. In some embodiments, the latex beads may be of different colors, such as white, blue, red, black, green, yellow, orange, or brown.

[0074] In some embodiments, the biomarker-capturing particles may include agglutinating beads. In some embodiments, the biomarker may include multiple epitopes, each of which can bind to a biomarker-capturing moiety. In some embodiments, the multiple epitopes may include two or more repeating epitopes. In some embodiments, the multiple epitopes may include two or more different epitopes. In some embodiments, the multiple epitopes may include any combination of epitopes. In some embodiments, the agglutinating beads may include multiple biomarker-capturing moieties so that the agglutinating beads can capture / bind to multiple biomarkers. In some embodiments, the agglutinating beads may aggregate or condense when one, two, three, four, five, six, seven, eight, nine, ten, or more agglutinating beads are bound to a single biomarker. In some embodiments, a biomarker may bind to multiple agglutinated beads, and some of these agglutinated beads may bind to multiple biomarkers, thereby forming a network comprising multiple biomarkers and multiple agglutinated beads. In some embodiments, agglutinated, aggregated, or precipitated agglutinated beads may be visually detectable, thereby enabling a rapid visual or point-of-care test in which the visual detection of the bead aggregates indicates the presence of a biomarker, e.g., a pathogen, disease, or antibody. In some embodiments, agglutinated, aggregated, or precipitated agglutinated beads may be detectable via detection, e.g., turbidity detection, UV-vis detection, infrared detection, light scattering, or microscopic detection. Referring to Figure 5, in the absence of virions, the biomarker-capturing particles remain monodisperse or a clear brownish liquid (501). However, in the presence of virions, the biomarker-capturing particles agglutinate, aggregate, or precipitate (502).

[0075] In some embodiments, the aggregated beads may include different colors (e.g., red, yellow, blue, or any color). In some embodiments, two or more different biomarkers may be visually detected in a single test, if each different colored bead (e.g., latex beads) contains one or more antibodies against different targets. For example, a bead having color A may contain a biomarker capture moiety specific to biomarker X indicating a disease, e.g., SARS-CoV-2; a bead having color B may contain a biomarker capture moiety specific to biomarker Y indicating another disease, e.g., influenza; and a bead having color C may contain a biomarker capture moiety specific to biomarker Z indicating a disease, e.g., RSV. When two or more types of beads are used, precipitation of beads having a particular color may indicate the presence of the corresponding biomarker. In some embodiments, the precipitated beads may include a blend of colors from two or more types of beads. For example, if a bead having color A precipitates, it may indicate the presence of biomarker X in the sample.

[0076] In some embodiments, the biomarker-capturing particles may comprise a first plurality of magnetic beads and a second plurality of latex beads or non-magnetic beads. In some embodiments, the second plurality of latex beads may exhibit color. In some embodiments, the first plurality of magnetic beads and the second plurality of latex beads may comprise different sizes or dimensions. In some embodiments, the magnetic beads may comprise a first capture portion, and the colored latex beads may comprise a second capture portion, with the first and second capture portions each capturing a first and second epitope on a single biomarker. If no biomarker is present, the magnetic beads may be removed, leaving 100% colored latex beads or non-magnetic beads in the solution for reference color, reference color intensity or darkness, reference concentration, etc. If a biomarker is present in the sample, it can be captured by magnetic beads and colored latex beads such that the magnetic beads capture both the antigen and the colored latex beads, forming a complex comprising the magnetic beads and colored latex beads crosslinked by the biomarker. When a magnet or magnetic field is applied, the complex can be withdrawn from the solution, resulting in a change in the color of the colored latex beads in the solution or supernatant, a decrease in color intensity, or a decrease in density. This change in the solution or supernatant indicates the presence of a biomarker. In some embodiments, the change in the solution or supernatant is readily detectable visually or using a densimeter, flow cytometer, turbidity detector, A600 absorbance meter, etc. This is similar to or analogous to coating a basketball with ping pong balls, where the ping pong balls are small latex or polystyrene nonmagnetic beads (e.g., in the range of 10-300 nm in various colors), and the basketballs are larger magnetic beads (e.g., in the size range of 200 nm-3,000 nm). In some cases, only the larger magnetic beads are attracted by the magnet.Small, non-magnetic latex or polystyrene beads are not attracted by magnets and are very colloidally stable because they are so small (e.g., they remain homogeneously dispersed with good colloidal stability and very slow sedimentation times, or they do not settle from the solution). In some cases, the main way that "non-magnetic" ping-pong balls move to a magnet is by interacting with each other and binding to a larger "magnetic" basketball, so that they move together to the magnet. This may require the basketball to be coated with a capture moiety or anti-target antibody or binding partner, and also require the ping-pong ball to be coated with a capture moiety or anti-target antibody or binding partner to the same target, biomarker, antigen, or analyte as the basketball, but ideally targeting a distal epitope such as an antibody pair commonly used today for sandwich immunoassays (i.e., the basketball binds to target epitope 1 and the ping-pong ball binds to target epitope 2).

[0077] In some embodiments, the surface of biomarker-capturing particles may be attenuated to reduce the density of captured portions per biomarker-capturing particle or per unit mass, so that fewer biomarkers are captured per biomarker-capturing particle, in order to increase the test sensitivity to differences in biomarker concentration. To enhance the assay sensitivity of POC, it may be important to control the number of ping-pong balls that can bind to each basketball. In this way, small changes in target or biomarker concentration can be detected by a large change in the residual concentration of ping-pong balls in the solution that are not bound to the target or biomarker. This may require maximizing the number of ping-pong balls that can bind to each basketball while minimizing or attenuating the number of targets or biomarkers that can bind to each ping-pong ball, or both basketballs and ping-pong balls may have attenuated surfaces or less antibody per unit surface area or per bead (ping-pong ball and basketball), or per mass of beads, for example, per microgram of bound antibody per mg of beads. Attenuation can be achieved in a primary coating step or via a secondary coating step by diluting or co-coating the antibody on the beads with a nonspecific antibody, streptavidin, BSA, or a low molecular weight protein such as a lysine-rich peptide, or a polymer containing an amine such as an NH2-PEGx compound such as JSR Blockmaster CE210 and / or CE510, or by coating streptavidin beads with a mixture of a biotinylated capture moiety and a small molecule such as biotin or biotin-fluorescein.In the case of primary covalent or hydrophobic coating or streptavidin coating, this is achieved by adding a protein solution of the capture moiety or binding partner, e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the solution with 95, 90, 80, 70, 60, 50, 40, 30, 20, or 10% of nonspecific proteins, peptides, polymers, or small molecules, and co-coating them onto the beads, thereby reducing or controlling the density of the specific capture moiety, binding partner. If the antibody and nonspecific proteins, peptides, polymers, or small molecules on the bead surface are also covalently labeled or recombinantly tagged with signal-generating molecules, proteins, or moieties, e.g., fluorescein, fluoropore, ALP, HRP, chemiluminescent, or luminol-based substrates (ABEI, luminol, isoluminol, acridinium ester), or ruthenium, this can also control or attenuate the signal response per bead. For secondary coating, the exact same approach can be used, but the specific capture moieties, binding partners, or antibodies, as well as nonspecific proteins or polymers, are tagged or labeled with biotin, or biotin-fluorescein or biotin signaling molecules, proteins, or moieties, and they are co-coated onto the streptavidin bead surface.

[0078] Conjugation reagents for attaching biotin to antibodies or proteins include, for example, amine-reactive labeling reagents such as sulfo-NHS-biotin, sulfo-NHS-LC-biotin, sulfo-NHS-LC-LC-biotin, sulfo-NHS-SS-biotin, NHS-PEO4-biotin, NHS-biotin, NHS-LC-biotin, NHS-LC-biotin, PFP-biotin, TFP-PEO-biotin, or NHS-iminobiotin trifluoroacetamide, for example, maleimide-PEO2-biotin This may include sulfhydryl-reactive biotin labeling reagents such as biotin-BMCC, PEO-iodoacetylbiotin, iodoacetyl-LC-biotin, or biotin-HPDP; carboxyl-reactive biotin labeling reagents such as biotin-PEO-amine or biotin-PEO-LC-amine; carbohydrate-reactive biotin labeling reagents such as biotin hydrazide, biotin hydrazide, or biotin-LC-hydrazide; or photoreactive biotin labeling reagents such as psoralen-PEO-biotin. Similar chemistry and linkers may be used to conjugate assay signal detection molecules such as ALP, HRP, luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, acridinium esters, acridinium ester derivatives, fluorophores, fluorescein, and enzymes to proteins and antibodies in order to prepare conjugates used in immunoassays (RIA, ELISA, CLIA, LF, PoC).

[0079] In some embodiments, the antibody pair is an antibody against an antigen or target such as an antibody pair against the spike protein of SARS-CoV-2 virions in an RT-PCR-positive saline oral rinse sample, where one antibody is biotinylated and the other is a 1.6 micron streptavidin magnetic target capture bead (biotin-labeled humanized monoclonal anti-SARS-CoV-2 spike protein RBD). An antibody pair against an antigen or target is created by coating an IgG antibody (where the other antibody is coated) with a fluorescent tag (e.g., iFluor488 or AlexaFluor488-labeled anti-SARS-CoV-2 spike protein NTD antibody), or by conjugating an EDC coated with streptavidin on a 70 nm white latex nonmagnetic carboxy bead. This produces "hot" fluorescent beads with a limited or attenuated amount of anti-RBD antibody per latex bead but a high relative fluorescence signal (RFU) per latex bead. In the presence of SARS-CoV-2 virions, magnetic target capture beads capture the virions via binding to the SARS-CoV-2 spike protein RBD, and this magnetic bead-anti-RBD-virion complex also binds to an anti-NTD-fluorescent conjugate or an anti-RBD-[latex bead]-fluorescent conjugate.If the magnetic capture beads separate on the magnet, the [anti-RBD magnetic target capture beads]-virion-[anti-NTD-fluorescent antibody] complex, or the [anti-RBD magnetic target capture beads]-virion-[anti-NTD-antibody fluorescent latex beads] complex will separate on the magnet, thereby, no signal or a significantly reduced signal in the supernatant is equivalent to a positive result (SARS-CoV-2 virions detected), and no change in the RFU signal or no significant reduction in the RFU signal is equivalent to a negative result (SARS-CoV-2 virions are not detected). Thereafter, the supernatant is aspirated and dispensed into a reading plate or container for fluorescence detection using an Agilent BioTek Synergy H1 Multimode Reader or similar.

[0080] In some embodiments, to improve detection sensitivity, a limited amount of fluorescence conjugate may be used so that low viral counts or low viral loads (high RT-PCR cycle threshold (Ct) counts, such as Ct>36) deplete the RFU signal. However, high viral loads (low Ct values, such as Ct<18) significantly bind all or most of the anti-RBD / NTD conjugate to the magnetic bead-virion complex, separating it to the magnet with the magnetic bead-virion complex, thereby significantly reducing or eliminating the supernatant RFU signal. However, if there are no viruses (virions) or the viral load is very low (Ct>42), the supernatant RFU signal does not change or changes only minimally to negative results. One way to prepare anti-NTD fluorescent conjugates to still have a very good, potent, and reproducible signal that is limited but above the background RFU signal is to serially dilute the anti-NTD fluorescent antibody conjugate, and dilutions in which 200 μL of the diluted conjugate read up to 500–1000 RFU can be used for testing. The same approach can be used for anti-RBD antibody-[fluorescent latex beads], thereby reading 500–1000 RFU from 200 μL. Using this approach, the conjugate is limited so that SARS-CoV-2 virions bind to most (if not all) of the SARS-CoV-2 virions at low SARS-CoV-2 viral loads (Ct>36), and the conjugate and RFU signal are significantly reduced from 500–1000 RFU to <400 RFU, or preferably to <100 RFU, or more preferably to <50 RFU, or most preferably to <10 RFU. Magnetic antibody-coated target capture beads coated with anti-RBD antibody should be in molar excess to bind to 95%–100% of virions in the sample, such as by adding 80–100 μg (0.08–0.10 mg) of target capture beads per 500 μL (0.5 mL) of undiluted saliva or 1000 μL (1.0 mL) of SOR.If target capture beads are bound, captured, or in excess of molar SARS-CoV-2 virions, but conjugation is limited, fluorescence, visual, or UV-vis OD600nm testing will show a significant decrease in RFU signal, a decrease in latex beads, or a decrease in OD600 absorbance in SARS-CoV-2 positive samples.

[0081] In some embodiments, the capture portion may be bound to a biomarker by a cleavable bond. The cleavable bond may include covalent or non-covalent bonds. Non-covalent bonds may include affinity, ionic, van der Waals (e.g., dipole / dipole forces or London forces), hydrogen bonds (e.g., between polynucleotide double chains), or hydrophobic interactions. In some embodiments, the non-covalent bond may be specific. In some embodiments, specific non-covalent binding may include: binding interactions between biotin and biotin-binding proteins (e.g., avidin, captavidin, SA, neutraavidin, fragments of SA, fragments of avidin, fragments of neutraavidin, or mixtures thereof); binding of biotinylated Fab, biotinylated immunoglobulin or fragments thereof, biotinylated small molecules (e.g., receptor hormones or ligands), biotinylated polynucleotides, biotinylated macromolecules (e.g., proteins or natural or synthetic polymers) to biotin-binding proteins, e.g., avidin, SA, neutraavidin, fragments of SA, fragments of avidin, fragments of neutraavidin, or mixtures thereof; binding of substrates to enzymes; binding of glycoproteins to glycoprotein-specific lectins; binding of ligands to ligand-specific receptors; binding of antibodies to antigens from which antibodies are produced; and double-strand formation between polynucleotides and polynucleotides that are complementary or substantially complementary to each other; etc.

[0082] In some embodiments, the biomarker-capturing particles may include a coating that can interact with the capture portion added to or present in the sample. In some embodiments, the capture portion added to or present in the sample may bind to a biomarker in the sample to form a biomarker-capturing portion complex, and when the biomarker-capturing particles are introduced, the biomarker-capturing particles may interact with the biomarker-capturing portion complex to capture the biomarker-capturing portion. In some embodiments, the coating may include a streptavidin coating. In some embodiments, the capture portion is added to or combined with the sample without the presence of capture particles and without being conjugated to the capture particles. In some embodiments, the capture portion is added to or combined with a sample having capture particles, and the capture portion is conjugated to the capture particles.

[0083] preparation In some embodiments, step (b) may further include incubating the sample containing the biomarker-capturing particles for 5 minutes to 24 hours. In some embodiments, the incubation time may be, for example, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours, 4 hours, 8 hours, or overnight. In some embodiments, incubation may be performed during shipment or transport of the sample. In some embodiments, incubation may be performed with or without mixing or stirring in a rocker, naturator, bottle roller, shaker, or plate mixer, etc., at room temperature or ambient temperature, at 2 to 8°C (e.g., in a cooler or on a cold pack), or at a heating temperature such as 30 to 50°C (e.g., 30, 37, or 42°C).

[0084] In some embodiments, the methods described herein may be carried out in less than one week, six days, five days, four days, three days, two days, one day, 24 hours, 20 hours, 16 hours, 12 hours, eight hours, four hours, two hours, one hour, 30 minutes, 15 minutes, 10 minutes, five minutes, or less. In some embodiments, the methods described herein may be carried out in less than one day.

[0085] In some embodiments, the method may further include removing the liquid phase from the sample after step (b). In some embodiments, the removal of the liquid phase may be performed after separating the liquid phase from the biomarker-capturing particles. Separation may include centrifugation or filtration. In some embodiments, a magnetic field or magnet may be applied to attract the biomarker-capturing particles, thereby separating them from the liquid phase. In some embodiments, a washing solution may be dispensed onto the sample or biomarker-capturing particles to remove or elute nonspecifically bound substances (a “clean” or “cleaning” process). To isolate or separate the biomarker-capturing particles (e.g., in a primary blood collection tube, a custom sample collection device, a secondary transfer tube or custom sample device, a pooled sample, or in a 96-well plate), a magnet-based device rapidly (less than 2 minutes, preferably less than 30 seconds) isolates the magnetic nanoparticles to the sides and / or bottom to form an essentially particle-free supernatant. The particle-free supernatant may then be aspirated, discharged, or otherwise removed without destroying the pellet containing the biomarker-capturing particles. In some embodiments, the pellet may be isolated or subjected to diagnostic testing. Another approach to isolating or separating magnetic particles is to use a disposable pipette tip equipped with a custom magnet inserted inside the disposable pipette tip to rapidly isolate the magnetic nanoparticles to the surface of the pipette tip, forming a sample supernatant that is essentially particle-free. The disposable pipette tip with the custom magnet can then be removed from the sample without destroying the pellet containing the particles. The disposable tip can then be inserted into a new tube for isolation and characterization of the particles in subsequent diagnostic testing (i.e., concentration). For example, a disposable tip with particles can be inserted into a secondary transfer tube containing a buffer. When the magnet is removed from the tip or turned off (e.g., by an electromagnet), the particles disperse freely in the buffer.

[0086] In some embodiments, the method may further include a step of eluting or cleaving the biomarker from the biomarker-capturing particles ("biomarker cleave process" or "biomarker cleaving process"). In some embodiments, the eluted biomarker may include a capture portion bound to / coupled with the biomarker, i.e., the capture portion-biomarker pair is eluted from the biomarker-capturing particles. In some embodiments, elution may be achieved after removal of the liquid phase. Elution of the biomarker from the biomarker-capturing particles may be achieved, for example, by adding an elution solution or cleaving reagent to the biomarker-capturing particles. The elution solution or cleaving reagent may cleave or elute the biomarker captured from the biomarker-capturing particles. After incubation time, the biomarker-capturing particles may be removed from the eluate by centrifugation, magnetic centrifugation, or filtration. The eluate can be measured by any method or system for the presence, concentration, and / or level of antibody-oligoconjugates or biomarkers in mass spectrometry (e.g., LC-MS, LC-MS / MS, MALDI-TOF, etc.), ELISA, CLIA, FIA, PCR, NGS, immunoPCR, or immunoPCR. In some embodiments, a neutralizing buffer may be added to the eluate before measurement.

[0087] In some embodiments, biomarkers are eluted, dissociated, or released from biomarker-captured particles (e.g., nanoparticles, microparticles) by an elution solution or cleavage reagent by disrupting binding interactions using an elution strategy such as pH. In some embodiments, elution may be performed by increasing the pH with a base such as sodium bicarbonate. In some embodiments, elution may be performed by decreasing the pH with an acid such as acetic acid, trichloroacetic acid, sulfosalicylic acid, HCl, or formic acid. In some embodiments, the elution solution may include a pH elution buffer such as 100 mM glycine*HCl at pH 2.5–3.0, 100 mM citrate at pH 3.0, 50–100 mM triethylamine or triethanolamine at pH 11.5, or 150 mM ammonium hydroxide at pH 11.5. In some embodiments, elution is performed using a displacer or displacing agent. Agents), competitive elution (e.g., >0.1M counterligands or analogs), ionic strength and / or chaotropic effects (e.g., NaCl, KCl, 3.5-4.0M magnesium chloride in 10mM Tris at pH 7.0, 5M lithium chloride in 10mM phosphate buffer at pH 7.2, 2.5M sodium iodide at pH 7.5, 0.2-3.0M sodium thiocyanate), surfactants, detergents, concentrated inorganic salts, denaturation (e.g., 2-6M guanidine*HCl, 2-8M urea, 1% deoxycholate, 1% SDS), organic solvents (e.g., pH 8-11.5) This can be carried out using alcohol, chloroform, ethanol, methanol, acetonitrile, hexane, DMSO, 10% dioxane, 50% ethylene glycol (or chaotropic), radiation or heat (temperature increase), conformational changes, disulfide bond reducing agents (2-mercaptoethanol, dithiothreitol, tris(2-carboxyethyl)phosphine), enzyme inactivation, chaotropic agents (urea, guanidinium chloride, lithium perchlorate), mechanical stirring, sonication, and protein digestive enzymes (pepsin, trypsin), as well as combinations thereof.

[0088] In some embodiments, biomarker-capturing particles may be captured on or by a feeder. A collection device having a neutralization buffer may be attached to the feeder. The captured biomarker may be rinsed with an elution buffer and eluted into the collection device for neutralization.

[0089] In some embodiments, after the washing process, the biomarkers are not subjected to a biomarker cleavage process for cleaving the biomarkers from the biomarker-capturing particles. Instead, the method may further include a step of generating a buffer solution containing the biomarker-capturing particles by adding a buffer to the biomarker-capturing particles containing the biomarkers after removing the liquid phase (a “concentrate” or “concentrating” process). In some embodiments, the volume of the buffer may be less than the volume of the sample. In some embodiments, the volume of the buffer may be substantially less than the volume of the sample.

[0090] Biomarker-captured particles can be dispersed, reconstituted, or resuspended in a buffer such as phosphate-buffered saline (i.e., PBS at pH 7.2) or LC-MS / MS-compatible buffer before the characterization or measurement steps. This means that the critical characterization or measurement steps of the biomarkers captured and concentrated by the particles occur in the buffer system rather than in an animal or human matrix.

[0091] In some embodiments, the method may further include a step of adding multiple conjugates to a buffer solution containing biomarker-capturing particles after the concentration process (a “conjugate” or “conjugating” process). Conjugates that bind to captured biomarkers on the biomarker-capturing particles may be added to detect, measure, or quantify one or more biomarkers on the biomarker-capturing particles. The conjugate may be specific to a single biomarker, or it may contain two or more antibodies or antigens specific to one captured biomarker or two or more captured biomarkers. The conjugate may be labeled with a signal detection portion, or two or more different conjugates may be labeled with different fluorophores (different luminescence / excitation) and pooled to create a multiple conjugate. In some embodiments, the conjugate may react with or bind to biomarkers captured on biomarker-capturing beads. Non-limiting examples of conjugates may include chemiluminescent substrates (isoluminol, luminol, ABEI, ruthenium, acridinium esters), fluorescent labels (fluorescein or other fluorophores and dyes), anti-alkaline phosphatase (ALP), anti-horseradish peroxidase (HRP), smaller magnetic beads, smaller non-magnetic beads, or smaller non-magnetic colored beads.

[0092] In some embodiments, the conjugate may be a triplex conjugate containing rabbit anti-human IgA, rabbit anti-human IgG, and rabbit anti-human IgM (Agilent DAKO, 5301 Stevens Creek Blvd., Santa Clara, California 95051), each labeled with a different fluorophore such as AlexaFluor® (ThermoFisher Scientific, 168 Third Avenue, Waltham, Massachusetts, USA 02451) 488, 555, or 647, or iFluor® (AAT Bioquest, Inc., 520 Mercury Drive, Sunnyvale, California 94085, USA) 488, 546, and 597. In some embodiments, the conjugate may be a 5-plex conjugate using five different iFluor having monoclonal anti-human IgA (total), as well as IgG subclasses IgG1, IgG2, IgG3, and IgG4, and / or IgM (Mabtech AB, Box 1233 SE-131 28, Nacka Strand, Sweden). By detecting two or more different human immunoglobulin classes or immunoglobulin subclasses, the sensitivity to detect a positive immune response to a pathogen (bacteria, virus, or fungus) or antigen, as well as antibody profiling that steps the progression of disease (acute or early infection versus chronic or late infection), can be improved. For example, if IgM is detected, it may typically indicate an early or acute infection; if IgG is detected, it may typically indicate a post-seroconversion infection, which is representative of a late or chronic infection; and if secretory IgA or SOR is detected in saliva, it may indicate an early or acute respiratory pathogen infection as a protective immunity or a first line of defense against prior infection or vaccination.

[0093] In some embodiments, biomarker-capturing particles may be coated with different recombinant or purified antigens or proteins for different pathogens, viruses, bacteria, or fungal species, and conjugates may contain the exact same recombinant or purified antigen coated on the biomarker-capturing particles, but each may be labeled or conjugated with a different fluorophore for multiple detection. Human IgG (bivalent or 2 Fabs), IgA (tetravalent or 4 Fabs), and IgM (bivalent or 10 Fabs) are polyvalent, so they can bind to the antigen they recognize or detect on the biomarker-capturing particles, but also to the exact same antigen labeled with a fluorophore. This approach enables multiple detection of one or more different pathogens, viruses, bacteria, or fungi, such as a tick-borne disease panel, an infectious disease panel, or a sexually transmitted infection panel (e.g., HIV, hepatitis A, B, and C, HSV1 and 2, HPV, etc.), with one or more different antigens or proteins for each pathogen used in both the antigen-coated biomarker-capturing particles and the fluorescently labeled conjugates.

[0094] In some embodiments, the conjugate may be washed before being added to a buffer solution containing biomarker-capturing particles. In some embodiments, washing may be performed using biomarker-capturing particles. In some embodiments, washing may be performed using interference-capturing particles. Washing may be important because some conjugates or polyclonal antibodies, especially if they are of human or animal origin, may nonspecifically bind to or cross-react with biomarker-capturing particles, capture moieties, or blockers, causing high background signals or noise in subsequent detection / measurement. By exposing or pre-incubating the conjugate with biomarker-capturing particles and removing the biomarker-capturing particles from the conjugate via filtration, magnetic separation, or centrifugation, some of the conjugate that may react with the biomarker-capturing particles is removed, and therefore, when the conjugate is used, the background signal is reduced or significantly reduced, thereby increasing the assay or test signal vs. noise and sensitivity. By adding small to large amounts of biomarker-capturing particles to the conjugate, the conjugate may be washed for biomarker-capturing particle-specific interference. In some embodiments, the amount of biomarker-capturing particles for conjugate washing may include, for example, 0.01 μg (0.00001 mg), 0.1 μg (0.0001 mg), 1.0 μg (0.001 mg), 10 μg (0.01 mg), or 100 μg (0.1 mg) of biomarker-capturing particles per 1 mL of conjugate, or preferably >1.0 μg (0.001 mg) of biomarker-capturing particles per 1 mL of conjugate, or most preferably >3.0 μg (0.003 mg) of biomarker-capturing particles per 1 mL of conjugate.

[0095] In some embodiments, after adding the conjugate to a buffer solution containing biomarker-capturing particles, the solution may be incubated for a period of time, for example, 5 minutes to 24 hours. In some embodiments, the incubation time may be, for example, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours, 4 hours, 8 hours, or overnight. In some embodiments, incubation may be performed during the shipment or transport of the sample. In some embodiments, incubation may be performed with or without mixing or stirring in a rocker, naturator, bottle roller, shaker, or plate mixer, etc., at room temperature or ambient temperature, at 2 to 8°C (e.g., in a cooler or on a cold pack), or at a heated temperature such as 30 to 50°C (e.g., 30, 37, or 42°C).

[0096] In some embodiments, the method may further include a step of removing excess conjugate. In some embodiments, the removal step may include separating the biomarker-capturing particles from the liquid phase containing the buffer solution and excess conjugate. Separation may be performed by centrifugation or filtration. In some embodiments, a magnetic field may be applied during separation and / or removal.

[0097] In some embodiments, the biomarker-capturing particles may be subjected to additional washing, incubation, and separation by adding a washing buffer to the biomarker-capturing particles.

[0098] In some embodiments, the method may further include the step of adding an elution buffer to biomarker-capturing particles to elute or cleave the conjugate from the biomarker-capturing particles (a "conjugate cleave process" or "conjugate cleaving process"). In some embodiments, the solution may be incubated for a period of 1 minute to 2 hours. In some embodiments, the incubation period may be, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, or 2 hours. In some embodiments, elution may be performed using acidic elution and incubation of 5 minutes or less, or preferably 2 minutes or less. Before neutralizing the pH of the eluate sample, use a neutralizing buffer such as adding 35 μL of neutralizing buffer (e.g., 300 mM Tween-20 at pH 10.0) to 220 μL of elution buffer (e.g., 100 mM glycine, 0.05% (w / v) Tween-20, pH 2.5) to neutralize the pH of the eluate sample to 7.2-7.5. For example, after washing biomarker-capturing particles (e.g., capture beads) with a washing buffer, for example 2 to 4 times, the captured biomarkers can be eluted from the biomarker-capturing particles using an acidic elution buffer (e.g., pH 2.5, 220 μL of 100 mM glycine, 0.05% Tween-20, or 180 μL of 40 mM acetic acid, 0.05% Tween-20, pH 3.05), and then neutralized with a neutralization buffer (e.g., pH 10.0, 35 μL of 300 mM Tween-20, or pH 10.5, 26 μL of 300 mM Tween-20) so that the biomarkers are purified into a final matrix-free buffer having a neutral pH of 7.0 to 8.0.

[0099] In some embodiments, the biomarker-capturing particles may be removed from the elution solution following the elution or cleavage of the conjugate from the biomarker-capturing particles. The elution solution containing the eluted conjugate may then be subjected to detection, measurement, and / or characterization ("characterize" or "characterizing" process). In some embodiments, detection may include fluorescence detection or microscopic detection. In some embodiments, a neutralizing buffer may be added to the elution solution before detection.

[0100] In some embodiments, the sample may include an untreated sample, such as a sample being collected. The sample may be collected in a sample collection tube, such as a serum / plasma primary collection tube, a saliva collection tube, a saline mouth rinse collection tube, a fecal collection tube, or a urine collection tube. Following sample collection, multiple biomarker-capturing particles may be added to the sample. This allows the entire collected sample to be processed and exposed to biomarker-capturing particles to (i) maximize the capture efficiency and recovery / yield of biomarkers in the sample, especially if they are low-abundance biomarkers, or (ii) maximize the recovery of biomarkers by freeing, releasing, dissolving, or cleaving the sample in the presence of biomarker-capturing particles, especially for low-abundance biomarkers or biomarkers that would otherwise be lost to the sample collection tube or device surface, for example via hydrophobic, ionic, or other nonspecific binding (NSB) mechanisms, or lost hydrophobically or via other binding mechanisms to other sample components such as other proteins, lipids, triglycerides, or molecules, or lost via sensitivity to protease or enzymatic digestion or degradation if released in solution and not bound or protected by a binding partner.

[0101] In some embodiments, the sample, biomarker-capturing particles, detection antibody, or a combination thereof can be pretreated to remove one or more interferences from the sample, biomarker-capturing particles, detection antibody, or a combination thereof. In some embodiments, the pretreatment may be performed before capture by the biomarker-capturing particles (a “conditioning” process). In some embodiments, the removal of one or more interferences may be performed before step (b).

[0102] In some embodiments, the interference includes one or more lipids, triglycerides, bilirubin, hemolytic products (e.g., hemoglobin, enzymes, or potassium), cholesterol, human anti-mouse antibodies (HAMA), rheumatoid arthritis interference (RF), manufacturing assay-specific interference (MASI), human anti-animal antibody (HAAA) interference such as mouse, goat, sheep, rabbit, and bovine immunoglobulins, free biotin interference, anti-streptavidin, anti-biotin interference, human anti-polyethylene glycol (PEG) or anti-polyethylene oxide (PEO) interference, anti-albumin, anti-histidine, and anti-polyhistidine tags. This may include (e.g., 6-his tags, 8-his tags), nonspecific binding, anti-polyvinylpyrrolidone (PVP), antipolymer, anti-alkaline phosphatase (ALP), anti-ruthenium, anti-fluorescein, anti-acridinium esters (including ABEI, luminol, isoluminol), autoantibodies, anti-horseradine peroxidase (HRP), anti-conjugation linkers such as LC, LC-LC, and PEOn, anti-amino acid tags, anti-polyhistidine tags, counter-over (OTC) supplements, herbal remedies, and / or therapeutic agents, or any combination thereof. In some embodiments, hemolysis may include the rupture (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into the surrounding fluid (e.g., plasma). In some embodiments, hemolytic products that are problematic and can cause interference and false results in chemical and immunoassay tests may include hemoglobin, potassium, enzymes such as lactate dehydrogenase (LDH), and / or fluids. Hemolytic interference can be caused by the release of intracellular substances, which falsely increases the serum / plasma concentrations of certain analytes, such as potassium and lactate dehydrogenase, while diluting other substances such as sodium. Potassium, LDH, AST, magnesium, or phosphorus are the parameters that have the greatest difference between the intracellular medium of RBCs and the extracellular medium, which already shows significant interference at low hemolytic levels. Human anti-animal antibody (HAAA) interference is mainly due to human heterophile antibodies specific to bovine, goat, mouse, rabbit, or sheep IgG, which are commonly used in immunoassays.In some embodiments, rheumatoid factor (RF) interference may be specific to the mouse IgG and Fc portions of antibodies used in immunoassays, while manufacturing assay-specific interference (MASI) may be directly or indirectly caused (i.e., to antibodies) by patient-specific interference from diet, dietary supplements, pharmaceuticals, or drug therapies / treatments that bind to or interact with the raw materials important to the immunoassay. In some embodiments, the interference may include small molecules or analogues of such small molecules.

[0103] In some embodiments, removing one or more interferences may involve bringing the sample into contact with the interference-trapping particles.

[0104] In some embodiments, the interference-trapping particle may include an interference-trapping portion that interacts with the sample interference such that the interference interacts and binds to the particle surface when exposed to the interference-trapping particle. In some embodiments, the trapping portion may interact with the interference via specific binding. In some embodiments, the trapping portion may interact with the interference via nonspecific binding.

[0105] In some embodiments, interference-captured particles may be stored in a storage diluent or buffer. The storage diluent or buffer may contain components or ingredients such as chemicals, salts, buffers for increasing or decreasing sample pH, detergents, surfactants, solvents, polymers, proteins, peptides, or blockers. In some embodiments, interference-captured particles may be added to the sample together with the storage diluent or buffer.

[0106] In some embodiments, interference-captured particles may be isolated or removed from the sample by magnetic centrifugation, centrifugation, or filtration. In some embodiments, a substantially particle-free sample may have a significantly lower level, concentration, threshold, or titer of interference so as not to interfere with the testing, measurement, or characterization of the sample. In some embodiments, the interference-captured portion on the interference-captured particles may contain human immunoglobulins (e.g., IgA, IgG, IgM, or IgE) that can target autoantibody interference. In some embodiments, the interference-captured portion may contain animal antibodies (e.g., mouse, goat, sheep, rabbit, cattle, llama, or alpaca) that can target heteroatomic interference such as HAMA, RF, and HAAA. In some embodiments, the interference-captured portion may contain polymerized antibodies or mouse antibody fragments (Fc, Fab, F(ab')2) that can target HAMA and RF interference. In some embodiments, the interference scavenging portion may include proteins, enzymes, or small molecules that can target anti-signal generation interference (e.g., ALP, HRP, fluorescein / fluorophores, luminol, isoluminol, acridinium esters, ABEI, ruthenium, or luciferin). In some embodiments, the interference scavenging portion may include streptavidin, avidin, or neutraavidin that can target biotin interference or biotin metabolite interference (e.g., bisnorbiotin or biotin sulfoxide). In some embodiments, the interference scavenging portion may include polyhistidines that can target anti-histidine or anti-polyhistag interference, or PEG or PEG that can target anti-PEG or anti-PEO interference. In some embodiments, the interference-capturing moiety may include antibodies, aptamers, antibody fragments, MIPs, or polymers that can target specific interferences via epitope binding, such as bilirubin, albumin, lipids, triglycerides, cholesterol, jaundice (e.g., bile pigments), hemoglobin, herbs (e.g., hemolytic products, stipulation, jaundice, tube additives, radioactive or fluorescent compounds, drugs, herbal medicines, and dietary supplements). In some embodiments, the interference-capturing moiety may include one or more interference-capturing moieties that can target one or more types, classes, or groups of interferences disclosed herein.

[0107] In some embodiments, the method may include a step of preparing the sample. In some embodiments, the preparation may be performed before or after removing one or more interferences. In some embodiments, the preparation may include modifying the chemical or physical properties of the sample. In some embodiments, the chemical or physical properties may include temperature, pH, color, salinity, conductivity, density, viscosity, surface tension, or protein content. In some embodiments, the preparation may include introducing into the sample a surfactant, detergent, cell lysant, antiprotease agent, protein-based or polymer-based blocking reagent, substitution agent, or agent for releasing the analyte or biomarker from the matrix or for removing interfering elements.

[0108] In some embodiments, the cell lysant may include a RIPA buffer for lysing cells such as exosomes, extracellular particles, virions, or bacteria. The lysant is compatible with interfering capture particles and does not damage or impair the activity or target binding of the antibody or capture moiety. The preanalytic lysis step may release or liberate biomarkers from cells such as exosomes for subsequent binding, capture, purification, detection, and / or measurement.

[0109] Figure 1 shows an exemplary sample preparation method. Sample 101 may contain multiple biomarkers, e.g., biomarkers 102, 103, and 104, and the multiple biomarkers may be of different types. For example, the multiple biomarkers may include antigens, antibodies, any other type of biomarker, or any combination thereof. In operation 100, multiple biomarker-capturing particles, e.g., biomarker-capturing particles 105, 106, and 107, are introduced into sample 101. After incubation time, the biomarker-capturing particles 105, 106, and 107 capture biomarkers 102, 103, and 104 to form multiple complexes 108, 109, and 111. In operation 110, a magnetic field or magnet is applied to attract the complexes 108, 109, and 111.

[0110] The liquid phase of sample 101 may be removed or discarded. The elution solution is then added to multiple complexes so that multiple biomarkers can be eluted or cleaved from multiple biomarker-capturing particles. The eluate containing the eluted biomarkers may be subjected to detection or measurement. In some embodiments, a neutralizing buffer may be added to the eluate before any detection or measurement.

[0111] In some embodiments, after removing the liquid phase of sample 101, multiple conjugates may be added to a complex and conjugate with the captured biomarkers. Subsequently, an elution solution is added to the complex containing the multiple biomarkers and multiple conjugates, and the multiple conjugates may be eluted or cleaved from the multiple biomarker-capturing particles. The eluate containing the eluted multiple conjugates may be subjected to detection or measurement. In some embodiments, a neutralizing buffer may be added to the eluate before any detection or measurement.

[0112] Figure 2A shows an exemplary method for detecting and / or measuring a biomarker, including capture, concentration, and biomarker cleavage. Figure 2B shows an exemplary method for detecting and / or measuring a biomarker, including capture, washing, concentration, and biomarker cleavage. Figure 2C shows an exemplary method for detecting and / or measuring a biomarker, including capture, washing, concentration, conjugation, and conjugate cleavage. Figure 2D shows an exemplary method for detecting and / or measuring a biomarker, including preparation, capture, washing, concentration, conjugation, and conjugate cleavage. The methods disclosed in Figures 2A to 2D may further include a characterization process. For example, Figure 2E shows an exemplary method for detecting and / or measuring a biomarker, including preparation, capture, washing, concentration, conjugation, conjugate cleavage, and characterization. A process such as that shown in Figure 2E may be referred to as a “7C” process.

[0113] In some embodiments, the method may further include the step of conjugating the captured biomarker with a detection antibody. In some embodiments, the method may further include the step of measuring the detection antibody. In some embodiments, the method may further include the step of comparing the detection antibody with a standard curve. In some embodiments, the standard curve is generated from biomarker-capturing particles conjugated to a known amount of biomarker.

[0114] In some embodiments, the method may further include the step of washing the detection antibody with interference-capturing particles or biomarker-capturing particles.

[0115] In some embodiments, the detection antibody may include an anti-human antibody. In some embodiments, the detection antibody may include an antibody against an antigen.

[0116] In some embodiments, the detection antibody may be conjugated to the detection reagent. In some embodiments, the detection reagent may include an enzyme or a label. In some embodiments, the label may include a fluorescent tag. Non-limiting examples of labels may include ALP, HRP, isoluminol, luminol, acridinium, ABEI, ruthenium, or antibody-oligoconjugates in immuno-PCR, or enzymes, proteins, or small molecules such as luciferin for ELISA (enzyme-conjugated immunoassay), CLIA (chemiluminescent immunoassay), or FIA (fluorescence immunoassay).

[0117] In some embodiments, the method may further include the step of multiplexing the capture particles with additional capture particles containing a second antigen, and the biomarker includes an antibody that binds to the second antigen. In some embodiments, the step of quantifying the biomarker may include multiplexing the detection antibody with a second detection antibody that recognizes the second antigen. In some embodiments, the method may further include the step of multiplexing the capture particles with additional capture particles containing a third antigen, and the biomarker includes an antibody that binds to the third antigen. In some embodiments, the step of quantifying the biomarker may include multiplexing the detection antibody with a third detection antibody that recognizes the third antigen.

[0118] In some embodiments, the method may further include the step of multiplexing the detection antibody with a further detection antibody that recognizes a biomarker in the sample.

[0119] In some embodiments, the method may further include the step of monitoring the biomarker over time to determine whether the amount of the biomarker in a second sample of interest has increased or decreased compared to a quantified biomarker.

[0120] In some embodiments, the biomarker capture particle may include multiple capture portions. In some embodiments, the multiple capture portions may include different types of capture portions, e.g., antigens, antibodies, any other type of capture portion, or any combination thereof, thereby enabling multiplexing or combo detection.

[0121] In some embodiments, the biomarker-capturing particles may contain multiple recombinant antigens or viral or bacterial lysates to improve the possibility and sensitivity of detecting human immunoglobulins, immunoglobulin subclasses, or combinations thereof, such as IgA, IgG, IgM, and IgE, or antigens.

[0122] In some embodiments, the biomarker capture particles may contain multiple antibodies to improve the likelihood and sensitivity of detecting an antigen or biomarker, or for the multiple detection of multiple antigens or biomarkers. This is an antigen test, which may be performed, for example, by a sandwich assay, complementary assay, piggyback assay, or delayed addition or delayed capture assay.

[0123] detection In some embodiments, detection and / or measurement of eluted biomarkers or conjugates may be performed using any suitable detection and / or measurement method, e.g., ELISA, CLIA, FIA, lateral flow, POCT, microarrays, lab-on-a-chip, HPLC (e.g., affinity chromatography using any of reversed phase, normal phase, ion exchange / anion exchange / cation exchange, hydrophobic interaction (HIC), hydrophilic interaction, partition, substitution, size exclusion, and isocratic or gradient elution), mass spectrometry (e.g., LC-MS, LC-MS / MS, MALDI-TOF), molecular analysis (e.g., PCR, RT-PCR, nucleic acid amplification assays, next-generation sequencing (NGS)), and chemical or bead-based aggregation, e.g., particle-enhanced turbidity immunoassay (PETIA). The methods and compositions of this disclosure may be used in conjunction with any suitable affinity assay or immunoassay, including, but not limited to, any suitable assay such as protein-protein affinity assays, protein ligand affinity assays, nucleic acid affinity assays, indirect fluorescence antibody assays (IFAS), enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and enzyme immunoassays (EIA), direct or indirect assays, competitive assays, sandwich assays, antibody-oligoconjugate assays in immunoPCR, homogeneous or proximity-based assays, etc.

[0124] In some embodiments, the concentration of eluted biomarkers or conjugates in a sample can be determined by using a bead-based calibration curve. The calibration curve can be generated using 2 to 10 different calibration beads, each having a different known amount of purified biomarker or conjugate captured or conjugated to the calibration bead. The signal detected after cleavage / elution from the calibrator beads is proportional, for example, to the known amount of purified biomarker or conjugate from each calibrator bead. If an unknown sample is detected, the concentration can be calculated based on the calibration curve and the detected signal. For example, the calibration curve can be generated using 7 different calibration beads, each having a different amount of purified human IgG conjugated to the calibration bead. When testing by fluorescence, the relative fluorescence signal is directly proportional to the amount of antigen-specific IgG immunoglobulin captured by the beads.

[0125] In some embodiments, the calibration beads may include multiple types of biomarkers for multiple calibration. For example, a calibration curve may be generated using seven different triplex calibration beads, each having different amounts of purified human IgM, IgG, and IgA conjugated to it. When tested by fluorescence, the relative fluorescence signal of each fluorophore is directly proportional to the amount of antigen-specific IgG, IgM, and / or IgA immunoglobulin captured by the beads.

[0126] Sample preparation For feces and semen, the samples include: light green top or green top plasma separatory tubes (PSTs) containing heparin sodium, heparin lithium, or heparin ammonium; pale blue top tubes containing sodium citrate (i.e., 3.2% or 3.8%) or citrate; red top tubes for serology or immunohematology for serum collection in glass (no additives) or plastic tubes (containing coagulation activators) containing theophylline, adenosine, dipyridamole (CTAD); red top tubes for chemistry for serum collection in glass (no additives) or plastic tubes (containing coagulation activators); and EDTA K2, EDTA K3, liquid EDTA solution (i.e., 8%), or EDTA for testing plasma in molecular diagnostics and viral load detection. Blood samples may be collected in primary blood collection tubes (PBCTs), secondary transfer tubes (SSTs), 24-hour (24-hour) urine collection devices, saliva collection tubes, blood spot filter paper, or any collection tube or device, including purple lavender top tubes containing K2 / gel tubes, pink top tubes for blood bank EDTA, gray top tubes containing potassium oxalate and sodium fluoride, sodium fluoride / EDTA, or sodium fluoride (without anticoagulants, resulting in serum samples), yellow top tubes containing ACD solution A or ACD solution B, royal blue top tubes (serum, with no additives or sodium heparin), white top tubes, or any other color or tube type, with or without additives for blood collection, with or without any combination thereof.

[0127] Primary blood collection tubes (PBCTs) and secondary transfer tubes (SSTs) are any commercially available standard or custom collection tubes (with or without gel separators) from companies such as Becton Dickinson (BD), Greiner, VWR, and Sigma Aldrich, glass tubes, plastic tubes, light green-top or green-top plasma separator tubes (PSTs) containing heparin sodium, heparin lithium or heparin ammonium, light blue-top tubes containing sodium citrate (i.e., 3.2% or 3.8%) or citrate, theophylline, adenosine, dipyridamole (CTAD), red-top tubes (no additives) or plastic tubes (containing coagulation activators) for serology or immunohemology for serum collection in glass, red-top tubes for chemistry for serum collection in glass (no additives) or plastic tubes (containing coagulation activators), and EDTA K2, EDTA K3, liquid EDTA solution (i.e., 8%), or EDTA for testing plasma in molecular diagnostics and viral load detection. The tubes may be purple lavender top tubes containing K2 / gel tubes, pink top tubes for blood bank EDTA, gray top tubes containing potassium oxalate and sodium fluoride, sodium fluoride / EDTA, or sodium fluoride (without anticoagulant, resulting in serum samples), yellow top tubes containing ACD solution A or ACD solution B, royal blue top tubes (serum, with no additives or sodium heparin), white top tubes for any application or diagnostic test type, without additives or any combination thereof, for blood collection, or any other color or tube type.

[0128] Saliva collection tubes (neat saliva, drol, spitz, sputum) or saline oral rinse (SOR) collection tubes (e.g., 3-5 mL of 0.9% sodium chloride in purified water, or phosphate-buffered saline) are available from Abclonal, Canvax Biotech, CD Genomics, DNA Genotek, Eagle Biosciences, IBI Scientific, Oasis Diagnostics, Omni International, Porex Life Sciences Institute & AG Industries, Ray Biotech, Salimetrics, Sarstedt, Stellar Scientific, Spectrum Solution, ThermoFisher, Thomas Scientific, and Zymo Any commercially available standard or custom collection tube (with or without preservatives) from companies such as Research, and the preservative may already be present in the saliva or SOR collection tube as a liquid, spray-drying reagent, or lyophilized reagent before saliva collection; or the preservative may be added to the saliva sample after saliva or SOR collection, for example by a collection tube cap, thereby containing a preservative reagent with a reagent release mechanism that breaks a membrane or barrier when the cap is placed on or screwed onto the collection tube; or the preservative may be added to the collected saliva or SOR sample by injection, distribution, dropping, or mixing with an exogenous preservative reagent.Saliva or SOR samples are collected in a collection tube and subsequently filtered into a filtrate tube using a filter or multiple filters, thereby each filter having the same or different porosity or molecular weight cutoff (MWCO), and preservatives are either already present in the saliva or SOR filtration tube as a liquid, or as a spray-dried reagent, or as a lyophilized reagent prior to saliva or SOR filtration, or preservatives are added to the filtered saliva or filtered SOR sample filtrate tube, for example by a filtration tube cap, the cap containing the preservative with a reagent release mechanism, thereby a membrane or barrier is broken when the cap is placed on or screwed onto the filtrate tube, or the exogenous preservative is added to the filtered saliva or SOR sample by addition, injection, distribution, dropping, or mixing.

[0129] A collection tube cap or filtrate tube cap, or a combination thereof including a preservative reagent or preservative, may contain magnetic particles coated with capture molecules or capture portions, thereby, when the magnetic particles are added to a collected saliva or SOR sample or to a filtered saliva or SOR filtrate, the magnetic particles may capture a specific analyte, target, or biomarker of interest, or two or more different analytes, targets, or biomarkers of interest from the entire sample, or the magnetic particles may capture interference of interest, or two or more different interferences of interest from the entire sample, thereby, when the magnetic particles are subsequently isolated or removed from the sample, for example after magnetic separation, filtration, or centrifugation, the concentration of the analyte, target, biomarker, or interference will be reduced, removed, or depleted from the sample or sample filtrate. One or more analytes, targets, biomarkers, or interferences captured by magnetic particles can subsequently be detected, measured, or quantified on the particle surface or on the one or more analytes, targets, or biomarkers, or the interferences can be cleaved, eluted, released, or dissociated from the particle surface for subsequent detection, measurement, or quantification. The magnetic particles may be washed with a washing buffer or diluent before the cleaving, eluting, release, or dissociation of one or more analytes, targets, biomarkers, or interferences from the magnetic particles for subsequent detection, measurement, or quantification, thereby removing sample matrix, sample components, or sample interferences that, if not removed, would obfuscate, interfere with, or adversely affect the accuracy or sensitivity of the detection, measurement, or quantification of one or more analytes, targets, biomarkers, or interferences. The cleaved, eluted, released, or dissociated analytes, targets, biomarkers, or interferences are subsequently prepared, quenched, or neutralized by adding another buffer or diluent to improve their stability before detection, measurement, or quantification.

[0130] Preservation reagents, particle preservation buffers, or diluents containing magnetic particles coated with capture molecules or capture moieties, or preservation reagents having magnetic particles coated with capture molecules or capture moieties, are used to alter, modify, or adjust the conductivity, density, viscosity, surface tension, or protein content of a sample, or to add surfactants, detergents, cell lysants, antiproteases, antiphosphatases, protein-based or polymer-based blocking reagents, substitutions, or agents for releasing analytes from the matrix, or agents for removing interfering elements such as RIPA buffers for lysing cells, extracellular particles, exosomes, neuroexosomes, virions, or bacteria. The sample preparation reagent may further include a substitution agent or substitution reagent, such as acidic pH, danazol, or 19-nortestosterone derivatives, or 8-anilino-1-naphthalenesulfonic acid (ANSA), which releases one or more bound analytes, targets, biomarkers, or interferences from within cells, extracellular particles, exosomes, virions, or bacteria, or from binding partners such as 25-hydroxyvitamin D (25OHD) derived from vitamin D-binding protein (VDBP), or dihydrotestosterone derived from testosterone, estradiol, or sex hormone-binding globulin (SHBG).

[0131] If the sample preparation reagent, lysis buffer, substitution agent, or substitution reagent also includes magnetic particles coated with a capture molecule or capture moiety, then free analytes, targets, biomarkers, or interferences from cells, extracellular particles, exosomes, neuroexosomes, virions, bacteria, or binding partners may be released into the collected sample or sample filtrate in the presence of magnetic beads, thereby maximizing the recovery and capture of one or more analytes, targets, biomarkers, or interferences of interest to improve the accuracy or sensitivity of the detection, measurement, or quantification of one or more analytes, targets, biomarkers, or interferences of interest, or to increase the sensitivity of the detection, measurement, or quantification of low-abundance, dilute, or very low-concentration analytes, targets, biomarkers, or interferences of interest.

[0132] In some embodiments, the subject may receive a sample collection device or receptacle by mail, from a testing facility, or at a point-of-care facility. The sample collection device may comprise a tube and a cap. The subject collects the sample into the tube and caps the tube. In some embodiments, the cap may contain biomarker-capturing particles or capture portions that are released into the sample upon capping. For example, the cap may be a screw cap that releases biomarker-capturing particles or capture portions when the subject screws the cap onto the container. The capture particles or capture portions may include capture portions conjugated to capture particles. The capture particles or capture portions may include capture portions that do not contain capture particles. The tube or cap may also contain a solution or buffer. For saliva collection, the subject may be provided with an oral rinse. The oral rinse may contain a buffer. The collected sample may then be delivered to a laboratory or testing facility. During transport, the biomarker-capturing particles or capture portions are incubated with any biomarkers from the collected sample.

[0133] How to use In some embodiments, the capture portion is immobilized, conjugated, or coated onto the surface of the biomarker-capturing particle, thereby such biomarker-capturing particle may be any antigen, therapeutic agent, drug, small molecule, peptide, protein, vaccine, or immunogen that is therapeutically administered or given to a subject, to which immunoglobulins can be bound (e.g., orally as a pill or liquid or solvent, as a shot, as a supplement or herbal remedy, as a food or liquid, as a lotion, as an IV injection, as a patch, as an enema, or the therapeutic agent may be administered or introduced to the subject by the skin, hair, etc., or by any external route including absorption, ingestion, and injection into the subject). In some embodiments, the immunoglobulin may include human immunoglobulin classes IgA, IgG, IgM, and IgE, as well as human immunoglobulin subclasses such as IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4, or species-specific antibodies such as IgG, and the biomarker-capturing particles may be used for the detection and monitoring of antibody production against antigens, therapeutic agents, drugs, small molecules, peptides, proteins, vaccines, or immunogens.

[0134] In some embodiments, the methods provided herein may be used for therapeutic monitoring to detect, measure or quantify any immune response or autoantibody response to a drug or immunogen that could be dangerous to a patient, cause health and / or safety problems, or increase the risk of adverse events or treatment responses, including disease, hospitalization, or death, where the absence of such antibodies indicates that the treatment is safe and effective without harmful immune responses.

[0135] In some embodiments, the methods provided herein may be used for vaccine efficacy testing and monitoring to 1) detect, measure, or quantify any immune response, antibody response, or human immunoglobulin class or subclass response to a vaccine or immunogen to determine whether the vaccine is functioning as intended; and 2) monitor the antibody response (timing, duration, level, and magnitude) by antibody class or subclass for antibody profiling, etc., to determine the antibody response, timing, or the time it takes for antibody production after the first vaccine dose, after the second vaccine dose, and after booster doses, and to establish a protective immune threshold or cutoff. Antibody levels may be monitored in circulation (blood, plasma, or serum), as a secretory response in oral fluids such as saliva or SOR, or in feces (produced in the intestines or GI system).

[0136] In some embodiments, the antibody may be a therapeutic antibody such as a monoclonal antibody, a humanized monoclonal antibody, an antibody fragment, an aptamer, an MIP, a nanobody such as a Lama or Alpaca-derived nanobody, or an animal-produced antibody or pesticide antibody, administered or given to a subject (e.g., a human patient or animal) for therapeutic, health improvement, or any health problem, in order to treat, cure, manage or mitigate a disease, disease onset, disease progression or spread, or to eradicate, eliminate or destroy a disease, infection, pathogen, cancer, or autoimmune disorder. In some embodiments, the therapeutic antibody is immobilized, coated, or conjugated onto a biomarker-capturing particle to monitor any immune response to the therapeutic antibody in a human patient or animal as described above. In some embodiments, therapeutic antibodies are developed in human patients or animals, and antigens or targets targeted against them, recognizing or binding to them, may be immobilized on biomarker-capturing particles. These biomarker-capturing particles can be used to test human patient or animal samples, and therapeutic antibody levels, concentrations, or titers for precision medicine and induction therapy can be monitored and measured. The method may be used to determine specific therapeutic antibody doses, circulating concentrations, or secretory concentrations. The method may be used in pharmacokinetic studies to determine the temporal normalization, steady state, equilibrium, and metabolism of therapeutic antibodies in circulation (e.g., blood, serum, or plasma) or secretion (e.g., oral fluids such as saliva and SOR, or feces, GI system).

[0137] In some embodiments, assay methods are disclosed herein that include the step of obtaining a sample of interest. The sample may be subjected to washing by removing assay interference from the sample having interfering particles. In some embodiments, the method includes capturing an antibody from the sample. In some embodiments, the method includes contacting the sample with biomarker-capturing particles containing the antigen of the antibody. In some embodiments, assay methods are disclosed herein that include the steps of obtaining a sample of interest subjected to washing by removing assay interference from a sample having interfering particles, and capturing an antibody from the sample by contacting the sample with biomarker-capturing particles containing the antigen of the antibody. In some embodiments, the antibody may include an epitope that binds to the antigen. In some embodiments, the method includes eluting the antibody from the biomarker-capturing particles. In some embodiments, the method includes determining the amount of antibody in the sample or subject. In some embodiments, determining the amount of antibody in the sample or subject includes determining the antibody mass per sample volume. In some embodiments, the method includes washing the sample by removing assay interference from the sample having interfering particles. In some embodiments, the method includes washing the biomarker-capturing particles with the interfering particles. In some embodiments, the interfering particles are removed to produce a clean sample. In some embodiments, the washed sample is substantially uninterfered. In some embodiments, biomarker-capturing particles are added to the washed sample to capture the biomarker of interest (e.g., an antibody).

[0138] In some embodiments, the subject is suspected of having the disease or has been vaccinated against the disease. In some embodiments, the antigen contains a pathogen or component of the disease. In some embodiments, the captured antibody contains an antibody against the pathogen or disease. In some embodiments, the vaccine contains an antigen. In some embodiments, vaccine efficacy is determined based on the amount of antibody in the sample or subject. In some embodiments, the vaccine is re-administered to the subject based on the efficacy of the vaccine. In some embodiments, the likelihood of the subject having the disease is identified based on the amount of antibody in the sample or subject.

[0139] Some embodiments include identifying whether the disease is active or acute. Some embodiments include administering disease treatment to a subject if the subject is identified as having an active or acute disease. Some embodiments include not administering or discontinuing treatment if the subject is identified as not having an active or acute disease. In some embodiments, administering treatment includes adjusting the dose or timing. In some embodiments, the subject is administered a therapeutic compound. In some embodiments, the therapeutic compound comprises a therapeutic agent. In some embodiments, the antigen comprises the therapeutic compound or a fragment thereof. In some embodiments, the captured antibody comprises an autoantibody against the therapeutic compound. Some embodiments include determining the level of safety of the therapeutic compound based on the amount of antibody in the sample or subject. Some embodiments include administering the therapeutic compound to a subject or adjusting the dose thereof based on the amount of antibody in the sample or subject. In some embodiments, the therapeutic agent comprises a therapeutic antibody. In some embodiments, the therapeutic antibody comprises an antibody-conjugated fragment. In some embodiments, the captured antibody comprises the therapeutic antibody. In some embodiments, the antigen comprises the antigen or target of the therapeutic antibody. Some embodiments include determining the pharmacokinetic profile of a therapeutic antibody based on the amount of antibody in a sample or subject. Some embodiments also include administering a therapeutic antibody to a subject or adjusting the dose based on the amount of antibody in a sample or subject.

[0140] In some embodiments, the captured antibody includes a secreted antibody. In some embodiments, the captured antibody includes IgA, IgG, or IgM, or a combination thereof. In some embodiments, the captured antibody is conjugated with a detection antibody. In some embodiments, the detection antibody is measured. In some embodiments, the detection antibody is compared to a standard curve. In some embodiments, the standard curve is generated from biomarker-capturing particles conjugated to a known amount of antibody. In some embodiments, the detection antibody is washed with interfering capture particles. In some embodiments, the detection antibody includes an anti-human antibody. In some embodiments, the detection antibody includes an antibody against an antigen. In some embodiments, the detection antibody is conjugated to a detection reagent. In some embodiments, the detection reagent includes an enzyme or label. In some embodiments, the label includes a fluorescent tag. In some embodiments, the capture particles are multiplexed with additional capture particles containing a second antigen, and the antibody includes an antibody that binds to the second antigen. In some embodiments, determining the amount of antibody in a sample or subject includes multiplexing the detection antibody with a second detection antibody that recognizes the second antigen. Some embodiments include multiplexing the detection antibody with an additional detection antibody that recognizes a biomarker in the sample. Some embodiments also include monitoring the antibody over time to determine whether the amount of antibody in a second sample of interest has increased or decreased.

[0141] Assay method using the capture portion and biomarker-capturing particles In some embodiments, assay methods are disclosed herein, comprising the steps of (i) providing a sample of interest, (ii) contacting the sample with a capture portion, and (iii) contacting the sample with biomarker-capturing particles. In some embodiments, the biomarker-capturing particles may include a coating, such as a streptavidin coating. In some embodiments, the capture portion may interact with a biomarker. In some embodiments, the capture portion may further include a portion that can interact with and / or bind to the biomarker-capturing particles. In some embodiments, step (ii) includes adding the capture portion to a sample in a sample collection device. In some embodiments, the capture portion may be present in the sample collection device before or during sample collection. In some embodiments, the capture portion may be a biotinylated capture portion. In some embodiments, the capture portion may form a biomarker-capturing portion complex. In some embodiments, the biomarker-capturing portion complex may include one or more biomarkers. In some embodiments, the biomarker-capturing portion complex may include one or more capture portions and one or more biomarkers. In some embodiments, step (iii) includes adding biomarker-capturing particles in a molar excess relative to the total moles of the capture portion. In some embodiments, step (iii) may be performed in a laboratory, testing facility, or point-of-care facility. The biomarker-capturing particles may bind to the capture portion and the biomarker-capturing portion complex.

[0142] In some embodiments, the sample may be subjected to interference removal by contacting the sample with an interference-capturing composition. In some embodiments, the interference-capturing composition is removed before contacting the sample with biomarker-capturing particles.

[0143] In some embodiments, prior to (ii), the method may include a step of contacting the sample with the interference-capturing composition to remove assay interference. In some embodiments, after contacting the sample with the interference-capturing composition, the interference-capturing composition is removed.

[0144] In some embodiments, the capture portion may be present in a reagent, for example, a liquid reagent or a solid reagent. In some embodiments, the liquid reagent may include a sample preservation reagent or stabilizer. In some embodiments, the liquid reagent may include a sample preparation reagent or reagent. In some embodiments, the liquid reagent may include a sample preservation reagent or stabilizer and a sample preparation reagent or preparation agent. In some embodiments, a solid reagent may be formed by lyophilizing, spray-drying, or pelletizing a liquid reagent. In some embodiments, the capture portion may be present in the solid reagent, for example, in a spray-dried, lyophilized, or pelletized form. In some embodiments, the solid reagent may be derived from a lyophilized, spray-dried, or pelletized liquid reagent as disclosed herein. In some embodiments, the sample preparation reagent or reagent may further include a solvent, cell lysant, substitution agent, or binding partner substitution or dissociation agent. If a biomarker is located inside a cell, extracellular particle, exosome, neuroexosome, virion, or bacterium, or is bound to a binding partner (e.g., vitamin D-binding protein, sex hormone-binding globulin, autoantibody, immune complex), the biomarker may be released or unleashed in the presence of the capture moiety to enhance the capture and retrieval of the biomarker by the capture moiety.

[0145] In some embodiments, the reagent containing the capture portion may already be present in the sample collection device before the sample or filtered sample is added to or collected in the sample collection device. In some embodiments, the reagent containing the capture portion may be added to the sample collection device after sample collection, or after sample collection and filtration. In some embodiments, the reagent containing the capture portion may be stored in a screw cap having a screw cap release mechanism, thereby allowing the reagent to be added and mixed with the sample after the screw is tightened and the barrier is broken to mix the sample and the reagent. In some embodiments, the reagent containing the capture portion may be stored in a separate tube, vial, bottle, ampoule, or container, and after the sample has been collected in the sample collection device, the storage device may be opened or broken and the reagent contained therein may be added to the sample or filtered sample by adding, pouring, discarding, dropping, flowing out, or mixing it with the sample. In some embodiments, the reagent containing the capture portion may be added dropwise from a dropper bottle, poured from a storage bottle after loosening or removing the cap, or poured / squeezed from an ampoule after removing the tab. In some embodiments, a solid reagent containing the capture portion may be added or dropped into a sample, where the pellet dissolves and releases the capture portion into the sample. In some embodiments, the capture portion may be stored inside a capsule, pellet, or pill that is soluble as a liquid or solid reagent, whereby a soluble or time-delayed soluble capsule, pellet, or pill is added to the sample, and as the capsule, pellet, or pill dissolves in the sample, the capture portion is released and mixed into the sample for biomarker capture.

[0146] In some embodiments, biomarker capture particles may include particles coated with streptavidin or anti-fluorescein antibodies, which may target or bind to a portion on the capture portion, such as biotin or fluorescein. In some embodiments, the portion may include a tag or fusion protein recombinantly attached to the capture portion, such as a His tag, 6His tag, or maltose-binding protein (MBP). The portion on the capture portion may enable rapid and efficient capture of the total capture portion or biomarker-capture portion complex from the sample. In some embodiments, the capture portion may be captured by a binding partner immobilized or coated on the biomarker capture particle, such as an anti-capture portion antibody. For example, if the capture portion is an animal-derived antibody (such as mouse, rabbit, goat, sheep, cattle, horse, llama, alpaca, camel, or pig), the biomarker capture particles may be coated with an anti-animal antibody, such as an anti-mouse, rabbit, goat, sheep, cattle, horse, llama, alpaca, camel, or pig antibody. If the capture portion is an aptamer or a molecularly imprinted polymer (MIP), or is tagged, conjugated, or labeled with an oligonucleotide, peptide, polymer, or other tag, the capture portion may be captured by biomarker capture particles coated with an anti-aptamer, anti-MIP, anti-oligonucleotide (complementary sequence), anti-peptide, or anti-polymer, or by biomarker capture particles coated with an anti-other binding partner.

[0147] In some embodiments, the assay method may further include a concentration process as disclosed elsewhere in this disclosure. In some embodiments, the assay method may further include a cleavage process as disclosed elsewhere in this disclosure. In some embodiments, the assay method may further include a washing process as disclosed elsewhere in this disclosure. In some embodiments, the assay method may further include a biomarker cleavage process as disclosed elsewhere in this disclosure. In some embodiments, the assay method may further include a conjugation process as disclosed elsewhere in this disclosure. In some embodiments, the assay method may further include a conjugate cleavage process as disclosed elsewhere in this disclosure. In some embodiments, the assay method may further include a characterization process as disclosed elsewhere in this disclosure.

[0148] In some embodiments, the assay method may further include the steps of isolating biomarker-capturing particles from a sample and removing or aspirating the sample matrix. In some embodiments, the biomarker-capturing particles may be isolated / separated from the sample by centrifugation, filtration, or magnetic separation. In some embodiments, the captured biomarker may then be detected directly on the biomarker-capturing particles with or without washing of the biomarker-capturing particles before detection. In some embodiments, the captured biomarker may be cleaved or eluted from the biomarker-capturing particles for subsequent detection and / or measurement via any preferred detection method presented herein. In some embodiments, the biomarker may be concentrated, enriched, purified, conjugated, and / or neutralized before detection / measurement. The assay method may improve biomarker binding kinetics and biomarker capture efficiency (including reduction of time) due to a homogeneous reaction between the capture portion and the biomarker.

[0149] In some embodiments, the capture portion and biomarker may include any biomarker, capture portion pair disclosed herein. In some embodiments, the biomarker may include multiple biomarkers. In some embodiments, the biomarker may include multiple types of biomarkers, and the capture portion may include multiple types of capture portions for multiple capture and detection / measurement.

[0150] Monitoring of vaccine efficacy and immune response The methods provided herein may be used to monitor vaccine efficacy and immune response. After vaccination, it is important to know whether antibodies are produced against the vaccine or immunogen, and to know the levels of these antibodies. Singleplex detection of antibodies such as total IgG, total IgM, or total IgA, or antibody profiling, for multiple detection of immunoglobulin classes IgG, IgM, and IgA, and / or immunoglobulin subclasses IgA1, IgA2, IgG1, IgG2, IgG3, and / or IgG4, may induce vaccine efficacy and immune response. In some embodiments, recombinant proteins or peptides containing epitopes in which the vaccine is generating an immune response may be detected by the methods provided herein.

[0151] The methods provided herein may be used to monitor any existing vaccine. Non-exclusive examples of vaccines include those for varicella (varicella), dengue fever, diphtheria, influenza (influenza), hepatitis A, hepatitis B, Hib (Haemophilus influenzae type b), HPV (human papillomavirus), measles, meningococcal disease, mumps, pneumococcal disease, polio (poliomyelitis), rotavirus, rubella (German measles), herpes zoster (herpes zoster), tetanus (Rocky), Hoping's cough (partssis), adenovirus, anthrax, cholera, Japanese encephalitis (JE), rabies, Smarpox, tuberculosis, herpes zoster, and yellow fever. Vaccines may include inactivated vaccines, live attenuated vaccines, messenger RNA (mRNA) vaccines, subunit, recombinant, polysaccharide, and conjugate vaccines, toxoid vaccines, Novavax vaccines, DNA and recombinant vector vaccines (also known as platform-based vaccines), and viral vector vaccines.

[0152] In some embodiments, the biomarker capture particle may contain one or more different types of biomarker capture moieties capable of binding to antibodies. The biomarker capture moieties may contain an antigen, a vaccine, or a vaccine fragment. After capture or capture and conjugate, the biomarker or conjugate may be eluted from the biomarker capture particle and detected and measured for the presence of a target antibody or multiple target antibodies. In some embodiments, the efficacy of the vaccine may be monitored over a long period to generate a vaccine response profile.

[0153] In another embodiment, the Disclosure provides a method for determining the efficacy of a vaccine, the method comprising the steps of (a) providing a sample of a subject to be administered the vaccine, (b) capturing a biomarker from the sample by contacting the sample with biomarker-capturing particles containing a biomarker-capturing portion, (c) quantifying the eluted biomarker, and (d) determining the efficacy of the vaccine. In some embodiments, the method may include the step of re-administering the vaccine to the subject based on the vaccine efficacy. In some embodiments, the sample may be subjected to interference removal by contacting the sample with an interference-capturing composition. In some embodiments, the interference-capturing composition is removed before contacting the sample with the biomarker-capturing particles. In some embodiments, before (b), the method may include the step of contacting the sample with an interference-capturing composition to remove assay interference. In some embodiments, the interference-capturing composition is removed after contacting the sample with the interference-capturing composition.

[0154] Monitoring of therapeutic drugs

[0155] The methods provided herein may be used to monitor the efficacy of a pharmaceutical product. In some embodiments, the pharmaceutical product may include a vaccine or a therapeutic agent. In some embodiments, the methods disclosed herein may be used for therapeutic agent monitoring or therapeutic antibody monitoring for companion diagnostics, precision medicine, or induction therapy by monitoring the level, concentration, or dose of the therapeutic agent in a patient, or by continuously monitoring the patient over time. In some embodiments, the therapeutic agent or a conjugate of the therapeutic agent may be immobilized, conjugated, or bound onto a capture particle (e.g., a therapeutic capture particle). After elution from the capture particle, the therapeutic agent may be detected and measured. The therapeutic agent may be monitored or characterized in a biological fluid. In some embodiments, the biological fluid may include whole blood, serum, plasma, urine, oral fluid (saliva, drol, oral mucosal exudate (OMT), or mouth rinse), peritoneal fluid, pleural fluid, cerebrospinal fluid (CSF), tissue, sweat, or tears. In some embodiments, clinical decisions may be made based on the level of the therapeutic agent detected in a patient sample.

[0156] In one embodiment, the present disclosure provides a method for determining the efficacy of a therapeutic agent, the method comprising the steps of (a) providing a sample of a subject to which the therapeutic agent has been administered; (b) capturing a biomarker from the sample by contacting the sample with biomarker-capturing particles containing a biomarker-capturing portion; (c) quantifying the eluted biomarker; and (d) determining the efficacy of the therapeutic agent. In some embodiments, the sample may be subjected to interference removal by contacting the sample with an interference-capturing composition. In some embodiments, the interference-capturing composition is removed before contacting the sample with the biomarker-capturing particles. In some embodiments, before (b), the method may include a step of contacting the sample with an interference-capturing composition to remove assay interference. In some embodiments, after contacting the sample with the interference-capturing composition, the interference-capturing composition is removed.

[0157] In some embodiments, a subject is administered a therapeutic compound. In some embodiments, the therapeutic compound may include a therapeutic agent or a therapeutic antibody. In some embodiments, the biomarker may include a therapeutic compound or a fragment thereof. In some embodiments, the captured biomarker may include an autoantibody against the biomarker. In some embodiments, the method may include a step of determining the level of safety of the therapeutic compound based on a quantified biomarker. In some embodiments, the method may further include a step of administering the therapeutic compound to a subject or adjusting the dose thereof based on the quantified biomarker. In some embodiments, the therapeutic antibody may include an antibody-conjugated fragment. In some embodiments, the captured biomarker may include a therapeutic antibody.

[0158] In some embodiments, the method may further include a step of determining the pharmacokinetic profile of a therapeutic antibody based on a quantified biomarker. In some embodiments, the method may further include a step of administering a therapeutic antibody to a subject or adjusting the dose thereof based on a quantified biomarker. In some embodiments, the method may include a step of adjusting the timing or duration of administration based on a quantified biomarker.

[0159] Monitoring of the safety and efficacy of therapeutic drugs The methods provided herein may be used to monitor the safety and efficacy of therapeutic agents by monitoring a patient to detect a possible immune response or autoantibody response to the therapeutic agent, or by continuously monitoring a patient over time. In some embodiments, the therapeutic agent is immobilized, conjugated, or bound to capture particles. In some embodiments, the immune response is monitored or characterized, including antibody profiling for multiple detection of immunoglobulin classes IgG, IgM, and IgA, and / or immunoglobulin subclasses IgA1, IgA2, IgG1, IgG2, IgG3, and / or IgG4 against the therapeutic agent. In some embodiments, the therapeutic agent may be a drug, pharmaceutical, small molecule, protein or peptide, antibody, humanized monoclonal or chimeric antibody, or therapeutic antibody. Detection of an immune response may help in administering or adjusting the dose of the therapeutic agent. In some embodiments, dose adjustment may include reducing the dose for patient safety. In some embodiments, dose adjustment may include increasing the dose to overcome antibody-binding complexes so that the active dose is effective even in the presence of autoantibodies.

[0160] Multiple detection and / or measurement of biomarkers from saliva samples In one embodiment, the Disclosure provides a method for detecting and / or measuring a biomarker from a saliva sample. The method may include the step of washing the saliva sample with interference-capturing particles (or washing beads) to capture, remove, deplete, reduce, or eliminate saliva-specific interferences that, if otherwise still present in the sample, would adversely affect the accuracy, specificity, and / or sensitivity of subsequent biomarker capture by the biomarker-capturing particles. In some embodiments, the method may include the step of preparing the saliva sample using a washing reagent. In some embodiments, the washing reagent may include a lysis reagent for lysing pathogens, exosomes, extracellular particles, or cells, thereby releasing or freeing biomarkers from pathogens, exosomes, extracellular particles, or cells. In some embodiments, the method may include the step of isolating or removing the washing beads from the washed or washed and prepared sample by magnetic separation, filtration, or centrifugation. In some embodiments, the method may include the step of adding biomarker-capturing particles (or capturing beads) to the washed or washed and prepared sample to capture biomarkers in the saliva sample. In some embodiments, the method may include washing the captured biomarkers on the capture beads and sample matrix with a washing buffer to remove or reduce nonspecific binding to the capture beads. In some embodiments, the method may include reducing the washing volume or elution buffer to concentrate the captured biomarkers. In some embodiments, the method may include cleaving or eluting the captured biomarkers into a buffer. In some embodiments, a neutralizing buffer may be added to the buffer. In some embodiments, the eluted biomarkers may be detected or measured by any preferred detection method or existing test methods, test systems, or assays.

[0161] In some embodiments, a saliva-based biomarker of interest present in normal or low abundances may be captured and enriched from the entire collected saliva or SOR sample using magnetic biomarker-capturing particles for biomarker purification, detection, measurement, or quantification. Biomarker-capturing particles, antigen-coated biomarker-capturing particles, or mixtures or pools of two or more different antibodies, antigens, or antibodies and antigen-coated biomarker-capturing particles may already be stored in the saliva collection tube cap, in a reagent vial or container, or in the collection tube together with a preservative, preparation reagent, or mixture of preservative and preparation reagent. The biomarker-capturing particles are released from the cap, added to the sample, or mixed with the collected sample, and as a result, the preservative and biomarker-capturing particles are introduced into the entire collected saliva or SOR sample as a biomarker-capturing and enrichment reagent, and the biomarker is then purified from the saliva or SOR for subsequent testing or measurement by a test, diagnostic test, or assay method. Biomarker-capturing particles can target a single biomarker, or a pool of different biomarker-capturing particles can target a panel of biomarkers (multiple capture). Since the biomarker-capturing particles are introduced into the entire collected saliva or SOR sample of 0.5–5 mL (e.g., 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL) or >5 mL, the entire saliva sample volume or total volume is the sample volume for biomarker capture by the biomarker-capturing particles. This approach is particularly important for capturing low-abundance or diluted biomarkers in saliva or SOR, thereby facilitating biomarker concentration or enrichment for subsequent detection, measurement, or quantification of the biomarker.For biomarkers that can be released or released in saliva from a binding partner (i.e., 25OH vitamin D from a vitamin D-binding protein, or testosterone from a sex hormone-binding globulin), or from particles or cells such as exosomes or extracellular particles, virions, or bacteria, the biomarker capture particle preservation buffer may also contain a sample preparation buffer or cell lysant, such as RIPA buffer, or a substitute or chemical, as well as a preservative. In this way, the biomarker is released or released in the presence of antibody and antigen-coated biomarker capture particles, maximizing the sensitivity of biomarker recovery and biomarker detection. When saliva or SOR collection tubes containing magnetic biomarker capture particles arrive in the laboratory, the magnetic beads can be easily isolated using a magnet, either by a magnet on the side of the tube with a strong magnet, or via a magnet in a tip approach, so that the contents of the tube or sample can be removed and discarded, or so that the beads are removed from the tube, placed in a new tube, and the beads are washed. Subsequently, one or more biomarkers purified from the saliva sample are eluted, neutralized, and tested or measured by ELISA, automated CLIA, chemical, multiplex testing (i.e., Luminex, FIA, mass spectrometry), or a method of choice. In some embodiments, magnetic biomarker-capturing particles are isolated to a magnet, the saliva or SOR sample is removed and discarded before washing the beads, and the captured biomarkers are eluted from the beads. This washing process removes most of the saliva matrix and saliva-based interference before testing, so that the eluted and neutralized biomarkers are in the buffer without all or most of such saliva-based interference. This approach significantly simplifies subsequent biomarker testing and increases the sensitivity of biomarker detection. In some embodiments, biomarkers captured by magnetic biomarker-capturing particles in the collected saliva sample can be measured directly on the biomarker-capturing particles using a conjugate after the magnetic biomarker-capturing particles are isolated to a magnet, the saliva or SOR sample is removed and discarded, and the beads are washed.

[0162] In some embodiments, the use of a modifier to lyse cells in the presence of biomarker-capturing particles, or to replace and release biomarkers, facilitates the capture of all biomarkers in saliva or SOR samples, or all free biomarkers, as well as biomarkers released or released from cells or binding partners.

[0163] In some embodiments, the target biomarker(s) may be captured in a saliva collection device such that the magnetic beads may subsequently be isolated and washed, and the captured biomarker(s) may be eluted and neutralized in a buffer for subsequent testing by existing tests or test platforms such as Luminex, ELISA, automated CLIA, and mass spectrometry.This approach overcomes several key challenges in working with and testing saliva, including: 1) improved sensitivity, or increased possibility of detecting low-abundance biomarkers, because the entire collected saliva sample is the sample for magnetic biomarker-capturing particles, and the eluted biomarkers can be much smaller in sample size due to their concentration / enrichment; 2) improved precision, or all the known challenges that exist in saliva testing, such as the saliva matrix, can be mitigated by simple magnetic isolation and washing of the biomarker-capturing particles by the laboratory before eluting the biomarkers into a simple (saliva matrix-free) test-easy buffer; 3) improved recovery, or because the biomarker-capturing particles are added to the saliva sample immediately after saliva sample collection, this maximizes biomarker capture and recovery from the sample before any possible biomarker loss via hydrophobic or nonspecific binding mechanisms, as the biomarker-capturing particles release the bound biomarker or intracellular biomarker in the presence of the capture beads. 4) Improved stability, or biomarker capture particles can be stored in a storage reagent, and biomarkers, proteins, and antigens are generally more stable when they are bound to or immobilized on a solid phase, and once the biomarker capture particles bind to their target biomarkers, the captured biomarkers are very stable until they are eluted from the biomarker capture particles for testing. 5) Improved laboratory workflow, or the biomarker capture process is already complete, as the biomarkers are already captured by magnetic beads in the saliva collection device before the laboratory receives the sample tube, eliminating the time and effort required to centrifuge the sample, remove the saliva matrix (which may be stored for other tests), wash the particles, and elute and neutralize the biomarkers for testing.This sample workflow can also be fully automated by any liquid handler, and buffer-based biomarker samples can be easily tested by any existing testing platform, including a fully automated CLIA analyzer, or by an analyzer on a track system, which may be difficult or impossible for viscous pure saliva samples.

[0164] Multiple detection and / or measurement of neuronal markers In one embodiment, the present disclosure provides a method for detecting and / or measuring neuronal markers in a sample. In some embodiments, the neuronal markers may be biomarkers for Alzheimer's disease or amyloid pathology (amyloid plaques), such as phosphorylated tau 181 (pTau181), amyloid β1-42 (Aβ42 or AB42), or amyloid β1-40 (Aβ40 or AB40) in cerebrospinal fluid, or phosphorylated tau 217 (pTau217) in plasma. In some embodiments, the sample may include plasma, saliva, or urine.

[0165] Methods for detecting / measuring neuronal markers may include washing a sample with interfering particles (or cleaning beads) to capture, remove, deplete, reduce, or eliminate interfering substances that, if still present in the sample, would normally adversely affect the accuracy, specificity, and / or sensitivity of subsequent biomarker capture by the biomarker capturing particles. In some embodiments, the method may include a step of preparing the sample using a washing reagent. In some embodiments, the washing reagent may include a lysis reagent for lysing pathogens, exosomes, extracellular particles, or cells, thereby releasing or freeing biomarkers from pathogens, exosomes, extracellular particles, or cells. In some embodiments, the method may include a step of isolating or removing the washing beads from the washed or washed and prepared sample via magnetic separation, filtration, or centrifugation. In some embodiments, the method may include a step of adding biomarker capturing particles (or capturing beads) to the washed or washed and prepared sample to capture biomarkers in the sample. In some embodiments, the method may include a step of washing the sample matrix of capture beads and captured biomarkers with a washing buffer. In some embodiments, the method may include a step of concentrating the captured biomarkers by reducing the washing volume or elution buffer. In some embodiments, the method may include a step of cleaving or eluting the captured biomarkers into a buffer. In some embodiments, a neutralizing buffer may be added to the buffer. In some embodiments, the eluted biomarkers can be detected or measured by any suitable detection method or existing test methods, test systems, or assays.

[0166] In some embodiments, the elution buffer may include an acidic buffer (low pH), such as 100-125 mM glycine at pH 2.5, or 0.15% (v / v) TFA trifluoroacetic acid (TFA), or 40 mM acetic acid, 0.05% Tween-20 at pH 3.05. In some embodiments, the eluted and purified p-tau 181 and Aβ42 can be enriched or concentrated by reducing the elution volume, or to a volume smaller than the original sample volume, or smaller than the final wash buffer volume. In some embodiments, the neutralization buffer may include 300 mM Tris or 1 M Tris at pH 8.0, or 300 mM TRIS, 0.05% Tween-20 at pH 10.5.

[0167] Sandwich competition assay In one embodiment, the disclosure provides a sandwich competitive assay (SCA) method for detecting and / or measuring a biomarker. The SCA method can test one or more samples in batches using a 96-well plate or a 384-well plate to improve test throughput.

[0168] The SCA method involves adding a sample such as serum, plasma, saliva, saliva diluted with a diluent such as 0.9% NaCl or PBS, or physiological saline mouth rinse, to the wells of a 96-well or 384-well plate. The sample can be tested as is, i.e., "as is," in the primary collection tube or secondary transfer tube, or the sample can be clarified before use or before testing, for example, after centrifugation or filtration of the sample, by removing cells, fragments, particulate matter, lipids, or other pre-analytical sample interfering substances that may precipitate as a pellet by centrifugation or be excluded or removed by filtration. Alternatively, the sample can be prepared or pre-incubated using a preparing agent, interference capture beads, or a preparing reagent containing interference capture beads before centrifugation or filtration of the sample.

[0169] The SCA method may include different addition orders or “assay formats.” In some embodiments, multiple larger magnetic biomarker capturing beads and multiple conjugates (e.g., smaller non-magnetic biomarker capturing beads or non-magnetic conjugate capturing portions such as detection antibodies) can be added to a sample to bind to the biomarker and form an immune complex. In some embodiments, multiple larger magnetic biomarker capturing beads can be added to the sample first, followed by the addition of multiple smaller non-magnetic biomarker capturing beads or non-magnetic conjugate capturing portions as a second reagent additive to form an immune complex. In some embodiments, multiple smaller non-magnetic biomarker capturing beads or non-magnetic conjugate capturing portions can be added to the sample first, followed by the addition of multiple larger magnetic biomarker capturing beads to form an immune complex. In some embodiments, a labeling and capturing portion, such as a biotin-labeled capturing portion, and a plurality of smaller non-magnetic biomarker capturing beads or non-magnetic conjugate capturing portions are added to a sample, followed by the addition of a plurality of larger magnetic anti-labeling beads, such as larger magnetic streptavidin-coated capturing beads, which can then bind to the biotin label or immunocomplex of the labeling and capturing portion.

[0170] The SCA method may further include incubation. In some embodiments, incubation can be carried out with mixing. In some embodiments, incubation can be carried out at a controlled temperature such as 2–8°C, room temperature or ambient temperature, 30°C, 37°C, or 42°C. The incubation time may be 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 4 hours, 8 hours, or overnight.

[0171] Larger magnetic trapping beads, or labeled trapping portions, are in molar excess across all biomarkers in the sample. This molar excess can be 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 40x, 100x, 1000x, or more than 1000x to capture 100% of the biomarkers, or up to 100% of the biomarkers within the incubation time.

[0172] Smaller non-magnetic capture beads or non-magnetic conjugate capture portions are limited to a molar ratio less than the total number of moles of biomarker in the sample, for example, the molar ratio of smaller non-magnetic capture beads or non-magnetic conjugate capture portions per mole of biomarker is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:40, 1:100, 1:1000, 1:2000, 1:5000, 1:10,000, or 1:100,000. In some embodiments, the smaller non-magnetic capture beads or non-magnetic conjugate capture portions may be any limited amount, mass, concentration, number of moles, or molar concentration so that the subsequent decrease in amount, mass, concentration, number of moles, or molar concentration in the sample can be detected by a reader such as a fluorometer, emission photometer, or UV / vis detector. In some embodiments, more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of the smaller non-magnetic capture beads or non-magnetic conjugate capture portions may be consumed or complexed with the larger magnetic capture beads-biomarker immunocomplex as a larger magnetic capture beads-biomarker-smaller non-magnetic capture beads complex or a larger magnetic capture beads-biomarker-non-magnetic conjugate capture portion complex.

[0173] In some embodiments, smaller nonmagnetic trapping beads or nonmagnetic conjugate trapping portions may be labeled with fluorescein or fluorophores for fluorescence detection by a fluorophotometer. In some embodiments, smaller nonmagnetic trapping beads or nonmagnetic conjugate trapping portions may be labeled with chemiluminescent substrates such as ABEI, luminol, isoluminol, and acridinium esters for emission detection by an emission photometer. In some embodiments, smaller nonmagnetic trapping beads or nonmagnetic conjugate trapping portions may be labeled with electrochemiluminescent substrates such as ruthenium for electrochemiluminescence (ECL).

[0174] In some embodiments, detection can be performed by a UV / vis detector at light wavelengths of 200 nm to 800 nm. In some embodiments, smaller nonmagnetic capture beads or nonmagnetic conjugate capture portions can be labeled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP).

[0175] After the formation of immunocomplexes, i.e., [larger magnetic capture beads]-biomarker-[smaller non-magnetic capture beads] complexes or [larger magnetic capture beads]-biomarker-[non-magnetic conjugate capture portion] complexes, the larger magnetic capture beads can be separated or isolated on one side, multiple sides, or the bottom of each well by placing the 96-well or 384-well plate on a 96-well or 384-well plate magnet of their respective or appropriate strength. Furthermore, since the biomarker, biomarker-[smaller non-magnetic capture beads] complex, or biomarker-[non-magnetic conjugate capture portion] complex is attached to, bound to, or composited with the larger magnetic capture beads, the complex can be moved to the magnet along with the larger magnetic capture beads, or separated, isolated, or removed from the sample supernatant.

[0176] The SCA method may further include the step of aspirating or dispensing the sample supernatant into a suitable 96-well or 384-well detection or reading plate to measure any residual signal of smaller non-magnetic capture beads or non-magnetic conjugate capture portions by a fluorometer, emission photometer, or UV / vis detector. A 100% conjugate signal or a high level of residual conjugate signal detected in the sample supernatant indicates a negative test result for the biomarker, i.e., the biomarker is not detected. A decrease in the conjugate signal, such as less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, or less conjugate signal detected in the sample supernatant, indicates a positive result, i.e., the biomarker is detected.

[0177] In some embodiments, the conjugate may comprise non-magnetic white biomarker-capturing beads or non-magnetic colored latex biomarker-capturing beads. In some embodiments, the conjugate may be large enough to be detected visually, for example, by the human eye. In some embodiments, the conjugate may comprise a mixture of two different colored non-magnetic latex beads, such as green and red, where one colored bead is also a biomarker-capturing bead (i.e., a "green" bead) and the other colored bead is an inactive, non-functional, or non-biomarker-binding bead (i.e., a "red" bead). If a biomarker is present in the sample, this biomarker will form an immunocomplex with larger brown magnetic-capturing beads (added to the sample in a molar excess relative to the biomarker) and smaller non-magnetic white biomarker-capturing beads, or a mixture of green non-magnetic-capturing beads and red non-magnetic beads (added to the sample in a limited molar ratio less than the total number of moles of biomarker). After magnetic separation, the brown magnetic capture beads are removed from the sample supernatant, either together with the non-magnetic white latex capture beads or the "green" non-magnetic latex capture beads (if a biomarker is present and an immunocomplex is formed), or without them (if a biomarker is not present). In the case of using white non-magnetic latex biomarker capture beads, a visual decrease, reduction, or absence of white beads (i.e., a clear solution) indicates a positive result, i.e., the biomarker was detected, while the presence or visual detection of white beads (i.e., a white solution, a "milky" solution, or a whitish, cloudy solution) indicates a negative test result, i.e., the biomarker was not detected. In the case of using green non-magnetic latex biomarker capture beads mixed with red non-magnetic latex beads, the "red" color of the solution after magnetic separation of the magnetic biomarker capture beads indicates a positive result, i.e., the biomarker was detected, while the "brown" color of the solution after magnetic separation of the magnetic biomarker capture beads indicates a negative test result, i.e., the biomarker was not detected. The SCA format can be used at point of care (POC).

[0178] kit Some embodiments relate to kits. A kit may include any of the embodiments described herein. In some embodiments, a kit may include a sample collection device for collecting biological samples disclosed herein. In some embodiments, a kit may include a sample container. In some embodiments, a kit may include biomarker capturing particles disclosed herein. In some embodiments, a kit may include a capturing portion disclosed herein. In some embodiments, a kit may include an interference capturing composition disclosed herein, such as washing particles. In some embodiments, a kit may include a buffer solution disclosed herein. In some embodiments, a kit may include a washing solution disclosed herein. In some embodiments, a kit may include a reagent for measuring biomarkers present in a sample. In some embodiments, a kit can be used in the manner disclosed herein. In some embodiments, a kit may include instructions for use, such as instructions for performing the method disclosed herein.

[0179] In some embodiments, the interference capture composition may include the interference capture portion disclosed herein. In some embodiments, the biomarker capture particles may include the biomarker capture portion disclosed herein.

[0180] In some embodiments, the kit may include a detection antibody.

[0181] In some embodiments, the interference capture portion may include human immunoglobulins, antibodies, animal antibodies, antibody fragments, polymerized antibodies, mouse antibody fragments, aptamers, proteins, enzymes, small molecules, streptavidin, avidin, neutraavidin, MIP, polymers, conjugation linkers, or any combination thereof.

[0182] In some embodiments, the biomarker capture portion may include antibodies, antibody fragments, polypeptide binders, monobodies, non-immunoglobulin binders, DARPin, aphibodies, antikalin, molecularly imprinted polymers (MIPs), aptamers, chimeric antibodies, therapeutic antibodies, antigens, proteins, small molecules, therapeutic agents, hormones, peptides, signaling peptides, exosomes, cells, disease-specific antigens, antibodies, biomarkers, or any combination thereof. [Examples]

[0183] The following examples are included for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0184] Example 1: Detection of 25OH Vitamin D 25OH vitamin D, or 25OHD, can be measured by immunoassay and / or LC-MS / MS tests to assess a patient's vitamin D sufficiency or deficiency. 25OHD exists in circulation in two distinct forms, 25OHD3 and 25OHD2, depending on diet, supplements, and UV exposure such as sunlight. 25OHD2 is more hydrophobic than 25OHD3 and is protected by binding to circulating vitamin D-binding protein (VDBP), and is also transported to cellular targets or the kidney for further hydrolysis to 25OH2D. For capture, detection, and measurement by immunoassay or LC-MS / MS, 25OHD may be released from VDBP. This release is achieved by a low acidic pH, e.g., less than 4.3, or by a substitution agent or chemical. The best 25OHD total assays liberate or release 25OHD2 and 25OHD3 in the presence of capture antibodies to achieve maximum recovery of the analyte, particularly the more hydrophobic 25OHD2, which would normally be lost hydrophobically to lipids, triglycerides, fats, or other proteins in the sample.

[0185] Biomarker-capturing particles coated with anti-25OHD2 and anti-25OHD3 antibodies, such as magnetic capture particles, are added to a saliva sample in a collection device along with a release agent to maximize recovery and enrich the biomarker by releasing or liberating potentially low amounts of total 25OHD in the presence of the capture antibodies. Upon arrival of the saliva collection device in the laboratory, the biomarker-capturing particles are separated either on a magnet or via centrifugation. The biomarker-capturing particles are then concentrated / enriched by washing to remove the sample matrix and NSBs and reduce the sample volume. The total 25OHD captured on the biomarker-capturing particles is then eluted and neutralized from the particles for subsequent immunoassay or LC-MS / MS measurement.

[0186] Example 2: Detection of nerve markers This involves the accurate and sensitive detection of neuronal markers in saliva-based samples from saliva or saline mouth rinses collected at home, or from saliva collections at clinics, pharmacies, or specialized clinics (neurologists). This is intended for age-based risk screening of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, or for diagnosing or early detection of these diseases, or for saliva-based testing in athletes suspected of having TBI or concussion, or for rule-in / rule-out of TBI or concussion in emergency rooms such as for patients or victims of car accidents, falls, or shaken baby syndrome.

[0187] Biomarker capture particles are added to saliva or saline oral rinse collection devices to maximize the recovery of these neuronal markers during or within the collection device. This is important for any neuronal markers that may be lost or become undetectable due to hydrophobic or NSB interactions with the collection device and tubing. By containing multiple different antibodies, the biomarker capture particles can bind to one or more different neuronal markers or panels of neuronal markers during pre-analytical capture. If these neuronal markers are located in neuronal exosomes, these exosomes can also be lysed in the presence of antibody-coated biomarker capture particles to maximize capture efficiency and yield from the sample.

[0188] Example 3: Aggregation study Magnetic beads (or biomarker-capturing particles, 550 nm) coated with streptavidin and co-coated with two different biotinylated monoclonal antibodies were tested for aggregation against 200 ng of IFN (protein), 200 ng of BSA (negative protein control 1), 200 ng of Gro (negative protein control 2), a triplex mixture of IFN, BSA, and Gro (200 ng each), and a buffered blank (matrix control - no protein).

[0189] The antibody pairs co-coated onto magnetic beads are shown in Table 1. The beads were prepared as follows: First, biotinylated antibody was added to a 5 mL sample tube, and streptavidin beads were (slowly) added dropwise to the biotinylated antibody while mixing (vortex mixing) to reduce bead aggregation or biotin-protein crosslinking of the streptavidin beads. The sample tube was then capped and incubated on a rocker at room temperature for 2 hours. After 2 hours, the beads were washed three times with TBS (10 mM TRIS, 150 mM NaCl, pH 7.4) containing 0.05% Tween-20 and 0.05% sodium azide. 2 mg of total magnetic beads were coated with 100 μg of total antibody (50 μg antibody / mg beads), i.e., 50 μg of each biotinylated anti-hIFNg antibody.

[0190] [Table 1]

[0191] Analytes containing the corresponding IFN, BSA, and Gro were added to 30 μL of buffer in an assay plate to prepare solutions containing 200 ng of IFN (Sample A), 200 ng of BSA (Sample B), 200 ng of Gro (Sample C), and a mixture of 200 ng of IFN, 200 ng of BSA, and 200 ng of Gro (Sample D). Sample E was a control sample (blank, buffer only). 20 μg of magnetic beads were added to samples A-E and mixed 10 times by pipetting.

[0192] After incubation, the magnetic beads were allowed to settle for 30 minutes, and the bottom of a clear, flat-bottomed well was imaged using a Cytation 5 cell imaging multimode reader.

[0193] Figures 4A to 4E show images of samples A to E, respectively. Figures 4F to 4J show images of samples A to E at high magnification, respectively.

[0194] Table 2 shows the cell count, total surface area, and total strength of samples A to E.

[0195] [Table 2]

[0196] The data show significantly larger aggregate formation for IFN or IFN-containing triplex mixtures compared to BSA or Gro.

[0197] Example 4: Multiplex detection and quantification of total IgA, IgG, and IgM against viral vaccine in serum. This example demonstrates multiplex detection of total IgA, IgG, and IgM immunoglobulins against more than 50 different HSV epitopes. Attenuated HSV viruses used as vaccine immunogens were conjugated to biomarker-capturing particles (or capture beads) by thiol-maleimide chemistry or biotin-PEG linker chemistry, or the immunogen or viral particles were digested by thiol-maleimide chemistry or biotin-PEG linker chemistry and then coated onto the biomarker-capturing particles. Regardless of the method used, all vaccine epitopes are reliably expressed on the capture beads, allowing for the detection and quantification of any immunoglobulin response to the vaccine, such as IgA, IgG, and / or IgM.

[0198] Example 5: Multiplex assay for active Lyme infection This example illustrates a multiplex assay for detecting active Lyme (Borrelia burgdorferi) infection. A single multiplex test for detecting active Lyme disease in serum or saliva combines a Borrelia antibody test and an antigen test. The Borrelia antibody test uses a pool or mixture of four different Borrelia antigen-coated capture beads (DpbA, OspA, OspC, and VIsE) to capture, detect, and quantify anti-Borrelia IgG, IgM, and IgA antibodies. Feasibility studies demonstrated 100% sensitivity and 100% specificity using a CDC validation panel (N=32) and Lyme disease biobank samples (N=19) supplied by the Bay Area Lyme Foundation.

[0199] Two testing protocols were used to determine whether patients had active Lyme disease infection. The first protocol used a 5-plex test that combined a Borrelia antibody test with an additional test measuring biomarkers of the inflammatory response to Lyme infection. This can also be used as a tick-borne disease screening test to determine whether patients showing signs and symptoms of Lyme disease have various tick-borne infections such as anaplasmosis, babesiosis, ehrlichiosis, Poissant virus disease, Borrelia miyamotoi disease, Borrelia mayonii disease, or Rocky Mountain spotted fever (RMSF). The second protocol quantified antibody levels (e.g., ng / mL or μg / mL) for antibody class profiling or "fingerprinting." This protocol mimics the algorithm of a serial cardiac troponin test used to rule in or rule out acute myocardial infarction (AMI). For example, longitudinal Borrelia antibody testing was performed on days 0 and 3 to determine whether there were rapid changes in IgM and / or IgG antibody levels or the IgG-to-IgM ratio, indicating active infection. Because obtaining serum samples over several days was difficult, saliva or saline oral rinse (SOR) was used as the sample type. This offers the further advantage of making the study more accessible to diverse patient populations and geographical characteristics by allowing saliva sample collection at home.

[0200] Figure 3A shows the first protocol combining the Borrelia antibody test with the inflammatory antigen test. The Borrelia antibody test can produce two results: negative and positive. The inflammatory antigen test can also produce two results: negative and positive. The combination of the Borrelia antibody test result and the inflammatory antigen test can indicate whether or not the subject has had Lyme infection. For example, a negative Borrelia antibody test and a negative inflammatory antigen test means that the subject has not had Lyme infection and has never had it. A negative Borrelia antibody test and a positive inflammatory antigen test means that the subject has not had Lyme infection and has never had it, but may be infected with other tick-borne diseases. A positive Borrelia antibody test and a negative inflammatory antigen test means that the subject has had Lyme disease infection in the past (past infection), but does not have active Lyme infection. A positive Borrelia antibody test and a positive inflammatory antigen test means that the subject has active Lyme infection. In contrast, antibody tests without other tests may yield false positive results or fail to capture other tick-borne infections.

[0201] Figure 3B shows the second protocol, a longitudinal Borrelia antibody test on days 1 and 3. This quantifies the change in anti-Borrelia IgG and / or IgM levels over time to determine whether an active infection is present and not a past infection. If no antibodies are detected on both days 1 and 3, it indicates a true negative (no Lyme infection). If antibodies are detected but do not increase over time, it indicates a past Lyme infection. If antibodies are detected and increase over time, it indicates an active Lyme infection.

[0202] The 7C process (shown in Figure 2E) was used for the study. In the preparation (C1) step, patient samples were prepared before analysis with paramagnetic interference capture particles, e.g., "washing beads," to perform sample-specific heterophilic antibody interference and / or autoantibody interference, as well as selective binding and removal of free biotin interference, anti-streptavidin interference, and anti-biotin interference. Subsequently, in the capture (C2) step, specific antibodies and / or antigens were captured by adding paramagnetic capture beads to the prepared samples. The capture beads consisted of paramagnetic streptavidin beads coated with biotinylated antibodies and / or biotinylated antigens. After binding the target antibody or antigen to the beads, the samples were washed by washing the beads and removing the sample matrix (C3). The samples were concentrated by reducing the sample volume (C4). A targeted fluorescent conjugate (C5) was added to the samples. Specific human immunoglobulins were detected by adding rabbit polyclonal anti-human IgG, anti-human IgM, and anti-human IgA multiplex fluorescent conjugates, and specific antigens were detected by adding fluorescently labeled monoclonal antibodies. After incubation of the samples, the capture beads were washed to remove excess conjugate. The conjugate was cleaved from the capture beads (C6) and characterized by fluorescence measurement (C7). The amounts of neutralizing antibodies and antigens detected in the samples were determined using bead-based calibration curves. Calibration curves were generated using seven different calibration beads, each conjugated with a different amount of purified human IgG. The relative fluorescence units were directly proportional to the amount of antigen-specific IgG immunoglobulin captured by the capture beads.

[0203] For Protocol 1, if two of the three IgG, IgM, or IgA antibodies are positive, the sample is antibody-positive; if two of the three IgG, IgM, or IgA antibodies are negative, the sample is antibody-negative. For Protocol 2, if both IgG and IgM are positive, the sample is positive; otherwise, the sample is negative. In either case, a positive IgG, IgM, or IgA test result occurs when the respective antibody class test signal exceeds the cutoff established for Lyme disease-negative samples. The results for Protocols 1 and 2 can be found in Table 3. The tested samples were either true-positive Lyme disease serum samples (containing anti-Borrelia antibodies, from patients clinically diagnosed with Lyme disease) or true-negative serum samples (not containing anti-Borrelia antibodies, from patients not clinically diagnosed with Lyme disease), as determined and validated by the CDC and the Bay Area Lyme Foundation. Prior to the inventors' analysis, the samples were also tested by IgM Western blot, IgG Western blot, and ELISA (IgG / IgM C-6 peptide ELISA) to confirm positive and negative results.

[0204] [Table 3]

[0205] Example 6: COVID-19 Neutralizing Antibody Test This example illustrates the development of a COVID-19 neutralizing antibody test.

[0206] Removing BSA-coated beads from the capture bead pool: BSA-coated beads (or biomarker-capturing particles) added to RBD, NTD, and Delta RBD capture bead pools as "null beads" to increase the total mass of beads per test demonstrated false-positive human serum results in troubleshooting studies. The mechanism of cross-reactivity is likely human anti-BSA heterophilic interference. To improve the efficacy and accuracy of the assay, BSA-coated beads were removed from the capture bead pool.

[0207] Figure 6 shows that the main response of capture beads was similar with and without BSA-coated beads. However, the population of samples located within the red circle demonstrates a subpopulation where capture beads with BSA were significantly increased compared to beads without BSA. The absence or reduction of signals specifically associated with the absence of BSA-coated beads may be signals related to antibodies directed towards Covid-19 RBD or NTD.

[0208] The triplex conjugate containing RBD / NTD / Delta RBD capture beads was washed to remove SARS-CoV-2 spike protein RBD and NTD cross-reactivity and reduce background signal. Agilent DAKO anti-human IgG polyclonal rabbit antibody raw material used in Alexa Fluor 555-labeled anti-human IgG conjugates exhibits SARS-CoV-2 spike protein RBD and NTD cross-reactivity. This cross-reactivity resulted in higher background signal and assay cutoff (LoQ) for the desired relative fluorescence units (RFU) in the test compared to Agilent DAKO anti-human IgM and anti-human IgA polyclonal rabbit antibody raw materials.

[0209] The triplex conjugate was treated to increase the mass of captured beads. The unit of the conjugate is 200 μL of the final conjugate, which is the volume applied to the dose per study in the Covid-19 study. Figure 7 shows the decrease in IgG background (measured using physiological saline as the sample) with increasing mass (μg) of captured beads per 1 mL of triplex conjugate.

[0210] In the current configuration, the background reduction was sufficiently low with 0.22 μg of beads per test dose conjugate. To use a logarithmic scale in the graph above, zero μg of capture beads was placed at 0.001 μg.

[0211] The amount of captured beads per test dose of the conjugate was determined to be 0.3 μg.

[0212] To improve assay sensitivity, replace the fluorophores in the current triplex conjugate with novel fluorescent dyes: Novel fluorophores from other manufacturers (iFluors from AAT Bioquest and CFluors from Biotium) may improve assay sensitivity compared to the current ThermoFisher Alexa Fluor 488, 555, and 647 fluorophores used to construct the triplex conjugate, or 488 anti-human IgM, 555 anti-human IgG, and 647 anti-human IgA conjugates.

[0213] Experiment 1: Molar excess vs. S / N of AlexaFluor, CF Biotium, and iFluor. Anti-IgG, IgA, and IgM rabbit polyclonal antibodies of the currently used DAKO brand were fluorinated. Various molar excess responses were used and compared to the current ThermoFisher AlexaFluor brand. An example of each comparison is shown in Table 4.

[0214] [Table 4-1]

[0215] [Table 4-2]

[0216] The data show that iFluors provide a superior average signal-to-noise ratio. AlexaFluor had limited advantages compared to CF or iFluor. Even though AlexaFluor clearly had a superior S / N ratio for IgG at 10X, the surrounding 5X and 25X responses suggest that the narrow range of good signal may be affected by manufacturing errors if the mixture is not handled well.

[0217] Experiment 2. Direct comparison of washed conjugates with AlexaFluor and iFluor using control and selected samples. Direct comparison of selected samples and controls was performed using the IFU protocol of PN500081. The following data demonstrate good equivalence between AlexaFluor and iFluor (Table 5).

[0218] [Table 5]

[0219] The data shows that iFluor is acceptable when combined with a washed conjugate using 0.3 μg of capture beads per dose of conjugate.

[0220] Experiment 3.1 Capture beads were prepared using the following mixture per dose. i. Wild-type 100 μg, Delta 33 μg, NTD 33 μg ii. Wild-type 100 μg, Delta 33 μg, Omicron 33 μg, NTD 33 μg

[0221] The underlying theory is that the presence of omicron variant RBDs on the capture beads results in a larger RFU signal. Samples collected at the early 2022 investor conference were tested, and the results are shown in Table 6.

[0222] [Table 6]

[0223] Experiment 3: Omicron mutant RBD capture beads may increase secretory IgA, IgG, or IgM responses in patient samples of saline mouth rinse from some patients compared to delta mutant RBD capture beads. Having both omicron mutant RBD capture beads and delta mutant RBD capture beads in a capture bead pool containing wild-type RBD and NTD capture beads would increase the overall sensitivity for detecting anti-SARS-CoV-2 spike protein RBD antibodies.

[0224] Experiment 4: The same capture bead mixture as in Experiment 3 is compared, but using recently collected positive samples. The following data (Table 7) compare the case with and without omicrons.

[0225] [Table 7]

[0226] The addition of Omicron RBD to the mixture did not significantly affect the control (with the notable exception that replication of IgG BR 15 increased from 228.9 to 353.5, while replication of its pair slightly decreased). However, two patient samples showed significant increases. S2 IgG, S3 IgA, and IgG increased by 52%, 44%, and 101%, respectively. Indeed, the change of S3 IgG from negative to positive is a significant change. The positive change in signal indicates that the addition of Omicron effectively increases the sensitivity of the assay. Omicron mutant RBD capture beads may increase secretory IgA, IgG, or IgM responses in patient samples of saline oral rinse from some patients compared to delta mutant RBD capture beads, and having both Omicron mutant RBD capture beads and delta mutant RBD capture beads in a capture bead pool containing wild-type RBD and NTD capture beads would increase the overall sensitivity for detecting anti-SARS-CoV-2 spike protein RBD antibodies.

[0227] The current test was validated as a semi-automated method on a laboratory bench. The test protocol involves numerous manual pipetting and mixing steps, as well as timely heating (37°C) incubation, which are not as well controlled as automation due to human error and inter-operator variability, and therefore may limit the pursuit of accuracy, precision, and continuous quality control. To improve the accuracy, reproducibility, sensitivity, and throughput of the assay, the test will be fully automated using a liquid handler such as the Beckman Coulter® Biomek® i7.

[0228] Manually, pipette mixing was used to break up the beads after pellet formation during magnetic washing and resuspend them. The i7 pipetter was unable to achieve this function. Several programmable pipetting techniques were attempted. Following these, the resuspending of the beads in the DW plate was visually inspected. In each case, large particles were observed, indicating insufficient resuspending. Subsequently, attempts to suspend by increasing the shaker speed on the QInstrument were proposed and tested. As an initial result, resuspending was achieved by increasing the shaker speed to 2,000 RPM. However, the built-in clamping device of the heater shaker was not sufficiently secure to keep the DW plate on the heater shaker, and the DW plate could fall. Subsequent tests showed that reducing the shaking speed to 1,500 RPM was barely sufficient for resuspending, and various attempts to demonstrate that the plate was secured resulted in a 1 in 10 failure to keep the plate on the heater shaker. After the installation of the adapter, the DW plate was stable during shaking at 1,500 RPM. Visual inspection confirmed that the beads were well suspended and dispersed.

[0229] For the manual cleaning method, a V&P plate magnet (PN VP 771BT-SF) fitted inside a BioTek plate washer was used. This plate magnet caused the problematic signal differences in alternating rows of deep-well plates. An example of the data is shown in Table 8.

[0230] [Table 8]

[0231] In this configuration, the odd-numbered rows had pellets located on the right-hand wall of the wells, while the even-numbered rows had pellets on the left-hand sidewall of each well. Simultaneously, the washer's dispensing nozzle was directed towards the left side of each well. This resulted in the washing buffer flowing directly over the even-numbered rows more directly than the odd-numbered rows, effectively washing their pellet-like beads.

[0232] We ordered two custom magnetic isolation plates from Alpaqua. Each plate was a custom version of the Magnum FLX and Catalyst 96 magnetic isolation plates already offered by Alpaqua, modified to fit the plate carrier of the ELx405DW washer. The Magnum FLX provides the strongest magnet to create circular bead pellets at the bottom of each well. Utilizing this magnet fully revealed the odd-even row issue. However, two problems were observed: A) Visual inspection of the washed plates showed that the left side of each circular pellet was washed away, and B) an unknown effect occurred where an unusually large number of wells in rows 8-11 had low signals. Thus, the overall variability increased. Table 9 shows the results for the Magnum FLX.

[0233] [Table 9]

[0234] Following the custom Magnum FLX, the custom Catalyst 96 was tested. These magnets are shaped such that each well has a pair of pellets on the opposite side of the well. Their orientation is adjustable. The inventors determined the orientation to be vertical so that there is a gap without beads on the sides of the well from which the washer dispenses the liquid. Photographs of the custom Catalyst 96 slotted magnets are shown in Figures 8 and 9.

[0235] Table 10 shows the accuracy data for the custom Catalyst 96.

[0236] [Table 10]

[0237] A custom Catalyst 96 plate magnet from Alpaqua provided an acceptable accuracy with a CV of 8% across the entire plate. Due to the reduced variability caused by the magnet and bead resuspension, pipetting parameter techniques can be further optimized.

[0238] We determined that the Span-8 pipetting parameter was the cause of variability due to a) slow delivery rate and b) delivery height. Visual evidence revealed that the pipetting technique resulted in the delivery of large drops from the top of the wells to the deep wells. Figure 10 shows an exemplary pipetting technique.

[0239] Instead of pipetting multiple 100 μL aliquots from a large primary aspiration, we programmed a different technique: single dispensing from a smaller volume tip. Furthermore, delivery was performed at the bottom of the well. After this programming was complete, we ran a complete plate using a single pooled sample using the IFU protocol, which runs entirely on the i7. Table 11 shows the antibody detection results.

[0240] [Table 11]

[0241] The Biomek® i7 was optimized for use with a custom Alpaqua Catalyst 96 magnet or a slotted magnet in the ELx405 deep-well washer. The washer programming demonstrated acceptable performance, and the pipetting parameters were optimized for acceptable performance. The Beckman Coulter® Biomek® i7 system has acceptable performance, has been validated in COVID-19 neutralizing antibody testing laboratory development tests according to the validation plan, and is ready for adoption.

[0242] Example 7: COVID-19 Neutralizing Antibody Test This specification discloses the neutralizing antibody test serum protocol (the "serum protocol") used in this antibody test. Serum samples were pretreated by analysis with paramagnetic "clean beads" to perform selective binding and removal of sample-specific heterophilic antibody interference and / or autoantibody interference. After incubation of the sample, the clean beads were separated with a magnet, the conditioned sample was aspirated, and transferred to a reaction plate (conditioning). Thereafter, neutralizing SARS-CoV-2 antibodies were captured by adding paramagnetic "capture beads" to the conditioned sample. The capture beads included paramagnetic streptavidin beads coated with biotinylated SARS-CoV-2 spike protein RBD-specific recombinant antigen and SARS-CoV-2 spike protein NTD-specific recombinant antigen (capture). After incubation of the sample, the capture beads were washed to remove the sample matrix and non-specific antibodies (washing), and the sample volume was reduced (concentration). Neutralizing SARS-CoV-2 antibodies were detected by adding rabbit polyclonal anti-human IgG fluorescence conjugate (conjugate). After incubation of the sample, the capture beads were washed to remove excess conjugate. For subsequent fluorescence detection and measurement, the conjugate was released from the capture beads (cleavage), aspirated, and dispensed into neutralizing buffer (characterization). The amount of neutralizing SARS-CoV-2 IgG antibody detected in the serum was determined using a bead-based calibration curve. A calibration curve was generated using seven different calibration beads (Figure 11) with different amounts of purified human IgG conjugated to each calibration bead. The relative fluorescence units were directly proportional to the amount of antigen-specific IgG immunoglobulin captured by the capture beads. IgG has the following assay limits. A blank upper limit (LoB) of 3.30 μg / mL or 1553 IU / mL, a limit of detection (LoD) of 4.46 μg / mL or 2099 IU / mL, and a limit of quantification (LoQ) of 4.46 μg / mL or 2099 IU / mL.

[0243] Clinical concordance study Frozen serum samples collected from unvaccinated patients who were positive for SARS-CoV-2 by the emergency use authorized Roche® Cobas® SARS-CoV-2 RT-PCR were tested according to the serum protocol. Table 12 summarizes the test results.

[0244] [Table 12]

[0245] Frozen serum samples collected from patients prior to November 2019 were tested according to the serum protocol. Three false positive samples were detected in clinical residues. False positives were not detected in normal human sera or in samples from the blood bank. The test results are shown in Table 13.

[0246] [Table 13]

[0247] The NISBC validation panel was tested according to the serum protocol. The test results are shown in Table 14.

[0248] [Table 14]

[0249] [[ID=三十五]] Finally, the performance of the serum protocol was compared to the results obtained from the GenScript® cPass™ SARS-CoV-2 Neutral Antibody Detection Kit (EUA201427). The results are shown in Table 15.

[0250] [Table 15]

[0251] These studies demonstrate that the serum protocol has been developed into a highly sensitive and specific test for detecting neutralizing antibodies against SARS-CoV-2 spike proteins RBD and NTD in serum.

[0252] Example 8: COVID-19 neutralizing antibody test on saliva samples This specification discloses the COVID-19 neutralizing antibody test SOR protocol ("SOR protocol") used in this antibody test to multiplex detection and quantification of levels of neutralizing SARS-CoV-2 antibodies (IgM, IgG, and IgA) against spike proteins RBD and NTD. The sample was a saline oral rinse (SOR) saliva sample.

[0253] The clinical performance of the SOR protocol was predictively evaluated using fresh saline mouth rinse samples from unvaccinated patients who tested positive for SARS-CoV-2 by the emergency use authorized OraRisk® COVID-19 RT-PCR (EUA200464). The test results are shown in Table 16.

[0254] [Table 16]

[0255] We tested pre-pandemic saliva samples (N=37) collected before November 2019 and SOR samples (N=11) that were SARS-CoV-2 negative by OraRisk® COVID-19 RT-PCR and met the following SOR inclusion criteria: (i) never having received a COVID-19 vaccine, (ii) never having tested positive for COVID-19, (iii) never having been exposed to a person who is COVID-19 positive, and (iv) not having experienced any symptoms of COVID-19 in the past 12 months. The test results are shown in Table 17.

[0256] [Table 17]

[0257] The concordance rates between serum and saliva (saline mouth rinse) samples were tested by the serum protocol and the SOR protocol. Matched serum and SOR samples were collected simultaneously from 27 different subjects. The comparison is shown in Table 18.

[0258]

Table 18

[0259] The data indicate that the SOR protocol has high specificity and sensitivity and may support the development of any low-abundance antibody detection platform. With the increase in sensitivity and selectivity, new types of samples such as SOR can be used for the detection of secreted antibodies.

[0260] Example 9: COVID-19 Antibody Test The clinical performance of the disclosed COVID-19 neutralizing antibody test was further evaluated against serum samples using the serum protocol (the "serum protocol"), and the results were compared with the method of the GenScript® cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (EUA201427). The serum protocol included collecting positive serum samples from patients who were tested positive for SARS-CoV-2 by Roche® Cobas® SARS-CoV-2 RT-PCR and also positive by the GenScript® cPass™ Neutralization Antibody Detection Kit. Presumptive negative samples were collected before November 2019 and independently tested negative for neutralizing antibodies against the RBD using the GenScript® cPass™ Neutralization Antibody Detection Kit.

[0261] In the serum clinical concordance rate study, the following human serum samples were used. (i) Retrospectively frozen (-20°C) serum samples (N=49) from patients who tested positive by Roche® Cobas® SARS-CoV-2 RT-PCR (4-28 days after symptom onset) and also tested positive by the GenScript® cPass® neutralizing antibody detection kit. (ii) Retrospectively frozen (-20°C) serum samples (N=137) collected from patients prior to December 2019 and negative by the GenScript® cPass® neutralizing antibody detection kit. These 137 negative serum samples included pre-pandemic normal human serum collected in Texas (N=50), blood bank samples collected in Florida (N=40), and clinical residual samples collected in Florida (N=47). (iii) Matched serum and saline mouth rinse (SOR) samples (N=28) prospectively collected from vaccinated patients (N=27) and unvaccinated patients (N=1) who were negative for COVID-19 by OraRisk® COVID-19 RT-PCR (EUA200464), had never been tested positive for COVID-19, had never been exposed to a person positive for COVID-19, and had never experienced symptoms of COVID-19. (iv) NISBC validation panel (WHO reference panel, first WHO international reference panel for anti-SARS-CoV-2 immunoglobulin, NISBC code: 20 / 268) (N=37). The first WHO international reference panel for anti-SARS-CoV-2 immunoglobulin consists of 0.25 mL equivalents of pooled plasma samples obtained from individuals who have recovered from COVID-19, and negative control plasma obtained from healthy blood donors prior to 2019.

[0262] GenScript® cPass® neutralizing antibody detection kit as a comparator protocol: (a) 137 retrospectively frozen serum samples collected from patients prior to December 2019 were tested using both the GenScript® cPass® neutralizing antibody detection kit and the serum protocol. (b) Forty-nine retrospectively frozen serum samples that were RT-PCR positive for COVID-19 by Roche® Cobas® SARS-CoV-2 RT-PCR and whose number of days since symptom onset was known were also tested for neutralizing antibodies against RBD using the GenScript® cPass® neutralizing antibody detection kit. These 49 samples were also tested according to a serum protocol, and the results were compared with those from the GenScript® cPass® neutralizing antibody detection kit. (c) Twenty-eight serum samples prospectively collected from matching sample studies were tested using both the GenScript® cPass® neutralizing antibody detection kit and the serum protocol. (d) 37 NISBC validation panel serum samples were tested using both the GenScript® cPass® neutralizing antibody detection kit and the serum protocol. All serum samples tested using the GenScript® cPass® neutralizing antibody detection kit and the serum protocol were compared to determine the sensitivity, PPA, specificity, and NPA of the serum protocol, as well as the correlation (r-value and p-value) between the two methods.

[0263] Negative concordance rate

[0264] Thirty-seven retrospectively frozen serum samples collected from patients prior to December 2019 were tested using the GenScript® cPass® neutralizing antibody detection kit and serum protocol. The results are summarized in Table 19. 95% confidence intervals (CIs) were calculated using the online clinical calculator (vassarstats.net / clin1.html).

[0265] [Table 19]

[0266] Positive match rate

[0267] Forty-nine retrospectively frozen serum samples that were RT-PCR positive for COVID-19 by Roche® Cobas® SARS-CoV-2 RT-PCR and also tested positive with the GenScript® cPass® neutralizing antibody detection kit were tested according to the serum protocol. The results are summarized in Table 20. 95% confidence intervals were calculated using the online clinical calculator (http: / / vassarstats.net / clin1.html).

[0268] [Table 20]

[0269] Overall agreement rate

[0270] In accordance with the FDA EUA's "Template for Test Developers for Serological Tests Detecting or Correlating Neutralizing Antibodies," serological protocols were compared with the GenScript® cPass® Neutralizing Antibody Detection Kit. This comparison used 137 retrospectively frozen serum samples collected from patients prior to December 2019, and 49 retrospectively frozen serum samples that were RT-PCR positive for COVID-19 by Roche® Cobas® SARS-CoV-2 RT-PCR and also tested positive with the GenScript® cPass® Neutralizing Antibody Detection Kit. The results are summarized in Table 21. 95% confidence intervals were calculated using the online clinical calculator (http: / / vassarstats.net / clin1.html).

[0271] [Table 21]

[0272] Consensus rate of predictive studies

[0273] Twenty-eight serum samples prospectively collected from matching sample studies were tested using the GenScript® cPass® neutralizing antibody detection kit and serum protocol. Results are summarized in Table 22. 95% confidence intervals (CIs) were calculated using the online clinical calculator (http: / / vassarstats.net / clin1.html).

[0274] [Table 22]

[0275] NISBC Verification Panel Match Rate

[0276] Thirty-seven NISBC validation panel serum samples were tested using the GenScript® cPass® neutralizing antibody detection kit and serum protocol. The results are summarized in Table 23. 95% confidence intervals (CIs) were calculated using the online clinical calculator (http: / / vassarstats.net / clin1.html).

[0277] [Table 23]

[0278] The correlation between quantitative IgG values ​​from serum protocols in CRM units (IU / mL) and semi-quantitative IgG values ​​from the GenScript® cPass® neutralizing antibody detection kit in CRM units (IU / mL) was compared, and the correlation between the two methods was evaluated. Linear regression and Passing-Bablok fitting were calculated using Analyze-It for Microsoft Excel (version 5.90, build 7870.29081) (N=113). The results are summarized in Figures 12A-12B, showing data for COVID-19 antibody testing (IU / mL) versus GenScript® cPass® neutralizing antibody detection kit (IU / mL). Figure 12A shows NIBSC IgG IU / mL against cPass semi-quantitative IU / mL with a fitting line. Figure 12B shows a summary of the data for different parameters, including 95% CI, SE, t, and p values.

[0279] Samples that showed high values ​​outside the measurement range (N=39) were high by both methods and needed to be excluded from this analysis (see Table 24).

[0280] [Table 24]

[0281] Prospectively collected, matched serum and SOR studies

[0282] To test the concordance rate between serum samples tested according to the serum protocol and saline mouth rinse samples tested according to the SOR protocol, matching serum and saline mouth rinse samples were simultaneously collected from 28 different healthy donors.

[0283] Study population and size

[0284] Matching serum and SOR samples were prospectively collected from healthy donors with an equal distribution of males and females (N=28). Twenty-seven healthy donors were vaccinated against COVID-19, and one healthy donor was negative for COVID-19 by OraRisk® COVID-19 RT-PCR, had not been exposed to anyone positive for COVID-19, and had not been tested for COVID-19 (N=1).

[0285] Prospectively collected, matched serum and SOR study protocols

[0286] Matching serum / SOR samples were assayed with the same lot's disclosed COVID-19 antibody test reagent using the serum protocol for serum samples and the SOR protocol for SOR samples, and the results were compared with the GenScript® cPass® neutralizing antibody detection kit as a comparator.

[0287] Comparator results from the GenScript (registered trademark) cPass (trademark) neutralizing antibody detection kit.

[0288] Twenty-eight matched serum and SOR samples were prospectively collected from vaccinated patients and unvaccinated patients who were negative for COVID-19 by OraRisk® COVID-19 RT-PCR, had never been tested positive for COVID-19, had not been exposed to a person positive for COVID-19, and had never experienced symptoms of COVID-19. These samples were tested using the disclosed COVID-19 antibody test and the GenScript® cPass® neutralizing antibody detection kit. Results are summarized in Tables 25 and 26. 95% confidence intervals were calculated using the online clinical calculator (http: / / vassarstats.net / clin1.html).

[0289] [Table 25]

[0290] [Table 26]

[0291] Overall summary

[0292] The IgG LoQ cutoff was validated for SARS-CoV-2-binding IgG antibodies based on sensitivity, specificity, PPA, NPA, correlation coefficient (r-value), and p-value, using correlation with the semi-quantitative GenScript® cPass® neutralizing antibody detection kit (EUA201427). The disclosed COVID-19 neutralizing antibody test demonstrated 94.1% PPA (95% CI 82.8–98.5%) and 99.3% NPA (95% CI 95.3–100%) compared to the GenScript® cPass® neutralizing antibody detection kit (EUA201427). The disclosed COVID-19 neutralizing antibody test IgG results (IU / mL) also demonstrated correlation with the GenScript® cPass® neutralizing antibody detection kit (r=0.87, p<0.001).

[0293] Example 10: Study on the clinical concordance rate of saliva samples The clinical performance of the disclosed COVID-19 neutralizing antibody tests was further evaluated by testing saline mouth rinse samples collected from RT-PCR-positive patients, as well as by testing presumably negative saline mouth rinse samples from individuals who should not have antibodies against SARS-CoV-2, and frozen saliva samples collected before November 2019 (pre-pandemic).

[0294] Study population and size

[0295] The following patient samples were used in the clinical study. (i) OraRisk® Prospectively collected saline mouth rinse samples (N=70) from SARS-CoV-2 patients who tested positive for COVID-19 RT-PCR. All 70 patients indicated on their questionnaires that they had never received a COVID-19 vaccine. (ii) Prospectively collected saline mouth rinse samples (N=11) from patients meeting the following inclusion criteria: a. Tested negative for COVID-19 by OraRisk® COVID-19 RT-PCR. b. Never been vaccinated. c. Never tested positive for COVID-19. d. Never been exposed to a person who is COVID-19 positive. e. Never experienced symptoms of COVID-19 in the past 12 months, including fatigue, flu-like symptoms, shortness of breath or difficulty breathing, or loss of taste or smell.

[0296] Frozen saliva samples collected before the pandemic were collected prior to November 2019 (N=32).

[0297] Clinical agreement rate study of saline oral rinse

[0298] The clinical sensitivity of disclosed COVID-19 neutralizing antibody tests was evaluated by testing saline mouth rinse samples that were RT-PCR positive by OraRisk® COVID-19 RT-PCR (EUA200464). Positive saline mouth rinse samples were prospectively collected from unvaccinated patients who tested positive for SARS-CoV-2 by OraRisk® COVID-19 RT-PCR at a known number of days after symptom onset. The results are shown in the table below. All samples were collected and tested on the same day by OraRisk® COVID-19 RT-PCR. The 95% confidence interval was calculated using an online clinical calculator (http: / / vassarstats.net / clin1.html). The positive percentage agreement rate (PPA) for the disclosed COVID-19 neutralizing antibody test SOR protocol (SOR protocol) for all tested saline mouth rinse samples collected 2 to 16 days after symptom onset was 95.5% (67 / 70, 95% CI 83.3–99.2%). The positive agreement rate results are shown in Tables 27 and 28.

[0299] [Table 27]

[0300] [Table 28]

[0301] The clinical specificity of the SOR protocol was evaluated by testing frozen saliva samples collected before November 2019 (pre-pandemic saliva samples) and by testing prospectively collected putative negative saline mouth rinse samples from patients who met the study inclusion criteria. The 95% confidence interval (CI) was calculated using the online clinical calculator (http: / / vassarstats.net / clin1.html). No false positives were detected in either the pre-pandemic saliva samples or the putative negative saline mouth rinse samples. The negative percent agreement rate (NPA) for the SOR protocol was 100% (48 / 48, 95% CI 90.8–100%). Table 29 shows the negative agreement rates.

[0302] [Table 29]

[0303] Example 11: Detection and quantification of multiplex secreted antibodies in saline oral rinse for SARS-CoV-2 vaccine efficacy testing. The disclosed COVID-19 neutralizing antibody test may include a bead-based, semi-automated fluorescence immunoassay intended for the multiplex quantitative detection of SARS-CoV-2 neutralizing IgG, IgA, and IgM secreted antibodies in saline mouth rinse. Saline mouth rinse samples were pre-treated with paramagnetic clean beads to selectively bind and remove sample-specific heterophilic antibody interference and / or autoantibody interference. After incubation of the samples, the clean beads were separated with a magnet, the prepared samples were aspirated, and transferred to a reaction plate. Subsequently, neutralizing SARS-CoV-2 antibodies were captured by adding paramagnetic capture beads to the prepared samples. The capture beads contained paramagnetic streptavidin beads coated with biotinylated SARS-CoV-2 spike protein RBD-specific recombinant antigen (wild-type and delta mutant) and SARS-CoV-2 spike protein NTD-specific recombinant antigen (wild-type). After incubation of the samples, the capture beads were washed to remove the sample matrix and nonspecific antibodies. SARS-CoV-2 neutralizing antibodies were detected by adding rabbit polyclonal anti-human IgA, anti-human IgG, and anti-human IgM triplex (Alexa Fluor 488, 555, and 647) fluorescent conjugates. After incubation of the samples, the capture beads were washed to remove excess conjugate. For subsequent fluorescence detection and measurement, the conjugate was eluted from the capture beads, aspirated, and dispensed into neutralizing buffer. The amount of SARS-CoV-2 neutralizing IgG antibody detected in serum or lithium heparinized plasma samples, or the amount of SARS-CoV-2 neutralizing IgG, IgA, and IgM secreted antibodies detected in saline oral rinses, was determined using bead-based calibration curves. Calibration curves were generated using seven different triplex calibration beads, each containing different amounts of purified human IgM, IgG, and IgA conjugated to the calibration beads. The relative fluorescence units of each fluorophore are directly proportional to the amount of antigen-specific IgG, IgM, and / or IgA immunoglobulin captured by the capture beads.

[0304] Saline mouth rinse samples were tested using the following disclosed COVID-19 neutralizing antibody test reagents. Reagent A - Clean Beads: Biotin-labeled animal antibodies (rabbit, goat, and cattle), as well as streptavidin-coated superparamagnetic nanoparticles bound to biotin-labeled human IgM, IgG, and IgA, and bovine serum albumin-coated superparamagnetic nanoparticles in TRIS buffer and 0.05% detergent. Preservative: 0.05% sodium azide. Reagent B - Capture Beads: Streptavidin-coated superparamagnetic nanoparticles bound to biotin-labeled SARS-CoV-2 spike protein RBD recombinant antigen (wild-type and delta mutant) produced in HEK293 cells and purified from the culture supernatant, and biotin-labeled SARS-CoV-2 spike protein NTD recombinant antigen (wild-type) produced in HEK293 cells and purified from the culture supernatant, in TRIS buffer, 10% goat serum, 0.1% bovine serum albumin, 0.001% polymer, and 0.05% detergent. Preservative: 0.05% sodium azide. • Reagent C-triplex conjugate: Rabbit polyclonal anti-human IgM antibody, anti-human IgG antibody, and anti-human IgA antibody, each labeled with a different fluorophore (Alexa Flour 488, 555, or 647), in TRIS buffer, 0.1% bovine serum albumin, and 0.05% detergent. Preservative: 0.05% sodium azide. • Reagent D - 25x wash buffer: TRIS buffer, 0.05% detergent, and less than 0.05% polymeric blocker. Preservative: 0.05% sodium azide. • Reagent E-elution buffer: Glycine buffer, 0.05% detergent. Preservative: 0.05% sodium azide. • Reagent F - Neutralizing buffer: TRS buffer. Preservative: 0.05% sodium azide. • Diluent A: Phosphate-buffered saline. Preservative: 0.05% sodium azide. • Diluent B: 0.9% physiological saline. Preservative: 0.05% sodium azide. Reagent G-Calibrator 0: Bovine serum albumin-coated superparamagnetic nanoparticles in TRIS buffer, 0.1% bovine serum albumin, and 0.05% detergent. Preservative: 0.05% sodium azide. • Reagents G-Calibrator 1 to Calibrator 6: Streptavidin-coated superparamagnetic nanoparticles bound to biotin-labeled human IgM, IgG, and IgA, as well as bovine serum albumin-coated superparamagnetic nanoparticles, in TRIS buffer, 0.1% bovine serum albumin, and 0.05% detergent. Preservative: 0.05% sodium azide.

[0305] Calibration beads for the triple human IgA, IgG, and IgM reagent G were prepared as follows, and a dose-response calibration curve was generated to quantify the antibody levels detected in the sample. • Preparation and biotinylation of IgA, IgG, and IgM: Human immunoglobulins A, G, and M were purified from pools prepared from serum of multiple donors. Individual immunoglobulins were then biotinylated with either NHS ester-PEG4-biotin reagent or TFP ester-PEG4-biotin reagent in pH 8 bicarbonate or phosphate-bicarbonate buffer in a molar excess of 10 moles of biotinylating reagent to 1 mole of protein. The biotinylating agent was solubilized in DMSO at a concentration of 20 mM. A sufficient volume of the biotinylation / DMSO solution was then introduced into the protein solution to produce a reaction in a molar excess of 10:1. The two solutions were thoroughly mixed and then incubated at ambient temperature for at least 2 hours with continuous mixing. Incubation could be extended up to 18 hours. At the end of incubation, the solution was desalted in neutral pH 7.4 PBS buffer by size exclusion chromatography to remove unreacted biotinylation. Preparation of streptavidin-coated paramagnetic microparticles (PMPs): 1.6 μm carboxyl-activated PMPs were obtained from Nippon Synthetic Rubber Co., Ltd. (JSR). These PMPs were washed with 25 mg PMP / mL in 40 mM MES pH 5.3 buffer. Lyophilized streptavidin purchased from Agilent was reconstituted to a concentration of 20 mg / mL in purified deionized water (resistivity of 18 MΩ). This solution was desalted in 40 mM MES pH 5.2 buffer by size exclusion chromatography to remove NaCl from streptavidin. This streptavidin MES solution was diluted to 10 mg / mL in 40 mM MES. Four volumes of a 25 mg / mL PMP suspension and five volumes of 10 mg / mL streptavidin were mixed to produce a mixture of 11.1 mg / mL PMP containing 5.56 mg / mL streptavidin in 40 mM MES pH 5.2. This mixture was thoroughly mixed and sonicated to ensure bead dispersibility. A solution of 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in 40 mM MES pH 5.2 was prepared such that when the PMP / streptavidin and EDC solution were mixed, there was 1 mg of EDC per 10 mg of PMP, i.e., 100 μL of EDC solution per 0.9 mL of PMP / streptavidin. Both PMP / streptavidin and EDC were introduced into MES at a constant rate of 18 mL of PMP / streptavidin per minute to 2 mL of EDC per minute to produce a mixture of 10 mg / mL of PMP, 5 mg / mL of streptavidin, and 1 mg / mL of EDC. This mixture was incubated at ambient temperature for at least 2 hours, but no more than 20 hours. The reacted PMP was magnetically separated, and streptavidin was removed from the supernatant. The pelletized beads were washed three times in PBS pH 7.4, then brought to volume in PBS pH 7.4 to a concentration of 10 mg / mL, and sonicated to ensure dispersibility. The protein coating the PMP was measured by a MicroBCA assay using ThermoFisher. A typical streptavidin concentration was 18 μg / mg of PMP. • Preparation of immunoglobulin-coated PMPs. Streptavidin-coated PMPs were washed three times in PBS, and then the volume was adjusted to produce a 20 mg / mL PMP mixture in PBS pH 7.4. Biotinylated immunoglobulins were prepared individually by diluting them to a concentration of 0.6 mg / mL in PBS pH 7.4. Each mixture was placed in a round-bottomed container with a central mixing blade driven by an overhead mixer mounted on a shaft. Both the container with PMPs and the container with biotinylated immunoglobulins were agitated at approximately 150 RPM by their mixing blades. The PMPs were sonicated to ensure dispersibility and pumped into the rapidly mixing immunoglobulin mixture at a rate of 20 mL / min. After pumping all the PMPs into the immunoglobulin mixture, the resulting mixture was incubated at ambient temperature for 2 hours. At the end of the 2 hours, the PMPs were magnetically separated and the remaining supernatant was removed. The pelletized PMP was washed three times with Tris (10 mM) buffered saline, Tween-20 (0.5%), and sodium azide (0.05%). Then, the PMP was volume-adjusted to a concentration of 10 mg / mL with the same TBS-Tween-sodium azide solution and sonicated to ensure dispersibility. • The allocation of immunoglobulin values ​​per 1 mg of PMP was: [Mass of immunoglobulin at the start of the coating process (0.6 mg / mL of the added solution as determined by A280 / 1.4) - Mass of immunoglobulin at the end of the coating process (concentration of immunoglobulin in the supernatant after magnetic separation as determined by A280 / 1.4)] / Number of mg of PMP to be coated. The amount was typically 16 μg of immunoglobulin per 1 mg of PMP. • Preparation of the Triplex Calibrator: A mixture of IgA, IgG, and IgM coated streptavidin PMPs was prepared in Tris-buffered saline containing Tween 20 and sodium azide, such that each 60 μL volume contained 500 ng of each immunoglobulin. Each individual immunoglobulin PMP was concentrated to obtain an immunoglobulin concentration of 25 μg / mL. Then, each individual immunoglobulin coated PMP was mixed in a 1:1:1 ratio with 25 μg / mL of immunoglobulin so that the resulting mixture had an individual immunoglobulin concentration of 8.333 μg / mL. To prepare a calibration curve from a stock of 500 ng / mL immunoglobulin PMP, PMP was mixed with 1.6 μm PMP coated with BSA to create seven individual PMP mixtures with concentrations of 500 ng / mL, 375 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, and 0 ng / mL. • Use of calibration PMPs in serological or human immunoglobulin IgG, IgM, and / or IgA assays: 60 μL of each of these triplex calibration PMPs was individually placed in a single column of wells on a 96-well microtiter plate, e.g., A1–G1 of a 96-well microtiter plate. These PMPs were then subjected to the same reagent and incubation procedures as the unknown samples, as well as the rest of the protocol. The conjugates eluted from the calibration PMPs were proportional to the immunoglobulin concentration. Curve fitting was achieved using a 4-parameter Log-Logit calculation. Figures 13A–13C show examples of calibration curves for human IgG, IgM, and IgA.

[0306] Saline mouth rinse samples were collected by having patients rinse and gargle with 5 mL of saline solution (0.9% sodium chloride in water) for 30 seconds and then having them squirt it into a collection tube. The collection tube was then sealed with a screw cap (e.g., a Falcon 50 mL conical centrifuge tube). Collection was supervised by a nurse practitioner, nurse, or healthcare provider.

[0307] This study includes the use of an external human plasma-based control supplied by Bio-Rad [VIROTROL SARS-CoV-2 Single Level Control (PN 200300A or 200305A)]. This study uses 1,000 μL of each control per assay (N=1 repeat). • Positive control 1: 15 μL of Bio-Rad VIROTROL SARS-CoV-2 Single Level Control (PN 200300A or 200305A), diluted in 985 μL of Diluent A included in the kit, as a positive control for neutralizing IgG and IgM antibodies against SARS-CoV-2. • Positive control 2: 60 μL of Bio-Rad VIROTROL SARS-CoV-2 Single Level Control (PN 200300A or 200305A), diluted in 940 μL of Diluent A included in the kit, as a positive control for neutralizing IgA antibodies against SARS-CoV-2. • Negative control: Diluent B included in the kit as a negative control for neutralizing IgG, IgA, and IgM antibodies against SARS-CoV-2.

[0308] The limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ) were determined using various saline mouth rinse samples to assess the levels of IgG, IgM, and IgA secreted antibodies against SARS-CoV-2. The LoB levels for IgG, IgM, and IgA were 43.6 ng / mL, 12.0 ng / mL, and 9.8 ng / mL, respectively. The LoD levels for IgG, IgM, and IgA were 67.5 ng / mL, 26.6 ng / mL, and 17.2 ng / mL, respectively. The LoQ levels for IgG, IgM, and IgA were 67.5 ng / mL, 39.1 ng / mL, and 34.7 ng / mL, respectively.

[0309] The analytical measurement interval was defined by Cal 6. Values are interpreted as "negative" (<LoQ) and "positive" (≧LoQ). Values beyond the measurement range are reported as >Cal 6 (lot-specific value assignment for IgG, IgA, and IgM). If the sample result exceeds the upper limit of the analytical measurement interval (ULMI), it can be diluted with Diluent B and retested. The recommended dilution is 1:4 (3 parts Diluent B and 1 part sample).

[0310] Accuracy was determined by testing four samples over three days in two runs per day. Four SOR samples were devised to include levels of IgG, IgM, and IgA detected from LoQ to samples at the upper limit of the clinical range. One lot of the disclosed COVID-19 neutralizing antibody test reagent was used to generate this data. Tables 30 - 32 show the accuracy of IgG, IgM, and IgA in saline oral rinses.

[0311]

Table 30

[0312] The within-run CV for IgG was 8.5% or less, the within-day CV was 14.3% or less (a CV of 15.7% was observed at a dose of 66.7 ng / mL or LoQ < 67.5 ng / mL), and the total CV was 18.1% or less.

[0313]

Table 31

[0314] The intrarun CV for IgM was ≤8.6% (14.7% CV was observed at a dose of 14.6 ng / mL or a LoQ <39.1 ng / mL), the intraday CV was ≤14.4% (27.7% CV was observed at a dose of 66.7 ng / mL, and 15.6% CV was observed at a dose of 26.1 ng / mL or a LoQ <39.1 ng / mL), and the total CV was ≤17.9% (40.7% CV was observed at a dose of 14.6 ng / mL or a LoQ <39.1 ng / mL).

[0315] [Table 32]

[0316] The intrarunning coefficient (CV) for IgA was 9.2% or less, the intraday CV was 15.3% or less, and the total CV was 18.3% or less.

[0317] Linearity was determined to demonstrate that the assay measures a proportional concentration of human immunoglobulin relative to the amount present in the sample. Saline mouth rinse samples were also pooled to prepare samples with high concentrations of IgG, IgM, and IgA secreted antibodies against SARS-CoV-2. These samples were then diluted with diluent B in 20% increments. The diluted samples were tested in double doses, and linearity results were calculated using Analyze-It for Microsoft Excel (version 5.90, build 7870.29081). For saline mouth rinse, linearity was demonstrated in the intervals of 67.5 ng / mL to 377 ng / mL for IgG, 39.1 ng / mL to 383 ng / mL for IgM, and 34.7 ng / mL to 244 ng / mL for IgA, with the total deviation from the straight line being within 15%. In this study, no endogenous saline oral rinse samples were obtained for IgG, IgM, or IgA levels exceeding the upper limit of the measurement range or Cal 6.

[0318] Recovery rates were determined to demonstrate that the assay measures a proportional concentration of human immunoglobulin relative to the amount present in the sample.

[0319] Twelve potential cross-reactive antibodies (Table 33) were evaluated. All serum samples were obtained prior to November 2019.

[0320] [Table 33]

[0321] Cross-reactivity to these viruses is not expected in saline oral rinses, but each sample of these 12 potential cross-reactive antibodies or ELISA antibody-positive serum samples for each disease state was tested by diluting the samples in 10% artificial saliva (Pickering, product number 1700 - 0316) in saline (0.9% NaCl in water). Since serum spiked with SOR can be recovered in excess, BioRad VIROCLEAR SARS-CoV-2 Single Level Control (product number 200500 or 200505) was also diluted as a negative control. The results of the cross-reactive samples were considered passing if read as <LoQ for IgG (<67.5 ng / mL), IgM (<39.1 ng / mL), or IgA (<34.7 mg / mL), or ≤ BioRad VIROCLEAR SARS-CoV-2 Single Level Control. The overall specificity obtained was 100%. The test results are shown in Table 34.

[0322] [Table 34]

[0323] Since most of the tested samples were below LoQ for IgM and IgA, cross-reactivity testing was repeated by spiking 1 μL of each of the 12 antibody-reactive samples into 999 μL of saline mouth rinse-positive samples for IgM (108 ng / mL), IgG (300 ng / mL), and IgA (66 ng / mL), as well as into 1 μL of positive serum control sample and 1 μL of Bio-Rad VIROCLEAR negative control. Cross-reactivity results were considered passable if 1) for IgM, IgG, and IgA, the positive serum control spike > VIROCLEAR SARS-CoV-2 Single Level Control, and 2) the cross-reactive sample read ≤ BioRad VIROCLEAR SARS-CoV-2 Single Level Control, or ≤ VIROCLEAR SARS-CoV-2 Single Level Control spike +15%. The test results are shown in Table 35.

[0324] [Table 35-1]

[0325] [Table 35-2]

[0326] The potential for microorganisms to interfere with assay results was assessed by spiking pooled saline mouth rinse samples with 13 different microorganisms. Table 36 shows the results of the microbial tests.

[0327] [Table 36]

[0328] Potential microbial interference from bacteria and viral microorganisms potentially present in oral fluid samples was tested. To prepare the samples, microorganisms were spiked into saline oral rinse antibody-positive samples and then tested using the SOR protocol. No false positive or false negative results were observed at the tested microbial concentrations (Table 37).

[0329] [Table 37-1]

[0330] [Table 37-2]

[0331] Potential sample interference unrelated to SARS-CoV-2 infection, which could affect the accuracy of the test results, was also tested (Table 38).

[0332] [Table 38]

[0333] Hemoglobin does not significantly interfere with the measurement of IgG, IgM, or IgA secreted antibodies against SARS-CoV-2 in saline oral rinse in this assay.

[0334] Biotin concentrations up to 3,500 ng / mL did not significantly interfere with the measurement of SARS-CoV-2 secreted antibodies (IgG, IgA, or IgM) in saline oral rinse tests for COVID-19 (Table 39).

[0335] [Table 39]

[0336] Various substances that may be present in saline mouthwash samples from food, alcohol, or bleeding / hemoglobin were spiked into the samples, and their effects on IgG, IgM, and IgA results were evaluated. Several exogenous interfering substances tested, such as toothpaste, mouthwash, antacids (tums), tobacco, and coffee, interfered with the measurement of saline mouthwash IgG, IgA, or IgM secreted antibodies against SARS-CoV-2 by more than 20% in this assay (Table 40, ctrl. = control). Patients were asked to refrain from eating, drinking alcohol, smoking, chewing gum, or brushing their teeth for at least 30 minutes prior to the collection of their saline mouthwash samples.

[0337] [Table 40]

[0338] Class specificity tests were performed to determine the ability of the triplex conjugate to distinguish between human IgG, IgM, and IgA immunoglobulins. Conjugate cross-reactivity was tested by testing individual calibration beads coated with purified human IgG, IgA, or IgM against the individual conjugates and the triplex conjugates. The class specificity of each conjugate (anti-human IgG, anti-human IgM, and anti-human IgA) and the triplex conjugate was assessed by testing the reactivity of 60 μg of each individual Cal 6 bead (IgG, IgM, and IgA coated beads), or 1.3 times more calibration beads than used in the assay to highlight potential cross-reactivity, against 200 μL of individual anti-IgA, anti-IgG, or anti-IgM fluorescent conjugates. The presence of conjugate cross-reactivity was determined by comparing the mean triplex signal to the mean relative fluorescence unit (RFU) signals of the individual conjugates. % cross-reactivity was calculated based on the ratio of the average triplex conjugate anti-IgG, anti-IgM, and anti-IgA RFU signals to the individual average anti-IgG, anti-IgM, and anti-IgA conjugate RFU signals when tested against each IgG, IgM, or IgA calibration bead. A cross-reactivity of ±10% is acceptable. Each conjugate demonstrated a strong fluorescence signal for its specific class and did not exhibit significant cross-reactivity to other classes. The cross-reactivity for each conjugate was less than 10%. The triplex conjugate correctly distinguishes between human IgG, IgM, and IgA immunoglobulins.

[0339] The analytical sensitivity of IgG, IgM, and IgA secreted antibodies against SARS-CoV-2 spike proteins RBD and NTD in saline oral rinse was determined based on the limit of quantification (LoQ) of IgG, IgM, and IgA secreted antibodies. If the IgG, IgM, and IgA antibody levels are below LoQ, the antibody result is reported as negative, and the interpretation of the test result is "IgG secreted antibodies against SARS-CoV-2 are not detected" and / or "IgM secreted antibodies against SARS-CoV-2 are not detected" and / or "IgA secreted antibodies against SARS-CoV-2 are not detected." If IgG and / or IgM and / or IgA antibody levels are LoQ or higher, the antibody result is reported as positive, the numerical result (ng / mL for IgG, ng / mL for IgM, ng / mL for IgA) is reported outside the laboratory, and the interpretation of the test result is "IgG secreted antibodies against SARS-CoV-2 were detected" and / or "IgM secreted antibodies against SARS-CoV-2 were detected" and / or "IgA secreted antibodies against SARS-CoV-2 were detected".

[0340] Interpretation of multiplex quantitative results in saline oral rinse for IgG, IgM, and IgA secreted antibodies against SARS-CoV-2 spike proteins RBD and NTD (Table 41).

[0341] [Table 41]

[0342] Example 12: Clinical trial to evaluate vaccine efficacy From June 2021 to April 2022, patient samples of saline mouth rinse were tested for secretory neutralizing IgA, IgG, and IgM against SARS-CoV-2 spike proteins RBD and NTD using COVID-19 neutralizing antibody testing. Longitudinal studies were also conducted on several patients to follow up and track secretory antibody levels before vaccination, after vaccination (Vaccine #1, Vaccine #2, and Vaccine #3), and after booster vaccination (Vaccine #1, Vaccine #2, and Vaccine #3), determining which levels of secretory IgA are protective by measuring antibody levels before and after breakthrough COVID-19 infection.

[0343] From June 30, 2021 to August 17, 2021, 55 unvaccinated patients with no known prior COVID-19 exposure or symptoms provided saline mouth rinse samples, which were tested according to the SOR protocol. Twenty-four patients had detectable neutralizing antibodies, and 24 / 24 (100%) had detectable secretory IgA indicating a previous COVID-19 infection. Three out of 24 (12.5%) also had detectable secretory IgM indicating a recent infection. Thirty-one patients who wanted to know if they had previously had COVID-19 or antibodies (asymptomatic) tested negative for secretory antibodies. These results demonstrate that asymptomatic patients with a previous COVID-19 infection primarily produce secretory IgA, but current or recent infections can produce both secretory IgA and IgM.

[0344] From June 30, 2021 to January 4, 2022, 286 fully vaccinated patients with no known prior COVID-19 exposure or symptoms provided saline mouth rinse samples, which were tested according to the SOR protocol. Fully vaccinated patients were defined as those who had received both the first and second doses of vaccine #1 or #2, with at least 14 days having passed since the second dose, or those who had received a single dose of vaccine #3, with at least 28 days having passed since that dose. A total of 286 fully vaccinated patients participated in this study (N=155 for vaccine #1, N=116 for vaccine #2, and N=14 for vaccine #3). Two additional patients who had received a booster dose of vaccine #1, resulting in a total of three vaccine doses, were also tested.

[0345] Table 42 shows the results of the vaccine efficacy trial for vaccine #1. Of 155 patients, 83 (53.5%) did not have detectable secretory IgA in saline mouth rinse samples, and 106 (68.4%) of the 155 patients did not have detectable secretory IgG. In contrast, 72 (46.5%) of the 155 patients had detectable secretory IgA, and 49 (31.6%) of the 155 patients had detectable secretory IgG. Looking at the number of days after vaccination, the positive predictive value (PPA) was 61.7% (66 positive out of 107 patients) more than 180 days after vaccination, and the PPA was 60.0% (93 positive out of 155 patients) for all days tested. Only 3 (1.9%) of the 155 patients also had detectable secretory IgM.

[0346] [Table 42]

[0347] Table 43 shows the results of the vaccine efficacy trial for vaccine #2. Of the 116 patients, 73 (62.9%) did not have detectable secretory IgA in the saline mouth rinse sample, and 92 (79.3%) of the 116 patients did not have detectable secretory IgG. In contrast, 43 (37.1%) of the 116 patients had detectable secretory IgA, and 24 (20.7%) of the 116 patients had detectable secretory IgG. Looking at the number of days after ...

Claims

1. A step of providing a substance to be cleaned by removing interference from the substance to be cleaned using an interference trapping composition, wherein the interference trapping composition includes an interference trapping portion, and the substance to be cleaned is (a) Samples derived from the subject, (b) Biomarker-capturing particles, (c) Detection antibody, and (d) Any combination of (a) to (c) A process selected from the group consisting of, A step of capturing a biomarker from the sample by contacting the sample with biomarker-capturing particles and optionally detecting an antigen using the detection antibody, wherein at least one of the sample, the biomarker-capturing particles, or the detection antibody includes the substance to be washed. Assay method including

2. A step of cleaning a substance by bringing the substance into contact with an interference-capturing composition to remove interference from the substance, wherein the interference-capturing composition includes an interference-capturing portion, and the substance is (a) Samples derived from the subject, (b) Biomarker-capturing particles, (c) Detection antibody, and (d) Any combination of (a) to (c) A process selected from the group consisting of, A step of capturing a biomarker from the sample by contacting the sample with biomarker-capturing particles and optionally detecting an antigen using the detection antibody, wherein at least one of the sample, the biomarker-capturing particles, or the detection antibody includes the substance to be washed. Assay method including

3. An assay method for determining the efficacy of a drug, The process of providing a sample of a subject who has been administered medicine, The process involves bringing the sample into contact with biomarker-capturing particles containing a biomarker-capturing portion to capture a biomarker from the sample. A step of quantifying the eluted biomarker, An assay method comprising a step for determining the efficacy of a drug.

4. The assay method according to claim 3, wherein the step of quantifying the eluted biomarker includes detecting the antigen using a detection antibody.

5. The process further includes a step of cleaning the substance by bringing the substance into contact with an interference-capturing composition to remove interference from the substance, wherein the interference-capturing composition includes an interference-capturing portion, and the substance is (e) The sample, (f) The biomarker capturing particles, (g) The detection antibody, and (h)(a) to (c) all combinations The assay method according to claim 3 or 4, selected from the group consisting of the following.

6. The assay method according to claim 3 or 4, wherein the pharmaceutical product comprises a vaccine, a therapeutic agent, or a combination thereof.

7. The assay method according to any one of claims 3 to 6, wherein the sample, the biomarker capturing particles, or a combination thereof are subjected to washing before capturing the biomarker from the sample, the washing comprising removing interference by contact with an interference capturing composition.

8. The method according to any one of claims 1 to 7, wherein the biomarker capturing particle includes a biomarker capturing portion.

9. The assay method according to any one of claims 1 to 8, wherein the biomarker capturing particle comprises an antibody, and the biomarker comprises an antigen that binds to the epitope of the antibody.

10. The assay method according to any one of claims 1 to 9, wherein the biomarker comprises an antibody, and the biomarker capturing particle comprises an antigen epitope that binds to the antibody.

11. The assay method according to any one of claims 1 to 10, comprising the step of detecting an antigen using the detection antibody, wherein the interference is removed by contacting the detection antibody with an interference capture composition, which includes removing interference or conjugate nonspecific binding (NSB) from the detection antibody.

12. The method according to any one of claims 1 to 11, wherein the interference capture composition comprises interference capture particles, biomarker capture particles, or a combination thereof.

13. The assay method according to any one of claims 1 to 12, comprising the steps of: contacting the detection antibody with the sample to bind a detection biomarker in the sample to the detection antibody; and detecting or measuring the detection antibody bound to the detection biomarker.

14. The assay method according to any one of claims 1 to 13, wherein the interference capture portion is in a liquid reagent.

15. The assay method according to claim 14, wherein the liquid reagent further comprises a sample preservation reagent or a stabilizer.

16. The assay method according to claim 14 or 15, wherein the liquid reagent further comprises a sample preparation reagent or a drug.

17. The assay method according to any one of claims 1 to 13, wherein the interference capture portion is in a solid reagent.

18. (b) The assay method according to any one of claims 1 to 17, wherein the step is to add the capture portion to the sample.

19. The assay method according to any one of claims 1 to 18, wherein the interference detection portion is present in the sample collection device before it comes into contact with the sample.

20. The assay method according to any one of claims 1 to 19, wherein the interference capture portion is biotinylated.

21. The assay method according to any one of claims 1 to 20, wherein the biomarker capturing particles are coated with streptavidin.

22. The assay method according to any one of claims 1 to 21, wherein the biomarker capture portion comprises an antibody or an antibody-binding fragment.

23. The assay method according to any one of claims 1 to 22, wherein the biomarker comprises one or more biomarkers selected from the group consisting of antigens, antibodies, proteins, small molecules, therapeutic agents, hormones, peptides, signaling peptides, exosomes, cells, or any combination thereof.

24. The assay method according to any one of claims 1 to 23, wherein the biomarker comprises an antibody, and the biomarker capture portion comprises the antigen of the antibody.

25. The assay method according to claim 24, wherein the antibody is selected from the group consisting of autoantibodies, therapeutic antibodies, immunoglobulin classes, immunoglobulin subclasses, circulating antibodies, secretory antibodies, alpaca-derived nanobodies, and animal-derived antibodies.

26. The assay method according to any one of claims 1 to 23, wherein the biomarker comprises an antigen.

27. The assay method according to claim 26, wherein the biomarker capture portion includes an antibody of the antigen.

28. The assay method according to claim 26 or 27, wherein the antigen comprises a therapeutic agent, a drug, a small molecule, a peptide, a protein, a vaccine, or an immunogen.

29. The assay method according to any one of claims 26 to 28, wherein the antigen comprises a repeating epitope, and the antigen binds to one or more biomarker-capturing particles.

30. The assay method according to any one of claims 26 to 29, wherein the antigen comprises at least a first type of epitope and a second type of epitope.

31. The assay method according to any one of claims 26 to 30, wherein the biomarker capturing particles are coated with at least a first type antibody specific to the first type of epitope and a second type antibody specific to the second type of epitope.

32. The assay method according to any one of claims 23 to 31, wherein the biomarker includes a disease-specific biomarker.

33. The assay method according to any one of claims 1 to 32, wherein the biomarker-capturing particles include aggregated particles, and the aggregated particles aggregate when they bind to the biomarker, thereby forming aggregated biomarker-capturing particles.

34. The assay method according to claim 33, wherein the aggregation is visually detectable or detectable by a detection technique.

35. The assay method according to claim 33 or 34, wherein the aggregated biomarker-capturing particles exhibit color.

36. The assay method according to any one of claims 1 to 35, further comprising the step of eluting the biomarker from the biomarker-capturing particles.

37. The assay method according to any one of claims 1 to 36, further comprising the step of quantifying the biomarker.

38. The assay method according to claim 37, wherein the step of quantifying the biomarker includes determining the mass of the biomarker per sample volume.

39. The assay method according to any one of claims 1 to 38, wherein the sample includes biological fluid.

40. The assay method according to claim 39, wherein the biological fluid includes blood, plasma, serum, saliva, or physiological saline oral rinse.

41. The assay method according to any one of claims 1 to 40, wherein the interference includes lipids, triglycerides, bilirubin, hemolysis products, cholesterol, human anti-mouse antibodies (HAMA), rheumatoid interference (RF), manufacturing assay-specific interference (MASI), human anti-animal antibody (HAAA) interference, free biotin interference, anti-streptavidin interference, anti-biotin interference, human anti-polyethylene glycol (PEG) interference, anti-albumin, nonspecific binding, anti-polyvinylpyrrolidone (PVP), anti-polymer, anti-alkaline phosphatase (ALP), anti-ruthenium, anti-fluorescein, anti-acridinium ester, autoantibodies, anti-horseradish peroxidase (HRP), anti-conjugation linkers, anti-amino acid tags, anti-histidine tags, anti-polyhistidine tags, over-the-counter (OTC) supplements, herbal remedies and / or therapeutic agents, or any combination thereof.

42. The assay method according to claim 41, wherein the hemolytic product comprises hemoglobin, lactate dehydrogenase, potassium, or a combination thereof.

43. The assay method according to claim 41, wherein the human anti-animal antibody (HAAA) interference comprises mouse immunoglobulin, goat immunoglobulin, sheep immunoglobulin, rabbit immunoglobulin, bovine immunoglobulin, or a combination thereof.

44. The assay method according to claim 41, wherein the anti-acridinium ester comprises ABEI, luminol, isoluminol, or any combination thereof.

45. The assay method according to claim 41, wherein the anti-conjugation linker comprises LC, LC-LC, PEOn, a chromogen, or any combination thereof.

46. The assay method according to any one of claims 1 to 46, further comprising the step of bringing the sample into contact with a conjugate.

47. The assay method according to claim 46, further comprising the step of washing the conjugate with the interference-capturing particles or the biomarker-capturing particles before bringing the sample into contact with the conjugate.

48. The assay method according to any one of claims 1 to 47, wherein the sample subject to the assay is subjected to one or more adjustments to the chemical or physical properties of the sample in order to eliminate one or more interferences.

49. The assay method according to claim 48, wherein the chemical or physical properties are selected from temperature, color, pH, salinity, conductivity, density, viscosity, surface tension, and protein content.

50. The one or more adjustments described above (i) Adding surfactants, detergents, cell lysants, antiproteases, protein-based or polymer-based blocking reagents, substitutions, drugs, or any combination thereof to the sample to release the analyte from the matrix or remove interfering elements, (ii) Diluting the sample, or The assay method according to claim 48 or 49, comprising any combination of (ii)(i) and (ii).

51. The assay method according to any one of claims 1, 2, or 5 to 50, wherein the interference capture portion interacts with the interference.

52. The assay method according to any one of claims 1 to 51, wherein the subject is suspected of having a disease or has been administered a vaccine for the disease.

53. The assay method according to claim 52, wherein the disease includes infectious diseases.

54. The assay method according to claim 53, wherein the infectious disease includes a viral infection, a bacterial infection, or a protozoan infection.

55. The assay method according to claim 52, wherein the disease includes tick-borne diseases.

56. The assay method according to claim 55, wherein the disease includes Lyme disease.

57. The assay method according to claim 52, wherein the disease includes severe acute respiratory syndrome.

58. The assay method according to claim 52, wherein the disease includes coronavirus infection.

59. The assay method according to claim 58, wherein the coronavirus infection includes coronavirus disease 2019 (COVID-19).

60. The assay method according to any one of claims 1, 2, or 5 to 59, wherein the washing comprises washing the sample by removing the interference.

61. The assay method according to claim 6 or 52, wherein the vaccine is configured to produce a pathogen or a component of a disease.

62. The assay method according to claim 36, wherein the eluted biomarker contains an antibody against a pathogen or disease.

63. The assay method according to claim 36 or 62, further comprising the step of determining the efficacy of the vaccine based on the level of a eluted biomarker.

64. The assay method according to claim 63, further comprising the step of re-administering the vaccine to the subject based on the effectiveness of the vaccine.

65. The assay method according to any one of claims 36 or 62 to 64, further comprising the step of identifying the possibility that the subject has the disease based on the eluted biomarker.

66. The assay method according to claim 36 or any one of claims 62 to 65, further comprising the step of identifying the possibility that the disease is active or acute.

67. The assay method according to claim 66, further comprising the step of administering disease treatment to the subject if the subject is identified as having an active or acute disease.

68. The assay method according to claim 67, further comprising the step of not administering or discontinuing disease treatment if the subject is identified as not having an active or acute disease.

69. The assay method according to claim 67, wherein the step of administering the disease treatment includes adjusting the dose or timing.

70. The assay method according to any one of claims 1 to 69, wherein the biomarker is secreted.

71. The assay method according to any one of claims 1 to 70, wherein the biomarker comprises IgA, IgG, IgM, or a combination thereof.

72. The assay method according to any one of claims 1 to 71, wherein the subject is administered a therapeutic compound.

73. The assay method according to claim 72, wherein the therapeutic compound includes a therapeutic agent.

74. The assay method according to claim 72 or 73, wherein the therapeutic compound or a fragment thereof is contained in the antigen.

75. The assay method according to any one of claims 72 to 74, wherein the captured biomarker includes an autoantibody against the therapeutic compound.

76. The assay method according to any one of claims 72 to 75, further comprising the step of determining the level of safety of the therapeutic compound based on the quantified biomarker.

77. The assay method according to claims 72 to 76, further comprising the step of administering a therapeutic compound to the subject or adjusting the dose thereof based on the quantified biomarker.

78. The assay method according to any one of claims 72 to 77, wherein the therapeutic agent comprises a therapeutic antibody.

79. The assay method according to claim 78, wherein the therapeutic antibody comprises an antibody-binding fragment.

80. The assay method according to claim 78 or 79, wherein the captured biomarker comprises the therapeutic antibody.

81. The assay method according to claim 78, wherein the antigen comprises the antigen or biomarker of the therapeutic antibody.

82. The assay method according to claim 78, further comprising the step of determining the pharmacokinetic profile of the therapeutic antibody based on the quantified biomarker.

83. The assay method according to claim 78, further comprising the step of administering the therapeutic antibody to the subject or adjusting the dose thereof based on the quantified biomarker.

84. The assay method according to any one of claims 1 to 83, wherein the subject is a human.

85. The assay method according to any one of claims 1 to 84, further comprising the step of binding the captured biomarker with a detection antibody.

86. The assay method according to any one of claims 1 to 85, further comprising the step of measuring the detected antibody.

87. The assay method according to claim 86, further comprising the step of comparing the detected antibody with a standard curve.

88. The assay method according to claim 87, wherein the standard curve is generated from biomarker-capturing particles bound to a known amount of the biomarker.

89. The assay method according to claim 86, further comprising the step of washing the detection antibody with the interference-capturing particles before detecting the biomarker using the detection antibody.

90. The assay method according to claim 86, wherein the detection antibody includes an anti-human antibody.

91. The assay method according to claim 86, wherein the detection antibody comprises an antibody against the antigen.

92. The assay method according to claim 86, wherein the detection antibody is conjugated to a detection reagent.

93. The assay method according to claim 92, wherein the detection reagent comprises an enzyme or a label.

94. The assay method according to claim 93, wherein the label includes a fluorescent tag.

95. The assay method according to any one of claims 1 to 94, further comprising the step of multiplexing the biomarker-capturing particles with additional biomarker-capturing particles containing a second antigen, wherein the biomarker includes an antibody that binds to the second antigen.

96. The assay method according to any one of claims 3, 4, or 37, wherein the step of quantifying the biomarker includes multiplexing the detection antibody with a second detection antibody that recognizes a second antigen.

97. The assay method according to claim 96, further comprising the step of multiplexing the detection antibody with an additional detection antibody that recognizes a biomarker in the sample.

98. The assay method according to any one of claims 1 to 97, further comprising the step of monitoring the biomarker in the first sample and the second sample over time to determine whether the amount of the biomarker in the target second sample has increased or decreased compared to the quantified biomarker in the first sample.

99. The assay method according to any one of claims 1 to 98, wherein the biomarker capturing particles include fine particles.

100. The assay method according to any one of claims 1 to 99, wherein the biomarker capturing particles include beads.

101. The assay method according to any one of claims 1 to 100, wherein the biomarker capturing particles contain a metal.

102. The assay method according to any one of claims 1 to 101, wherein the biomarker capturing particles are magnetic.

103. The assay method according to any one of claims 1 to 102, wherein the biomarker capturing particles include a plurality of biomarker capturing particles comprising (i) a plurality of magnetic beads and (ii) a plurality of non-magnetic beads, the plurality of magnetic beads being larger in size than the plurality of non-magnetic beads, and one or more of the plurality of magnetic beads and one or more of the non-magnetic beads forming a complex with the biomarker.

104. The assay method according to claim 103, wherein the concentration of the plurality of non-magnetic beads decreases when the complex is removed.

105. The assay method according to any one of claims 1, 2, or 5 to 104, wherein the washing comprises washing the biomarker-capturing particles by removing the interference.

106. The assay method according to any one of claims 1, 2, or 5 to 105, wherein the washing comprises washing the detection antibody by removing the interference.

107. The assay method according to any one of claims 1 to 106, wherein the assay sensitivity is at least 76%.

108. The assay method according to any one of claims 1 to 107, wherein the assay sensitivity is at least 99%.

109. The assay method according to any one of claims 1 to 108, wherein the assay sensitivity is 100%.

110. The assay method according to any one of claims 1 to 109, wherein the assay specificity is at least 76%.

111. The assay method according to any one of claims 1 to 110, wherein the assay specificity is at least 99%.

112. The assay method according to any one of claims 1 to 111, wherein the assay specificity is 100%.

113. a. An interference-trapping particle including an interference-trapping portion, b. Biomarker-capturing particles containing a biomarker-capturing portion A kit that includes this.

114. The kit according to claim 113, further comprising a detection antibody.

115. The kit according to claim 113 or 114, wherein the interference capture portion comprises human immunoglobulin, antibody, animal antibody, antibody fragment, polymerized antibody, mouse antibody fragment, aptamer, protein, enzyme, small molecule, streptavidin, avidin, neutraavidin, MIP, polymer, conjugation linker, or any combination thereof.

116. The kit according to any one of claims 113 to 115, wherein the biomarker capture portion comprises an antibody, an antibody fragment, a polypeptide binder, a monobody, a non-immunoglobulin binder, DARPin, an afibody, an antikalin, a molecular imprinted polymer (MIP), an aptamer, a chimeric antibody, a therapeutic antibody, an antigen, a protein, a small molecule, a therapeutic agent, a hormone, a peptide, a signaling peptide, an exosome, a cell, a disease state-specific antigen, an antibody, a biomarker, or any combination thereof.

117. A kit according to any one of claims 113 to 116 for use in the method according to any one of claims 1 to 104.

118. The kit according to any one of claims 113 to 117, further comprising a sample container.

119. A kit according to any one of claims 113 to 118, further comprising an instruction manual.

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