Enrichment of antigen-specific antibodies for analytical and therapeutic use

Abstract

Providing enriched antigen-specific antibodies for analytical and therapeutic use. [Solution] The present invention relates to a method for eliminating interference as described herein, or for enriching or isolating biomarkers, particularly antibodies, before diagnostic testing, or for use for prophylactic or therapeutic purposes, using particles (e.g., fine particles, nanoparticles; magnetic, nonmagnetic) having a surface containing the capture portion described herein. A simple, efficient, and cost-effective method for conditioning biological samples to control and mitigate a number of known sample-specific interferences that can result in erroneous test results and increased risks to patient safety, such as heterophilic antibodies in patients treated with monoclonal mouse antibodies or patients who have received them for diagnostic purposes, is described herein.

Description

Technical Field

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[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 008,472, filed Apr. 10, 2020, the entire content of which is incorporated herein by reference.

[0002] Field of the Invention The present disclosure relates to methods for using particles (e.g., microparticles, nanoparticles; magnetic, non - magnetic) comprising a surface comprising a capture moiety described herein for isolating antigen - specific antibodies for subsequent analytical, prophylactic or therapeutic use.

Background Art

[0003] Background Laboratory tests play an important role in health assessment, health management, and ultimately public health, affecting people at all life stages. Nearly everyone will undergo one or more laboratory tests over their lifetime. In the United States alone, an estimated 7 to 10 billion laboratory tests are performed each year, and laboratory tests influence approximately 70% of medical decisions.

[0004] <> Furthermore, the Centers for Medicare & Medicaid Services (CMS) implemented a new Clinical Laboratory Fee Schedule (CLFS) required by the Protecting Access to Medicare Act (PAMA) on January 1, 2018, and PAMA has reduced reimbursement for laboratory tests. Even more important is that laboratory results are accurate from the start, reducing troubleshooting efforts or time and not affecting the laboratory workflow.

[0005] Interference is a substance present in a patient sample that can alter the accurate value of a diagnostic test result, for example, by interfering with antibody binding, or by increasing or decreasing the assay signal through cross-linking, steric hindrance, or autoantibody mechanisms. While immunoassays are known to be susceptible to interference, clinical laboratories may still report erroneous results if such results are not recognized and flagged by the instrument (analyte) or laboratory, or if the physician does not notify the laboratory that the patient's results do not fit the clinical picture. False results can occur unexpectedly in any sample for which there is no practical means of identifying potentially problematic samples beforehand. As a result of such interference, erroneous results may lead to false-negative and false-positive test results, which can impact patient care and lead to unnecessary invasive, diagnostic, or therapeutic procedures, or failure of patient treatment.

[0006] Clinical laboratories are a source of many sample-specific interferences, including sample type (i.e., plasma), carryover, freeze / thaw, stability, hemolysis, jaundice, dyslipidemia, anticoagulant effects, sample storage, binding proteins, drugs and drug metabolites, and cross-reactivity. However, heterophilic antibody interference, such as human anti-animal antibody (HAAA) and human anti-mouse antibody (HAMA) interference, is cumbersome and problematic because it is difficult to detect and can affect patient management.

[0007] Despite the complexities arising from interference, biomarker screening and diagnostic testing can be challenging, for example, due to their presence or low abundance in biological samples.

[0008] Therefore, while biomarkers found in the body can be used to detect, predict, or manage disease, many are too scarce to be detected using currently available tests. There is an unmet clinical need for new diagnostic techniques to improve test accuracy, perform measurements that are difficult to detect, reduce costs, and ultimately prepare clinical samples to save lives.

[0009] Biotin, also known as vitamin B7, is a water-soluble B vitamin often found in multivitamins and over-the-counter health and beauty supplements. In vitro laboratory diagnostic tests using the streptavidin-biotin binding mechanism have the potential to be affected by high circulating biotin concentrations. Biotin can covalently bind to a wide range of targets, from large antibodies to steroid hormones, while minimizing the impact on specific non-covalent binding with avidin, streptavidin, or neutraavidin proteins. Therefore, biotin is frequently used in detection systems of various forms of immunoassays.

[0010] Immunoassays are generally classified into either sandwich immunoassays (non-competitive) or competitive inhibitory immunoassays. Generally, streptavidin-biotin conjugation is used to conjugate biotinylated antibodies in sandwich immunoassays or biotinylated antigens in competitive immunoassays to a streptavidin-coated surface during assay incubation. If a biological sample contains excess biotin, the biotin competes with the biotinylated antibody or antigen for binding to the streptavidin-coated surface, reducing the capture of the biotinylated antibody or antigen. Excess biotin can lead to falsely low results in sandwich immunoassays because the assay signal is directly proportional to the analyte concentration. Excess biotin in competitive immunoassays can lead to falsely high results because the assay signal is inversely proportional to the analyte concentration.

[0011] Normal circulating concentrations and metabolism of dietary biotin are too low (less than 1.2 ng / mL) to interfere with biotinylated immunoassays. However, intake of high-dose biotin supplements (e.g., 5 mg or more) can result in significantly higher blood concentrations that may interfere with commonly used biotinylated immunoassays. Several studies have shown that serum biotin concentrations can reach up to 355 ng / mL within one hour after biotin ingestion in subjects taking 20 mg of biotin supplements per day, and up to 1160 ng / mL in subjects after a single dose of 300 mg of biotin. According to the FDA, biotin in blood or other samples taken from patients taking high levels of biotin may, depending on the assay design, cause falsely high or falsely low results in biotin-based immunoassays.

[0012] Biotin levels in blood or other samples taken from patients ingesting high levels of biotin may, depending on the assay design, cause falsely high or falsely low results in biotin-based immunoassays. These false test results may lead to inadequate patient management and misdiagnosis.

[0013] Biotin interference thresholds vary significantly between assays, even on a single platform. Tests with biotin interference thresholds below 51 ng / mL are considered high-risk tests or vulnerable immunoassays and competing assays.

[0014] Biotin-based tests are also susceptible to interference mechanisms related to the use of streptavidin in test designs to capture biotin conjugated to antibodies, proteins, or antigens, or to anti-streptavidin interference. Anti-streptavidin antibodies and proteins can significantly interfere with certain laboratory tests and lead to erroneous test results. Similar to biotin interference, which can cause reduced test signal and falsely low or falsely high patient outcomes depending on assay design and format, anti-streptavidin interference also results in reduced test signal, but through a different mechanism and can therefore be mistaken for biotin interference. The cause of anti-streptavidin antibodies is not fully known and is debated, but one possible cause may originate from the bacterium S. avidinii. Many people are exposed to these bacteria in their daily lives, and it is thought that this can lead to an immunological response to them.

[0015] Therefore, there is a clinical need for a simple, inexpensive, automateable, and effective solution to eliminate or minimize sample interference and enrich biomarker concentrations before diagnostic testing, without impacting laboratory workflow and turnaround time. [Overview of the Initiative] [Means for solving the problem]

[0016] Summary of the Invention This specification describes a simple, efficient, and cost-effective method for conditioning biological samples to control and mitigate a number of known sample-specific interferences that can result in erroneous test results and increased risks to patient safety, such as heterophilic antibodies in patients treated with monoclonal mouse antibodies or those who have received them for diagnostic purposes. The method described herein can also control and mitigate sample-specific interferences that may arise from biotin, which may result from over-the-counter (OTC) supplements, multivitamins, and herbal medicines taken by consumers for health and beauty and weight loss, or for the treatment of multiple sclerosis, for example.

[0017] Methods for enriching or increasing the concentration of biomarkers in biological samples are also described herein. In particular, the biomarkers may be antigen-specific antibodies, viral structural proteins such as the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In various embodiments, the spike protein is the complete protein, the S1 subunit, the S2 subunit, or its antigenic fragment, e.g., the receptor-binding domain (amino acids 331-524, etc.) and / or the N-terminal domain of the S1 subunit (e.g., amino acid residues 1-260). The spike protein or its antigenic fragment can be biotinylated and bound to streptavidined beads, which can then function as a capture reagent for SARS-CoV-2 neutralizing antibodies. Typically, when both the N-terminal domain and the receptor-binding domain are used, their fragments are bound to separate beads, which are then mixed together to function as a capture reagent.

[0018] In one embodiment, a method for isolating an antigen-specific antibody from a biological sample is provided herein, comprising: a) combining the sample with particles containing a capture moiety to provide a mixture; b) mixing the mixture to provide a particle complex with the antibody; and thereby isolating the antibody from the biological sample. In some embodiments, the capture moiety is the SARS-CoV-2 spike protein. In some embodiments, the capture moiety is the S1 subunit of the SARS-CoV-2 spike protein, or its receptor-binding domain and / or N-terminal domain. The structure of the SARS-CoV-2 spike protein is known in the art (Walls et al., Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein, Cell 180:1-12, 2020, which is incorporated herein by reference in its entirety). In various embodiments, the biological sample may be blood, plasma, serum, or other antibody-containing biological fluid.

[0019] In some embodiments, the isolated antibodies were detected in serological assays. , quantified, or characterized.

[0020] In some embodiments, the capture-molecule-antibody complex is cleaved from the particle. In other embodiments, the antibody is eluted from the capture-molecule, particularly while the capture-molecule remains bound to the particle. The enriched or isolated antibody can then be subjected to protein chemistry analytical methods, including mass spectrometry and Edman degradation. The enriched or isolated antibody can be used for passive immunization for prophylactic or therapeutic purposes. For example, if antibodies that recognize the SARS-CoV-2 spike protein are isolated, they can be administered to COVID-19 patients as therapeutic agents, or prophylactically to healthcare workers or other people at risk of infection with SARS-CoV-2 due to exposure to COVID-19 patients.

[0021] In one embodiment, a method for removing interference from a biological sample is provided herein, comprising: a) combining the sample with particles containing a capture portion to provide a mixture; b) mixing the mixture to provide a particle complex for interference; and c) removing or eliminating the particle complex to provide a depleted solution, thereby reducing or mitigating the amount of interference (e.g., mass, volume, molar concentration, concentration). [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1 shows a scheme for confirmation and disqualification assays based on the removal (or depletion) of interference from biological samples by particles as described herein.

[0023] [Figure 2] Figure 2 shows a scheme for a depletion assay based on the removal (or depletion) of interference from a biological sample using lyophilized particles as described herein.

[0024] [Figure 3] Figure 3 shows a scheme for a depletion assay based on the removal (or depletion) of interference from a biological sample by a magnetized pipette tip described herein.

[0025] [Figure 4] Figure 4 is a graph showing the biotin concentration over time after biotin uptake.

[0026] [Figure 5] Figure 5 is a graph showing biotin depletion.

[0027] [Figure 6] Figure 6 is a graph showing biotin depletion.

[0028] [Figure 7] Figure 7 is a graph showing the biotin concentration over time after biotin uptake.

[0029] [Figure 8] Figure 8 is a graph showing the biotin concentration after the intake of different biotin doses.

[0030] [Figure 9] Figure 9 is a graph showing biotin depletion.

[0031] [Figure 10] Figure 10 is a graph showing the PTH concentration.

[0032] [Figure 11] Figure 11 shows calibration curves for IgA, IgG and IgM generated using triple calibrator beads.

[0033] [Figure 12]Figure 12 shows the total SARS-CoV-2 neutralizing antibody levels in five PCR-positive patients for whom serial samples were available and who were initially tested negative in the SARS-CoV-2 neutralizing antibody assay.

[0034] [Figure 13] Figure 13 shows the total SARS-CoV-2 neutralizing antibody levels in 37 PCR-positive patients for whom serial samples were available. [Modes for carrying out the invention]

[0035] Detailed description of the invention A method for depleting or enriching a biological sample is described herein, which includes combining the particles described herein with the biological sample described herein.

[0036] In one embodiment, a method for isolating a biomarker from a biological sample is provided herein, comprising: a) combining the sample with particles containing a capture portion to provide a mixture; b) mixing the mixture to provide a particle complex with the biomarker; and thereby isolating the biomarker from the biological sample. In some embodiments, the biomarker is an antigen-specific antibody. In some embodiments, the antigen-specific antibody recognizes the spike protein of the SARS-CoV-2 spike protein, e.g., the S1 subunit, or its receptor-binding domain and / or N-terminal domain.

[0037] In some embodiments, the method further comprises subjecting the particle complex to a diagnostic test. In some embodiments, the method further comprises subjecting a biomarker cleaved or eluted from the particle complex to a diagnostic test. In some embodiments, the biomarker is a pathogen-specific antibody. In some embodiments, the pathogen-specific antibody is an anti-SARS-CoV-2 antibody. In some embodiments, the anti-SARS-CoV-2 antibody comprises an antibody that recognizes the receptor-binding domain, the N-terminal domain, or both.

[0038] SARS-CoV-2 neutralizing antibodies exist that target both the receptor-binding domain and the N-terminal domain. Antibodies that bind to either of these domains sterically block the interaction between the S1 spike and the viral receptor (angiotensin-converting enzyme 2 (ACE2)). Therefore, antibodies that bind to these domains are considered neutralizing antibodies. IgM, IgG, and IgA isotype antibodies that recognize either of these domains are all considered to neutralize the virus. Therefore, capturing and quantifying both types of antibodies may be advantageous to accurately assess the degree of neutralizing antibodies in a biological sample. Similarly, when capturing and using both types of antibodies for prophylactic or therapeutic purposes, a stronger passive immunity can be established. For example, when an antibody that recognizes both the receptor-binding domain and the N-terminal domain is used, a variant virus with a mutation in one of these domains is less likely to escape neutralization than when an antibody that recognizes one of these domains is used.

[0039] To capture SARS-CoV-2 neutralizing antibodies, SARS-CoV-2 S1-RBD antigen and S1-NTD antigen are used as capture reagents. In some embodiments, these antigens are biotinylated and coated onto streptavidin-modified magnetic beads.

[0040] In one embodiment, a method for removing interference from a biological sample is provided herein, comprising: a) combining the sample with particles containing a capture portion to provide a mixture; b) mixing the mixture to provide a particle complex for the interference; and c) removing or eliminating the particle complex to provide a depleted solution, thereby reducing or mitigating the amount of interference (e.g., mass, volume, molar concentration, concentration).

[0041] In some embodiments, this method further includes subjecting the depleted solution to characterization (e.g., diagnostic testing).

[0042] In some embodiments, the particles are freeze-dried products (e.g., LyoSphere). Provided as (Trademark) (BIOLYPH LLC)).

[0043] In one embodiment, a method for improving the accuracy of a diagnostic test is provided herein, comprising: a) providing a mixture of a biological sample and particles containing a capture portion; b) mixing the mixture to provide a particle complex for interference; c) removing or eliminating the particle complex to provide a depletion solution; and d) subjecting the depletion solution to a diagnostic test, thereby improving the accuracy of the diagnostic test.

[0044] In some embodiments, at least 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% of interference are removed compared to biological samples not subjected to this method. In some embodiments, an amount of interference sufficient to provide less than 100 ppm of interference in the biological sample is removed. In some embodiments, an amount of interference sufficient to provide less than a detectable level of interference in a diagnostic test is removed.

[0045] In some embodiments, the capture portion is a human anti-animal antibody (e.g., mouse IgG, sheep IgG, goat IgG, rabbit IgG, bovine IgG, pig IgG, horse IgG). In some embodiments, the capture portion is heterophile antibodies (e.g., FR(Fc-specific), Fab, F(ab)'2, polymerized IgG (types 1, 2a, 2b IgG and IgG fragments, serum components). In some embodiments, the capture portion is assay-specific conjugates (e.g., biotin, fluorescein, anti-fluorescein poly / Mab, anti-biotin poly / Mab, streptavidin, neutraavidin). In some embodiments, the capture portion is assay-specific signaling molecules (e.g., HRP, ALP, acridinium ester, isoluminol / luminol, ruthenium, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) / cyclic ABEI). In some embodiments, the capture portion is assay-specific blockers (e.g., BSA, fish skin gelatin, casein, ovalbumin, PVP, PVA). In some embodiments, the capture portion is assay-specific conjugate linkers (e.g., LC, The capture moiety is LC-LC, PEO4, PEO16). In some embodiments, the capture moiety is an antigen autoantibody (e.g., free T3, free T4). In some embodiments, the capture moiety is a protein autoantibody (e.g., MTSH, TnI, TnT, non-cardiac TnT (skeletal muscle disease)). In some embodiments, the capture moiety is a chemiluminescent substrate (e.g., luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, ruthenium, acridinium ester) or a fluorescent label (e.g., fluorescein or other fluorophores and dyes). In some embodiments, the capture moiety is streptavidin, neutraavidin, avidin, polyA, polyDT, aptamer, antibody, Fab, F(ab')2, antibody fragment, recombinant protein, enzyme, protein, biomolecule, or polymer. In some embodiments, the capture moiety is biotin, fluorescein, Po1yDT, PolyA, antigen, etc.

[0046] In some embodiments, removal or elimination is separation. In some embodiments, separation includes physical separation. In some embodiments, separation includes magnetic separation. In some embodiments, the magnet for magnetic separation is a multi-magnet device containing 2 to 12 magnets in a rack designed to hold 1 to 12 sample preparation tubes in a large pipetting machine. Examples of such pipetting machines include, but are not limited to, those constructed by Hamilton or Tecan. In some embodiments, the magnet for magnetic separation is a multi-magnet device containing 96 or 384 magnets designed to provide magnetization to a 96-well or 384-well microtiter plate. In some embodiments, separation includes chemical separation. In some embodiments, removal or elimination is pellet To provide the pellet and supernatant, the process includes centrifugation at 1000 × g or more for at least 1, 2, 3, 4, or 5 minutes, and removal of the supernatant. In some embodiments, removal or exclusion includes filtration (e.g., by a filter). In some embodiments, the filter has a porosity or molecular weight cutoff (MWCO) that is sufficiently smaller than the diameter of the particles (e.g., nanoparticles, fine particles). In some embodiments, filtration is by gravity, vacuum, or centrifuge. In some embodiments, removal or exclusion includes magnetization. In some embodiments, magnetization is induced using a strong magnet (e.g., a neodymium magnet) to provide the pellet and supernatant. In some embodiments, the magnet is located in the centrifuge rotor. In some embodiments, the magnet is located in a disposable pipette tip, cover, or sheath.

[0047] In one embodiment, a method for isolating a biomarker from a biological sample is provided herein, comprising: a) combining the sample with particles containing a capture portion to provide a mixture; b) mixing the mixture to provide a particle complex containing the biomarker; c) removing the particle complex from the mixture; and d) adding a cleavage reagent or release (elution) agent to the mixture to obtain an isolate containing the biomarker, thereby isolating the biomarker from the biological sample. In some embodiments, the biological sample is pretreated or washed according to the methods disclosed herein before isolating the biomarker. In some embodiments, the biomarker is an antigen-specific antibody. In some embodiments, the antigen-specific antibody recognizes the spike protein of the SARS-CoV-2 spike protein, e.g., the S1 subunit, or its receptor-binding domain and / or N-terminal domain. In some embodiments, the method for isolating the biomarker from the biological sample is performed before performing a diagnostic test on the biological sample.

[0048] In some embodiments, the washing reagent contains human immunoglobulins, e.g., IgG, IgA, and / or IgM, as the capture portion. In some embodiments, the washing reagent contains animal immunoglobulins, e.g., rabbit, goat, or mouse IgG, as the capture portion. In some embodiments, the washing reagent contains BSA. Preferably, the washing reagent(s) are the same microparticles as the biomarker capture reagent. In this way, one or more heterophilic interferences(s) specific to the analyte antibody and assay reagents (e.g., streptavidin and the beads themselves) can be removed.

[0049] In one embodiment, a method for determining whether a biomarker is present in a biological sample is provided herein, comprising: a) combining the sample with a capture portion to provide a mixture; b) combining the mixture with particles containing the capture portion to provide a tertiary complex; c) removing the tertiary complex from the mixture to provide an isolate; and d) determining whether an indicator of the tertiary complex is present in the isolate, thereby determining whether a biomarker is present in the biological sample.

[0050] In one embodiment, a method for determining whether a biomarker is present in a biological sample is provided herein, comprising: a) providing a mixture by combining the sample with particles containing a capture portion; b) mixing the mixture to provide a particle complex for interference; c) removing or eliminating the particle complex to provide a depletion solution; d) combining the depletion solution with second particles containing a second capture portion to provide a second mixture; e) mixing the second mixture to provide a second particle complex containing a biomarker; f) removing the second particle complex from the second mixture; and g) adding a cleavage reagent or release agent to the second mixture to provide an isolate containing a biomarker, thereby isolating the biomarker from the biological sample.

[0051] In some embodiments, the method further includes washing the particle complex with a diluent.

[0052] In some embodiments, the cleavage reagent is a disulfide bond reducing reagent.

[0053] In some embodiments, the method further includes performing a diagnostic test on a biomarker.

[0054] In one embodiment, a method for enriching the amount of a biomarker in a sample is provided herein, comprising: a) adding particles containing a capture portion to the sample to provide a mixture; b) mixing the mixture to obtain a particle complex; c) separating the particle complex to obtain a pellet and a supernatant; e) removing the supernatant from the pellet; f) washing the pellet with a diluent; and g) eluting the biomarker from the pellet to provide an enriched sample, thereby enriching the amount of a biomarker in the sample. In some embodiments, the method for enriching a biomarker from a biological sample is performed before performing a diagnostic test on the biological sample.

[0055] In some embodiments, the biomarker is an autoantibody against a tumor antigen, such as p53. In some embodiments, the tumor antigen is a neoantigen. Some tumor antigens are expressed in developmentally inappropriate ways, for example, in tissues or at maturation stages where they would normally not be expressed at all, or at levels as high as they would be. This can lead to the production of antibodies that recognize the tumor antigen. Other tumor antigens involved in the carcinogenic process may be mutated, and the mutations make the tumor antigen immunogenic (neoantigen). Again, antibodies that recognize the mutated tumor antigen can be produced. Detection of such autoantibodies may be useful in the detection and diagnosis of cancer, including early detection or changes in malignancy, and is useful in selecting appropriate treatment. Such tumor antigens can be used in capture reagents for antitumor antigen autoantibodies.

[0056] Further autoantibodies against tumor markers that may be useful for the early detection of cancer include those that recognize cancer antigen 15-3 (CA15-3), carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9), c-Myc, p53, heat shock protein (Hsp) 27 and Hsp70, eukaryotic translation initiation factor 3 subunit A (EIF3A), and lung cancer (LC). These tumor antigen-specific autoantibodies have long half-lives and are produced in large quantities in response to low circulating or low-abundance oncoproteins, making them promising biomarkers for the early detection of cancer.

[0057] In some embodiments, the biomarker is an indicator of traumatic brain injury (TBI). In some embodiments, the biomarker is s-100β, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), neurofilamentous light chain (NFL), cleaved tau protein (C-tau), and ubiquitin C-terminal hydrolase-L1 (UCH-L1). In some embodiments, the biomarker is an indicator of Alzheimer's disease (AD). In some embodiments, the biomarker is amyloid beta, BACE1, and soluble Aβ precursor protein (sAPP). In some embodiments, the biomarker is an indicator of sexually transmitted diseases (STDs). In some embodiments, the STD is chlamydia, gonorrhea, syphilis, trichomoniasis, HPV, herpes, hepatitis B, hepatitis C, or HIV. In some embodiments, the biomarker is an indicator of bacterial infection. In some embodiments, the biomarker is a capture portion for bacteria. In some embodiments, the biomarker is cleaved from the complex by a cleavage reagent. In some embodiments, the presence of a biomarker is M It is determined by ALDI-MS. In some embodiments, the presence of the biomarker is determined by molecular diagnostic methods. In some embodiments, the presence of the biomarker is determined by immunoassays.

[0058] In some embodiments, the interference is fibrinogen, removal or exclusion is separation, such as physical separation by centrifugation, and the particle complex is trapped in a clot.

[0059] Referring to Figure 1, a scheme for confirmation and rejection assays based on the removal (or depletion) of interference from a biological sample by the particles described herein is shown. The biological sample is aspirated from a primary blood collection tube (PBCT) and dispensed into a secondary transfer tube (STT). The particles described herein, for example, particles with a surface containing a capture portion for free biotin and / or heterophilic antibodies, are added to the STT to bind to and deplete the sample interference.

[0060] Figure 2 shows a scheme for a depletion assay based on the removal (or depletion) of interference from a biological sample by lyophilized particles as described herein. A PBCT containing lyophilized particles (e.g., particles as described herein) receives the biological sample, resulting in the resuspension and dispersion of the particles by the biological sample.

[0061] Figure 3 shows a scheme for a depletion assay based on the removal (or depletion) of interference from a biological sample using a magnetized pipette tip as described herein. A pipette tip containing a magnet is added to the biological sample to remove the interference or biomarker described herein from the biological sample.

[0062] Separation method The particles described herein can be added to a collection device such as a primary blood collection tube, a 24-hour urine collection device, a urine collection device, a saliva collection tube, a stool collection device, a semen collection device, a blood collection bag, or any sample collection tube or device before adding the biological sample.

[0063] The particles described herein may also be added to a sample after the sample has been collected in a sampling device, or after the sample has been transferred from the primary sampling device to a storage or transfer device, such as a plastic or glass tube, vial, bottle, beaker, flask, bag, can, microtiter plate, ELISA plate, 96-well plate, 384-well plate, 1536-well plate, cuvette, reaction module, reservoir, or any container suitable for holding, storing, or processing liquid samples.

[0064] In some embodiments, the particles described herein are added to a collection device containing a biological sample. In some embodiments, the particles described herein are added to the collection device before the biological sample is added.

[0065] In some embodiments, particularly those involving preparative rather than analytical application, biological samples from multiple donors are pooled before particle addition. At least 10 liters can be processed at once.

[0066] In one embodiment, a device for releasing particles of a biological sample (i.e., a collection device according to this specification that includes a screw cap that drives a release mechanism, such as on a urine collection device) is described herein. For example, the device is a tube equipped with a screw cap that releases the particles described herein when the screw cap is closed.

[0067] In one embodiment, the present specification includes a device that chemically releases particles into a container containing a biological sample (i.e., an enclosed composition or a composition that dissolves in a solution at a specified rate or time). As described in the book. In some embodiments, the devices described herein are configured to delay the addition of the particles described herein, for example, to provide sample pretreatment before enrichment or isolation of a biomarker, or to perform a diagnostic test.

[0068] In some embodiments, the samples described herein may be pretreated with chemicals, proteins, blockers, surfactants, or combinations thereof before the addition of the particles described herein to, for example, adjust the pH, deplete or compete for sample-specific interference, and / or manage matrix-specific challenges, and then the nanoparticles may be added, introduced, dispersed, or mixed into the sample to improve the specificity and binding kinetics of the nanoparticles to the target biomarker(s). Delayed addition of nanoparticles to the sample after sample pretreatment can be physically controlled by adding the nanoparticles to the sample after sample pretreatment. Nanoparticles may also be present in the sample during sample pretreatment if the nanoparticles are encapsulated, shielded, or protected by a chemical, polymer, or sugar shell, coating, or polymerization, such that the chemical, polymer, or sugar needs to be dissolved before the nanoparticles can be released, added, dispersed, or mixed into the sample. Delayed release of nanoparticles can be achieved using chemistry known to those skilled in the art, such as that used today in delayed drug release techniques.

[0069] Preparative affinity separation is a commonly used column chromatography technique. Magnetic particle separation techniques can avoid column clogging problems that can occur with some samples. For example, magnetic particle techniques allow for the processing of whole blood or cell-containing blood fractions. Magnetic particle separation techniques can also be achieved in a shorter time than typical column-based affinity separations. Yet another advantage of particle separation techniques is that the elution of captured ligands (biomarkers) can be achieved in smaller volumes, resulting in more concentrated molecules without further processing.

[0070] Method for magnetic separation of particles

[0071] In one embodiment, methods for removing interference from a biological sample (e.g., before diagnostic testing or before enrichment or isolation of a biomarker), or for isolating or separating magnetic particles (e.g., in a primary blood collection tube, in a custom sample collection device, in a secondary transfer tube or custom sample device, or in a pooled sample) are provided herein. For example, a magnet-based device rapidly (less than 2 minutes; preferably less than 30 seconds) isolates magnetic nanoparticles to the sides and / or bottom to form an essentially particle-free supernatant. The particle-free supernatant can then be removed by aspiration, drainage, or other means without destroying the particle-containing pellet and dispensed into a separate transfer tube for diagnostic testing. In some embodiments, the pellet is isolated or subjected to diagnostic testing.

[0072] Devices for magnetic separation of particles

[0073] Provided herein are devices comprising particles described herein that can be used by means of the method herein to remove or deplete biomarkers, for example, for diagnostic testing. In some embodiments, the device includes a physical mechanism for delaying the combination of the particles described herein with the sample described herein. In some embodiments, the device described herein includes a timed release mechanism for delaying the combination of the particles described herein with the sample described herein.

[0074] Magnetic tube holder. A custom magnetic tube holder that can be removed from its stand and inserted into a sample rack for subsequent diagnostic testing of the particle-free supernatant. The custom magnetic tube holder has a physical opening or transparent / clear plastic (where the magnet or magnetic array is located) in its design. (If not present) it can be designed to have a sample tube barcode that can still be detected and read by the analyzer, or index tests such as lipidemia, hemolysis, cell debris / clot detection, and level detection can still be performed by the analyzer. The sample tube may be a custom sample tube designed to fit only into a custom magnetic tube holder in a specific position, having notches or tongue-like structures and grooves to ensure that the analyzer can view and read the barcode through the opening (space) of the magnetic tube holder or through the clear / transparent plastic, and / or perform index tests such as lipidemia, hemolysis, cell debris / clot detection, level detection.

[0075] In some embodiments, the use of magnets(s) that can be attached to the sample rack via adhesive, Velcro®, or other means. When a sample tube containing magnetic nanoparticles is inserted into the sample rack position(s) using magnets(s), the magnetic nanoparticles rapidly separate to the sides(s) and / or bottom of the sample tube, forming an essentially particle-free sample supernatant for diagnostic testing by a sample rack-specific testing platform or analyzer.

[0076] The sample rack itself functions as a custom magnetic sample rack to fit a given analyzer (e.g., specific to Abbott ARCHITECT, Siemens ADVIA Centaur XP, Roche cobas e411 / e601 / 602 / e801, Beckman Coulter Access 2 / DxI 400 / DxI 800, DiaSorin LIAISON / LIAISON XL, etc.). For example, all tube positions in the rack have numerous magnets designed to rapidly separate magnetic nanoparticles to the sides and / or bottom of the sample tube, forming an essentially particle-free sample supernatant for diagnostic testing.

[0077] In one embodiment, a device (e.g., a separation device) is provided herein that includes a holder for a rack of test tubes (e.g., a test tube holder), the holder including a magnet.

[0078] Disposable pipette tip. In one embodiment, the device is a disposable pipette tip comprising a custom magnet inserted inside the disposable pipette tip, which rapidly isolates magnetic nanoparticles on the surface of the pipette tip to form an essentially particle-free sample supernatant. The disposable pipette tip with the custom magnet can then remove the pellet containing particles from the sample without destroying it. The disposable tip containing particles can be discarded if it is not necessary to measure or characterize what the particles have captured (i.e., interference depletion), or it can be inserted into a new tube for isolation and characterization of the particles in a subsequent diagnostic test (i.e., enrichment). For example, the disposable tip containing particles can be inserted into a secondary transfer tube containing a buffer. When the magnet is removed from the tip, or when the magnet is turned off (e.g., an electromagnet), the particles disperse freely in the buffer.

[0079] In one embodiment, the herein provides a device comprising a disposable pipette tip, the tip comprising a magnet.

[0080] Methods for the physical separation of particles

[0081] In one embodiment, a method for removing the particles described herein by physical force (e.g., gravity) is described herein. In some embodiments, the particles described herein are separated, isolated from a biological sample by physical force (e.g., by centrifugation), and The particles are removed. In some embodiments, this method is used, for example, in a primary blood collection tube, a custom sample collection device, a secondary transfer tube, or a custom sample device before applying the diagnostic test method described herein. In some embodiments, the method for removing particles is filtration.

[0082] For example, magnetic nanoparticles specific to fibronectin and / or other coagulation factors, or subsequent capture or binding of clot components / constituents for "clots" (in serum), cell debris (i.e., erythrocyte membrane specific), and / or capture or binding of cell debris (in serum or plasma) can increase centrifugation speed and efficiency by integrating powerful magnets or magnetic technology in the centrifuge rotor and / or tube holder (reducing rotation time to improve laboratory efficiency, workflow, and throughput). This combination with magnetic separation of RCF or Gs and magnetic nanoparticle complexes (i.e., clots + magnetic beads, cell debris + magnetic beads) from centrifugation allows for much faster and more efficient separation and supernatant formation at the side or bottom of the sample tube to clarify the sample for subsequent analysis. For example, this centrifugation step in most laboratories takes 4 minutes or more, and can be reduced to 2 minutes or less (preferably 1 minute or less) by combining centrifugation with magnetic separation / isolation of clot / cell debris complexes with magnetic nanoparticles.

[0083] Furthermore, if the nanoparticles or multiple magnetic nanoparticles are also specific to one or more different sample interference mechanisms, such as one, five, ten, twenty, thirty, or more different interference mechanisms, these interferences, if present, are captured by the nanoparticles and removed from the sample after physical separation from centrifugation, or removed from the sample by a combination of centrifugation and magnetic separation as described above.

[0084] These magnetic nanoparticles do not need to have specificity to clots or cell debris isolated by centrifugation or a combination of centrifugation and magnetic separation in a centrifuge, but their surfaces can be co-coated or immobilized with two or more antibodies and / or antigens, one or more of which will be specific to the clots and / or cell debris, while other antibodies and / or antigens will be specific to the sample interference. In this regard, the nanoparticles will bind specifically to both the sample interference and the clots and / or cell debris for subsequent physical separation or isolation by centrifugation or a combination of centrifugation and magnetic separation.

[0085] The use of nanoparticles specific to clots and / or cell debris increases the clotting rate of speed through specific binding by magnetic nanoparticles, and enables magnetic separation and Everything is pulled magnetically for isolation. This bead-based pellet, formed by the magnetic field and its intensity, also accelerates clot formation based on forced proximity of clots or clotting factors specifically captured by nanoparticles and subsequent magnets.

[0086] Chemical separation methods for particles

[0087] In some embodiments, the particles described herein are separated, isolated, or removed from a biological sample by a chemical separation method. In some embodiments, the chemical separation method is used, for example, in a primary blood collection tube, a custom sample collection device, a secondary transfer tube, or a custom sample device, before the application of a diagnostic test method.

[0088] In one embodiment, a method for the chemical separation of particles is provided, comprising providing one or more of a salt, a solvent, a polymer, or a detergent.

[0089] In some embodiments, a chemical separation method, such as liquid-liquid phase separation, involves distributing particles into phase A, and a sample without nanoparticles into phase B, where phase B is tested. The agents for liquid-liquid phase separation (chemical phase separation) include salts, soluble polymers, and detergents. It could be due to...

[0090] For example, liquid-liquid phase separation can be performed by adding a nonpolar solvent such as hexane to a polar aqueous sample, thereby distributing the particles into the nonpolar phase and leaving an aqueous phase free of nanoparticles for testing according to the diagnostic test described herein. In some embodiments, the separation method described herein provides nanoparticles in an organic phase. In some embodiments, the separation method described herein provides nanoparticles in an aqueous phase.

[0091] A method for isolating particles in a biological sample, comprising: providing the particles and the biological sample with a nonpolar solvent and an aqueous polar solvent to provide a nonpolar solvent layer and a polar solvent layer; removing the nonpolar solvent layer containing the nonpolar solvent; and isolating the aqueous polar solvent containing the particles, thereby isolating the particles.

[0092] Sample recovery can be adjusted or corrected by adding and using internal standards, such as deuterated internal standards for LC-MS / MS, before aspirating and discarding the nonpolar phase.

[0093] In some embodiments, the separation is physical separation used in combination with magnetic separation. For example, in one embodiment, a device is provided (e.g., a magnetized centrifuge or centrifuge with magnets that assist in separation by both gravity and the magnetic force of magnets). In one embodiment, a device for separating particles described herein, comprising magnets and a centrifuge, is provided herein. In some embodiments, the device significantly reduces the centrifugation time.

[0094] Methods for eliminating interference Methods for eliminating or minimizing interference are described herein, and these methods include depleting (e.g., mitigating, reducing, or controlling) known pre-analysis and analysis sources of test errors (e.g., interference) caused by other interfering substances such as hemolysis, dyslipidemia, jaundice, bilirubin, microfibrin clots, cellular debris, blood cells, fibrinogen, drugs, metabolites, supplements, herbs, and multivitamins. In some embodiments, the methods described herein provide methods for eliminating interference due to matrix effects or differences in sample type (e.g., animal species, human species). In some embodiments, the methods described herein provide methods for eliminating interference before a diagnostic test (e.g., a diagnostic or biomarker test in a clinical trial). In some embodiments, the biomarker elimination methods described herein are used in clinical trials to improve the accuracy and reliability of diagnostic tests of the biomarkers described herein. For example, the methods described herein can be used for patient selection or screening, for example, for selection or exclusion criteria. In some embodiments, outliers in clinical data or clinical trial results can be identified using the methods described herein or the elimination or depletion methods. For example, outliers in clinical data or clinical trial results include the identification of false positives or false negatives of the biomarkers described herein.

[0095] Depletion is defined as complete when a sufficient amount of interference is captured and / or removed for subsequent quantitative, semi-quantitative, or qualitative analysis without or with reduced interference. Depletion is defined as partial when a sufficient amount of interference(s) or interference mechanism(s) is captured and / or removed for subsequent semi-quantitative or qualitative analysis, or when a sufficient amount of interference(s) or interference mechanism(s) and internal standard(s) is captured and / or removed for subsequent semi-quantitative or qualitative analysis, or for LC-MS and LC-MS / MS (i.e., deuterated internal standard) and HPLC (C14 or Partial is also defined as being captured for quantitative, semi-quantitative, or qualitative analysis by a measurement method that can use an internal standard to coordinate the recovery of target analytes (or biomarkers) such as tritiated internal radioisotopes (internal standards).

[0096] Depletion does not mean 100% removal of interference from the sample, but rather that residual interference will no longer lead to erroneous results. However, pre-treatment depletion of a sample can result in 100% removal of interference when required for specific assays or purposes, such as subsequent elution and analysis by LC-MS / MS, or for sample pretreatment, nucleic acid purification and concentration for molecular diagnostics, or for biomarker enrichment from difficult sample types such as urine, saliva, and stool.

[0097] In some embodiments, the methods described herein are carried out in a period of 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less. In some embodiments, the methods described herein are carried out in a period of less than 1 day.

[0098] interference The methods provided herein reduce, minimize, or eliminate interference in biological samples. Interference is a substance present in a patient sample that can alter the precise value of a diagnostic test result by interfering with antibody binding, for example, or increase or decrease the assay signal through cross-linking, steric hindrance, or autoantibody mechanisms. As used herein, “interference” includes blood, plasma, serum, CFS, urine, feces, saliva, semen, amniotic fluid, or immunoglobulins (IgG, IgM, IgA, IgE, IgD), proteins, antigens, lipids, triglycerides, cellular components, foreign substances, chemicals, drugs, drug metabolites, supplements, vitamins, herbs, and foreign bodies (viruses). This refers to bacteria (Gram-positive, Gram-negative), fungi, yeasts, and any foreign matter, food or food-related waste, or any endogenous or exogenous substance or combination of endogenous and / or exogenous substances in other bodily fluids or sample matrices that may interfere with the test and result in false test results through specific or nonspecific interactions with the test material, formulation, biological and synthetic components, test design, and / or test format. Interferences may include, but are not limited to, autoantibodies, rheumatoid factor (RF), human anti-mouse antibodies (HAMA), human anti-animal antibodies (HAAA), polyclonal and / or monoclonal antibodies of goats, rabbits, sheep, cattle, mice, horses, pigs, and donkeys, as well as assay-specific interferences used in the test design or assay formulation, such as chemiluminescent substrates (luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, ruthenium, acridinium esters), fluorescent labels such as fluorescein or other fluorophores and dyes, and capture moieties (streptavidin, neutraavidin, avidin, C aptAvidin, PolyA, PolyDT, aptamers, antibodies, Fab, F(ab')2, antibody fragments, recombinant proteins, enzymes, proteins, biomolecules, polymers) and their binding partners (i.e., biotin, fluorescein, Po1yDT, PolyA, antigens, etc.), conjugation linkers (LC, LC-LC, PEO, PEO), bovine serum albumin, human serum albumin, ovalbumin, gelatin, purified poly and monoclonal IgG, e.g., mouse, goat, sheep, and rabbit, polyvinyl alcohol (PAA), polyvinylpyrrolidone (PVP), Tween®-20, Tween®-80, Triton® Triblock copolymers such as X-100, Pluronic®, and Tetronic, as well as commercially available blockers, blocking proteins, and polymer-based blocking reagents from Surmodics and Scantibodies, are used in antibody-based diagnostic tests, non-antibody-based diagnostic tests, or mass spectrometry (i.e., HPLC, MS, LC-MS, LC-MS / M). S) Typically used in the design of sample preparation methods and devices for subsequent analysis using radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), chemiluminescence immunoassays (CLIA), molecular diagnostics, lateral flow, point-of-care (PoC), CLIA and CLIA exclusion tests, and devices.

[0099] In one embodiment, a method for removing interference (e.g., biotin) from a biological sample includes providing particles derivatized at a capture portion (bound to inference). A method for doing so is provided herein. In some embodiments, the interfering agent is biotin.

[0100] In another embodiment, a sample can be pretreated with particles (e.g., nanoparticles, fine particles) to deplete sex hormone-binding globulin (SHBG) or sex steroid-binding globulin (SSBG) from serum or plasma, and the SHBG-depleted sample can then be tested to measure free or bioavailable hormones or steroids (i.e., free testosterone). In some embodiments, the interference is sex hormone-binding globulin (SHBG) or sex steroid-binding globulin (SSBG).

[0101] In some embodiments, the interference is biotin, HAMA, RF, heterophile, or anti-SAv.

[0102] Methods for the removal or enrichment of biomarkers Methods for enriching or increasing the concentration of biomarkers in biological samples are described herein. "Enrichment" refers to complete or partial particle capture and the enrichment of biological samples (e.g., human or animal serum, plasma, blood, whole blood, processed blood, urine, saliva, feces (liquid and solid), semen or semen fluid, cells, tissues, biopsy material, DNA, RN) Enrichment is defined as the binding of a target analyte(s) or biomarker (which may be derived from a liquid or solid) to particles. In some embodiments, enrichment includes washing and concentrating a biological sample, for example, by washing biomarker-specific nanoparticles and then isolating them to remove or minimize interference before biomarker characterization and measurement steps.

[0103] In some embodiments, the methods described herein are used to isolate and purify specific targets (e.g., biomarkers) in biological samples for subsequent elution and testing or other use, or to enrich or increase the concentration of biomarkers before diagnostic testing, further purification, formulation or other use.

[0104] After washing or isolating biomarker-specific particles, they can be dispersed, reconstituted, or resuspended in a buffer such as phosphate-buffered saline (i.e., PBS pH 7.2) or LC-MS / MS compatible buffer before the characterization or measurement step. This means that the critical characterization or measurement step of the biomarker captured and enriched by the particles is performed in the buffer system, not in the animal or human matrix, where matrix effects or biases are introduced or caused between the same biomarker measured in animal blood, plasma, serum, or urine compared to the same biomarker measured in blood, plasma, serum, or urine using the same characterization, measurement, or test method or system. Washing allows for the washing away of sample matrix, components, proteins, and cellular components, as well as associated interferences or matrix effects. Similarly, isolated biomarkers can be removed from the animal or human matrix and released into formulation buffers for therapeutic or prophylactic use.

[0105] Enrichment is defined as complete when a sufficient amount of analyte(s) is captured for subsequent diagnostic testing, such as quantitative, semi-quantitative, or qualitative analysis. If a target analyte(or biomarker) is captured for subsequent semi-quantitative or qualitative analysis, it is defined as partial; or if a sufficient amount of target analyte(or biomarker) and internal standard(or internal standard) is captured for quantitative, semi-quantitative or qualitative analysis by an assay method that can use the internal standard to prepare for the recovery of the target analyte(or biomarker) or biomarker, such as LC-MS and LC-MS / MS (i.e., deuterated internal standard) and HPLC (C14 or tritiated internal radioisotope internal standard). If a sufficient amount of captured species is obtained for subsequent use in prophylactic or therapeutic products, enrichment is defined as preparative.

[0106] A method for enriching a sample with biomarkers before a diagnostic test is provided herein, comprising: a) adding particles (e.g., nanoparticles, microparticles) to the sample; b) mixing the sample with the particles (e.g., nanoparticles, microparticles); c) incubating the particles (e.g., nanoparticles, microparticles) with the sample to bind and capture biomarkers to the particles (e.g., nanoparticles, microparticles); d) separating or removing the particles (e.g., nanoparticles, microparticles) from the sample; e) storing the particles (e.g., nanoparticles, microparticles); f) washing the particles (e.g., nanoparticles, microparticles) with a suitable washing diluent to remove nonspecific substances; and g) measuring the amount, mass, volume molar concentration, concentration, or yield of biomarkers captured by the particles (e.g., nanoparticles, microparticles) using a qualitative, semi-quantitative, or quantitative diagnostic test specific to the biomarkers. In some embodiments, the diluent includes water (e.g., deionized water, water for injection, saline solution, buffered aqueous solution).

[0107] In some embodiments, the enrichment method described herein includes a washing step. The washing step removes the interference described herein and / or provides the desired washed, purified, or isolated biomarker (e.g., the biomarker described herein). In some embodiments, the enrichment method described herein reduces matrix effects or species effects. In some embodiments, the enrichment method described herein is used before a diagnostic test comparing two biological samples of different origins. In some embodiments, the enrichment method described herein is used before a diagnostic test comparing an animal sample and a human sample. In some embodiments, the enrichment method described herein is used before a diagnostic test comparing a serum sample and a plasma sample. In some embodiments, the enrichment method described herein is used for high-viscosity samples.

[0108] In some embodiments, the enrichment method includes combining a first biological sample enriched with a biomarker with a second biological sample enriched with a biomarker.

[0109] Provided herein are methods for measuring the amount, mass, volumetric molar concentration, concentration, or yield of a target biomarker captured and enriched by particles (e.g., nanoparticles, microparticles), thereby eluting the biomarker by cleavage reagents described herein, for example, pH (e.g., increasing pH with a base such as sodium bicarbonate, decreasing pH with an acid such as acetic acid, trichloroacetic acid, sulfosalicylic acid, HCl, formic acid, common pH elution buffers such as 100 mM glycine·HCl, pH 2.5-3.0, 100 mM citrate, pH 3.0, 50-100 mM triethylamine or triethanolamine, pH 11.5, 150 mM ammonium hydroxide, pH 10.5), displacers or displacing agents, competitive elution (e.g., counterligands or analogs greater than 0.1 M), Ionic strength and / or chaotropic effects (e.g., NaCl, KCl, 3.5-4.0 M magnesium chloride in 10 mM Tris pH 7.0, 5 M lithium chloride in 10 mM phosphate buffer pH 7.2, 2.5 M sodium iodide pH 7.5, 0.2-3.0 M sodium thiocyanate), surfactants, detergents, concentrated inorganic salts, denaturations (e.g., 2-6 M guanidine HCl, 2-8 M urea, 1% deoxycholic acid, 1% SDS), organic solvents ( For example, particles (e.g., nanoparticles, microparticles) are eluted, dissociated, or released by disrupting binding interactions using elution strategies such as alcohol, chloroform, ethanol, methanol, acetonitrile, hexane, DMSO, 10% dioxane, 50% ethylene glycol (pH 8-11.5, also 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.

[0110] In some embodiments, 100 mM glycine, pH 2.5 is used as the elution buffer to release the captured IgA, IgG, and / or IgM antibodies, which are complexed with a complexed anti-IgA, IgG, and / or IgM detection antibody (e.g., AlexaFluor488 anti-human IgG, AlexaFluor555 anti-human IgM, AlexaFluor647 anti-human IgA) or a labeled detection antibody, from the capture beads. Subsequently, the magnetic beads are isolated by ferromagnetism, and the eluate is then transferred to a new well with a neutralization buffer, e.g., 300 mM Tris pH 10.0, to neutralize the pH and improve the stability of the fluorophores for subsequent detection by fluorophotometer or fluorescence reader. This neutralization of the acidic elution pH may be important to improve the accuracy and reproducibility of the assay.

[0111] Unless otherwise specified or implied by this disclosure, any embodiment described herein in relation to any particular method or composition may be used in combination with any other embodiment described herein.

[0112] The methods and compositions of various embodiments include any suitable assay known in the art, 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, CLIA or CLIA-exempt tests (CLIA waved tests), LC-MS / MS, analytical assays, etc. These can be used in combination with any suitable affinity assay or immunoassay known in the art, but are not limited to these.

[0113] A method for both depleting sample interference and enriching biomarkers from the same sample before a diagnostic test, comprising: a) adding a chemical and / or biological reagent, additive or composition to the sample to block or deplete sample-specific interference before adding biomarker-specific particles (e.g., nanoparticles, microparticles) to the sample; b) adding biomarker-specific particles (e.g., nanoparticles, microparticles) to the sample after pretreatment or incubation with the sample with the chemical and / or biological reagent, additive or composition; c) incubating the biomarker-specific particles (e.g., nanoparticles, microparticles) with the sample to bind and capture the target biomarker(s) to the particles (e.g., nanoparticles, microparticles); d) washing the particles (e.g., nanoparticles, microparticles) or isolating the particles from the sample and the chemical and / or biological reagent, additive or composition; and e) characterizing the biomarker(s) captured and enriched by the particles (e.g., nanoparticles, microparticles) using a diagnostic test.

[0114] For example, in one embodiment, particles bound to CaptAvidin will bind to biotin in the sample at a neutral pH. The biotin bound to the CaptAvidin particles will release when the pH rises to 10.

[0115] biomarkers Methods for isolating, or isolating or enriching, biomarkers present in biological samples are described herein. “Biomarkers” as used herein are defined as characteristic biological or biologically derived indicators (e.g., metabolites) of a process, event, or state, such as aging or disease. Biomarkers may be endogenous and / or exogenous analytes, antigens, small molecules, large molecules, drugs, therapeutic agents, metabolites, xenobiotics, chemicals, peptides, proteins, protein digests, viral antigens, bacteria, cells, cell lysates, cell surface markers, epitopes, antibodies, antibody fragments, IgG, IgM, IgA, IgE, IgD receptors, receptor ligands, hormones, hormone receptors, enzymes, enzyme substrates, single-stranded oligonucleotides, single-stranded polynucleotides, double-stranded oligonucleotides, double-stranded polynucleotides, polymers, and aptamers. In some embodiments, the biomarker is an interference as described herein (a substance present in a patient sample that can alter the precise value of a diagnostic test result by interfering with antibody binding, for example, or increase or decrease the assay signal by crosslinking, steric hindrance, or autoantibody mechanisms). In some embodiments, the biomarker is an antibody against an infectious disease antigen, such as a viral antigen. Antibodies against infectious disease antigens can indicate exposure to infection by an infectious pathogen and recovery from said infection. In such cases, the antibody can be used for passive immunization for therapeutic or prophylactic purposes. In some embodiments, the antibody against an infectious disease antigen recognizes the spike protein of the SARS-CoV-2 spike protein, such as the S1 subunit, or its receptor-binding domain and / or N-terminal domain.As used herein, “interference” may include, but is not limited to, autoantibodies, rheumatoid factor (RF), human anti-mouse antibodies (HAMA), human anti-animal antibodies (HAAA), polyclonal and / or monoclonal antibodies of goats, rabbits, sheep, cattle, mice, horses, pigs, and donkeys, as well as assay-specific interferences used in the test design or assay formulation, such as chemiluminescent substrates (luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, ruthenium, acridinium esters), fluorescent labels such as fluorescein or other fluorophores and dyes, and capture moieties (streptavidin, neutra). Avidin, avidin, poly(A), poly(DT), aptamers, antibodies, Fab, F(ab')2, antibody fragments, recombinant proteins, enzymes, proteins, biomolecules, polymers) and their binding partners (i.e., biotin, fluorescein, Po1yDT, PolyA, antigens, etc.), conjugation linkers (LC, LC-LC, PEO, PEO), bovine serum albumin, human serum albumin, ovalbumin, gelatin, purified poly and monoclonal IgG, e.g., mouse, goat, sheep, and rabbit, polyvinyl alcohol (PAA), polyvinylpyrrolidone (PVP), Tween®-20, Tween®-80, Triton® Triblock copolymers such as X-100, Pluronic®, and Tetronic, as well as commercially available blockers, blocking proteins, and polymer-based blocking reagents such as those from Surmodics and Scantibodies, are typically used in the design of sample preparation methods and devices for antibody-based diagnostic tests, non-antibody-based diagnostic tests, or subsequent analysis by mass spectrometry (i.e., HPLC, MS, LC-MS, LC-MS / MS), radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), chemiluminescence immunoassays (CLIA), molecular diagnostics, lateral flow, point-of-care (PoC), CLIA and CLIA exclusion tests, and devices. In some embodiments, biomarkers are found in the biological samples described herein.

[0116] Fibrinogen. Fibrinogen is converted to fibrin by thrombin during tissue and blood vessel injury, subsequently leading to the formation of fibrin-based clots. In some embodiments, the particles described herein used in the method herein (e.g., particle-induced antifibrinogen (e.g., mouse antifibrinogen)) are in whole blood It binds to fibrinogen, enabling the separation of fibrinogen (e.g., chemical separation). Particles that bind to the clot via fibrin can be isolated and removed from the serum after centrifugation for a serological test that does not contain particles. In some embodiments, the biomarker is fibrinogen. In some embodiments, the method described herein uses particle-derived anti-fibrinogen to eliminate the need for centrifugation of the sample (e.g., blood sample).

[0117] Traumatic brain injury. In one embodiment, the biomarkers relate to traumatic brain injury. There are nine biomarkers associated with the severity and size of acute brain injury and the integrity of the blood-brain barrier (BBB), but they are present at very low circulating concentrations in the blood and are extremely difficult to detect and quantify using existing immunoassay technologies and test platforms. The Banyan BTI test (FDA approved February 14, 2018) measures only two of these biomarkers, but the methods and devices described herein (e.g., enrichment method; enrichment device) enable the simultaneous measurement of all nine biomarkers in a patient, aiding in the diagnosis and prognosis of most patients. Particles derivatized with a capture portion for each of the nine biomarkers can be added to a biological sample from a patient suspected of having TBI. In some embodiments, the traumatic brain injury biomarkers are selected from the group consisting of S100B, GFAP, NLF, NFH, γ-enolase (NSE), α-II spectrin, UCH-L1, total tau, and phosphorylated tau. In some embodiments, the traumatic brain injury biomarker is selected from GFAP and UCH-L1.

[0118] In some embodiments, the presence of 1, 2, 3, 4, 5, 6, 7, 8, or 9 traumatic brain injury biomarkers selected from the group consisting of S100B, GFAP, NLF, NFH, γ-enolase (NSE), α-II spectrin, UCH-L1, total tau, and phosphorylated tau is isolated or enriched using the methods described herein (e.g., enrichment methods).

[0119] Alzheimer's disease. In one embodiment, the biomarker is related to Alzheimer's disease. There are two biomarkers related to the severity and scale of Alzheimer's disease. In some embodiments, the Alzheimer's disease biomarker is selected from the group consisting of amyloid beta, BACE1, and soluble Aβ precursor protein (sAPP). In some embodiments, the Alzheimer's disease biomarker is selected from the group consisting of β-amyloid (1-42), phospho-tau (181p), and total tau. In some embodiments, the presence of one, two, or three Alzheimer's disease biomarkers selected from the group consisting of amyloid beta, BACE1, and soluble Aβ precursor protein (sAPP) is isolated or enriched using a method described herein (e.g., enrichment method). In some embodiments, the biomarker is amyloid beta, BACE1, or soluble Aβ precursor protein (sAPP). In some embodiments, the Alzheimer's disease biomarker is found in a biological sample (e.g., CSF).

[0120] Sexually Transmitted Infections. In one embodiment, the biomarkers are related to sexually transmitted infections (STDs). There are at least 10 biomarkers characteristic of STD transmission. In some embodiments, the STD biomarkers are biomarkers for Chlamydia, Gonorrhea, Syphilis, Trichomonas, HPV, Herpes 1 and 2, HSV, Hepatitis A, Hepatitis B, Hepatitis C, HIV 1 and 2. In some embodiments, using the methods described herein (e.g., enrichment methods), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 STD biomarkers: Chlamydia, Gonorrhea, Syphilis, Trichomonas, HPV, Herpes 1 and 2, HSV, Hepatitis A, Hepatitis B, Hepatitis C, HIV 1 and 2, and The presence of HIV antibodies is isolated or enriched. In some embodiments, the biomarker is present in urine (e.g., Chlamydia, gonorrhea, Trichomonas). In some embodiments, the biomarker is present in blood, serum, or plasma (e.g., syphilis, HPV, herpes simplex virus 1 and 2, HSV, hepatitis A, hepatitis B, hepatitis C, HIV 1 and 2, HIV antibodies).

[0121] Bacterial infection. In one embodiment, the biomarker is related to a bacterial infection, e.g., sepsis. The current gold standard test for bacterial infection is a blood culture, which can take 24-48 hours before a positive result can be reflected in confirmatory tests such as molecular diagnostics. When time is critical to successfully treat a patient to prevent or manage sepsis, there is a rule-in / rule-out method for ruling out bacterial infection in as little as 30 minutes or less, e.g., 60 minutes or less (e.g., 50 minutes, 40 minutes, 30 minutes, 20 minutes or less). The method is described herein. There are at least 30 biomarkers characteristic of bacterial infections. In some embodiments, the bacterial biomarker is selected from the group consisting of biomarkers of bacterial species that cause sepsis (e.g., Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus). In some embodiments, the biomarker is a biomarker for Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. In some embodiments, the biomarker is a biomarker for Gram-positive or Gram-negative bacteria. In some embodiments, the biomarker is a biomarker for yeast pathogens (e.g., yeast pathogens associated with bloodstream pathogens).

[0122] In some embodiments, the Gram-positive bacteria are Enterococcus, Listeria monocytogenes, Staphylococcus aureus, Streptococcus, Streptococcus agalactiae, Streptococcus pneumoniae, or Streptococcus pyogenes.

[0123] In some embodiments, Gram-negative bacteria include Acinetobacter baumannii, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Enterobacteriaceae, Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, or Serratia. It is marcescens.

[0124] In some embodiments, the yeast pathogens are Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis.

[0125] In some embodiments, mass spectrometry follows. A method is envisioned in which a cleaving agent (e.g., a reducing agent (e.g., DTT or TCEP)) is added to the bacterial-particle binding complex to cleave the linker (i.e., the linker that conjugates the particle to the surface-trapping portion). The resulting bacteria are grown in culture or analyzed by MALDI-TOF mass spectrometry.

[0126] A cleavage agent (e.g., a reducing agent (e.g., DTT or TCEP)) is used in the bacterial-particle binding complex. One proposed method involves adding the substance to cleave the linker (i.e., the linker that conjugates the particle to the surface trapping portion). The resulting bacteria are then grown in culture, analyzed by MALDI-TOF mass spectrometry, or analyzed by molecular diagnostics such as the FilmArray® Blood Culture Identification (BCID) panel by BioFire Diagnostics.

[0127] Pathogen-specific antibodies. In some embodiments, the biomarker is an antibody that recognizes a pathogen, particularly a structural or surface-exposed antigen of the pathogen. A pathogen-specific antigen is used as the capture site on magnetic particles. Whole blood, blood fraction, plasma, or serum is mixed with magnetic particles so that the antigen-specific antibody can bind to the capture site (pathogen-specific antigen). The particles are magnetically separated from the biological fluid. The magnetic particles are then reacted in a release buffer to elute the antibody from the capture site. This can be done on an analytical scale, and the antibody can be subjected to mass spectrometry (e.g., LC-MS for separating multiple species of possible antibodies), Edman degradation, and other protein chemistry analytical methods to determine the protein sequence of the antibody. Using the sequence information, monoclonal antibodies (paratopes) with the same specificity (one or more) can be constructed. This can also be done on a preparative scale, and the enriched or isolated antigen-specific antibodies are used in a clinical setting for treatment or prevention. In some embodiments, the antibody that recognizes the pathogen-specific antigen recognizes the spike protein of the SARS-CoV-2 spike protein, for example, the S1 subunit, or its receptor-binding domain and / or N-terminal domain and / or receptor-binding domain.

[0128] Thyroid function. TSH levels are measured as part of thyroid function tests in patients suspected of having an excess (hyperthyroidism) or deficiency (hypothyroidism) of thyroid hormones. Methods described herein in some embodiments are used to assess thyroid function. In some embodiments, the biomarker is an antigen (e.g., TSH). In some embodiments, the capture portion is an autoantibody (e.g., free autoantibody, complexed autoantibody) that has specificity for the antigen (e.g., TSH).

[0129] In some embodiments, the interfering factors (which affect the measurement of thyroid-stimulating hormone, free thyroxine, and free triiodothyronine) are macro-TSH, biotin, anti-streptavidin antibodies, anti-ruthenium antibodies, thyroid hormone autoantibodies, or heterophile antibodies.

[0130] Cardiac function. Methods described herein in some embodiments are used to assess cardiac function. Elevated levels of troponin circulating in the blood are a biomarker of cardiac disorder, e.g., myocardial infarction. Cardiac I and T are specific indicators of myocardial injury. Subunits of troponin are also markers of cardiac health. Specifically, cTnI and cTnT are biomarkers of acute myocardial infarction (AMI), e.g., type 1 and type 2 myocardial infarction, unstable angina, postoperative myocardial trauma, and related diseases. In some embodiments, the biomarkers are free cTnI, free cTnT, binary cTnI-TnC, or ternary cTnI-TnC-TnT. In some embodiments, the biomarkers are indicators of heart failure. In some embodiments, the biomarker is an indicator of stroke (for example, as described in https: / / www.ahajournals.org / doi / 10.1161 / STROKEAHA.117.017076 and https: / / www.360dx.com / business-news / roche-test-helps-differentiate-bleeding-risk-stroke-risk-patients-considering#.W1jz0thKhcA, which are incorporated in their entirety by reference). In some embodiments, the biomarker is an indicator of fibrosis (for example, as described in http: / / www. (As described at onlinejacc.org / content / 65 / 22 / 2449). In some embodiments, the biomarkers are for the diagnosis of acute coronary syndrome (ACS). In some embodiments, the biomarkers relate to cardiac troponins (I, IC, ICT, T) and other cardiac troponin fragments, natriuretic peptides (BNP, ANP, CNP), N-terminal fragments (i.e., NT-proBNP, NT-proCNP), glycosylated, unglycosylated, CRP, myoglobin, creatinine kinase (CK), CK-MB, sST2, GDF-15, and galectin-3.

[0131] In some embodiments, the accuracy and precision of testing large sample volumes (i.e., 1 mL, 10 mL, 100 mL, 1000 mL, etc.) is improved to enhance the accuracy of detecting very dilute or low-concentration biomarkers (multiple) that are typically impossible to test today or require dilution of the sample before testing (which impairs the sensitivity, precision, and accuracy of the test), as well as very small sample volumes (i.e., neonates, children, the elderly). In some embodiments, the biological sample is a volume of 1 mL, 10 mL, 100 mL, 1000 mL, or more. In some embodiments, the biological sample is a volume of 0.5 mL, 0.25 mL, 0.1 mL, 0.05 mL, or less.

[0132] Furthermore, this specification also provides a method or test method that uses particle sample pretreatment to assist in enriching biomarkers before a diagnostic test by enabling a washing step or particle isolation and subsequent selective release or elution of the captured biomarker(s), or a selective release or elution of the captured portion-biomarker complex from the particles before a biomarker characterization step.

[0133] The use of "cleavage reagents" or "release agents" to break the bond between the capture portion on the particle surface and the biomarker, such as acidic or basic pH, high volume molar concentration salts, sugars, chemical displacers, detergents, surfactants, and / or chelating agents, or combinations thereof, by removing only the biomarker without replacing or eluting the capture portion. After washing or isolating particles from the sample matrix using a magnet(s), the particles can then be treated with an elution solution containing a release agent(s) to selectively release the biomarker and / or labeled detection reagent into the solution. The particles can be rapidly isolated (less than 2 minutes; ideally less than 30 seconds) to the sides(s) and / or bottom of the sample device (vial, test tube, etc.) to form a sample supernatant that is essentially particle-free. Without destroying the pellet containing the particles, the particle-free supernatant can then be aspirated and dispensed into a separate transfer tube, or injected directly into an analytical system (i.e., LC-MS / MS or MALDI-TOF) to test the biomarker. In some embodiments, the supernatant containing the eluted components is transferred to a neutralizing buffer to re-establish less harsh conditions (such as pH) and preserve the biomarker and / or label from degradation or denaturation by the elution solution. For example, the cleavage reagents or release agents described herein include, for example, pH (e.g., raising pH with bases such as sodium bicarbonate, lowering pH with acids such as acetic acid, trichloroacetic acid, sulfosalicylic acid, HCl, formic acid, and general pH elution buffers, e.g., 100 mM glycine·HCl, pH 2.5-3.0; 100 mM citric acid, pH 3.0; 50-100 mM triethylamine or triethanolamine, pH 11.5; 150 mM ammonium hydroxide, pH 10.5), displacers or substitution agents, competitive elution (e.g., counterligands or analogs greater than 0.1 M), ionic strength and / or chaotropic effects (e.g., NaCl, KCl, 3.5-4.0 M magnesium chloride in 10 mM Tris, pH 7.0; 5 M lithium chloride in 10 mM phosphate buffer, pH 7.2; 2.5 M sodium iodide, pH 7.5; 0.2-3.0 M sodium thiocyanate), surfactants, and detergents. Dissolution strategies such as concentrated inorganic salts, denaturation (e.g., 2-6M guanidine HCl, 2-8M urea, 1% deoxycholic acid, 1% SDS), organic solvents (e.g., alcohol, chloroform, ethanol, methanol, acetonitrile, hexane, DMSO, 10% dioxane, 50% ethylene glycol pH 8-11.5 (also 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, are used to break the specified binding interactions or cleavable bonds between the particles and the capture portions described herein. In some embodiments, the supernatant containing the eluted components is transferred to a neutralizing buffer to re-establish less harsh conditions (such as pH) and preserve the biomarker and / or label from degradation or denaturation by the elution solution.

[0134] Characterization Method Methods for depleting and / or enriching biomarkers for subsequent characterization or diagnostic testing are described herein. Characterization of biomarkers (e.g., interferences) described herein includes identification and / or quantification of biomarkers (e.g., interferences) described herein.

[0135] Characterization may include the detection and / or quantification of biomarkers, such as antigen-specific antibodies. By conjugating antigen-specific antibodies to particles, they can be isolated from other specificities, released into a smaller volume than the original sample, and concentrated as needed. Typical diagnostic assays for specific antibodies, for example in relation to whole serum, detect them without first isolating the antibodies. This makes absolute quantification difficult. Antibody activity is often characterized as titer based on how much the serum can be diluted, but still retains activity. The amount of specific antibody present can be quantified using a simple protein assay by capturing the species of interest and separating it from the rest of the immunoglobulin in the serum. Furthermore, each isotype of interest can be quantified separately in parallel aliquots or, if conjugated to separate labels, in single aliquots, by multiplexing with a standard curve generated using a known amount of bead-conjugated immunoglobulin using isotype-specific reagents. The standard curve may generally be for immunoglobulin or for specific isotypes, the latter of which may be generated separately or in multiplexed form. IgM is typical of the initial response, while IgG and IgA are typical of a more mature and effective immune response. This easy absolute quantification allows for direct comparison of the amount of antigen-specific antibodies in serum from one serum sample to the next. In some embodiments, the antigen-specific antibody recognizes the SARS-CoV-2 S1 subunit, or its receptor-binding domain and / or N-terminal domain and / or receptor-binding domain.

[0136] Particles of the present invention Particles for the isolation, depletion, and / or enrichment of biological samples are described herein. In some embodiments, the particles include cleavable binding and capture moieties (e.g., a particle surface functionalized to present one capture moiety). In some embodiments, the particles include non-cleavable binding and capture moieties (e.g., a particle surface functionalized to present one capture moiety). In some embodiments, the particles described herein include capture moieties (e.g., capture moieties having high specificity for the biomarkers described herein). In some embodiments, the particles described herein (e.g., the surface of the particles described herein, the particle surface not bound to the capture moieties described herein) are inert (e.g., showing significant binding to the biomarkers described herein). (None). In some embodiments, the particles described herein can be used in the diagnostic tests described herein without further modification of the particles or the diagnostic tests. In some embodiments, the particles described herein can be added to and removed from a sample without altering the sample (for example, without adding or removing additional biomarkers (e.g., interferences)).

[0137] The particles described herein are sufficiently small, with an average diameter of 0.050 micrometers to a maximum of 3.00 micrometers, preferably 0.100 micrometers to 1.1 micrometers, more preferably 0.200 micrometers to 0.600 micrometers, or even more preferably 0.100 micrometers to 0.500 micrometers.

[0138] In some embodiments, the particles described herein (e.g., fine particles, nanoparticles) comprise a core or support, the core or support being a paramagnetic or superparamagnetic material selected from the group consisting of iron oxide, ferromagnetic iron oxide, Fe2O3 and Fe3O4, maghemite, or combinations thereof.

[0139] In some embodiments, the particle surface comprises an organic polymer or organic copolymer, and the organic polymer or organic copolymer is hydrophobic. In some embodiments, the surface of the particles (e.g., nanoparticles, fine particles) includes, but is not limited to, organic polymers or copolymers such as ceramics, glass, polymers, copolymers, metals, latex, silica, gold, silver, or colloidal metals such as alloys, 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 materials selected from the group consisting of these copolymers, organic polymers or copolymers, silicone or silica, methylolmelamine, dextran or poly(ethylene glycol)-dextran (PEG-DEX), or combinations thereof.

[0140] As used herein, “blocker” refers to a protein, polymer, surfactant, detergent, or combination thereof. In some embodiments, the binding of the capture portion on the particles described herein (e.g., nanoparticles, fine particles) 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, gamma 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 and (PEO)n-maleimide, triblock copolymers such as Pluronic® F108, F127, F68, Triton® X-100, Polysorbate 20 (Tween®-20), Tween® Nonionic detergents such as 80 (nonionic), amphoteric detergents such as CHAPS, ionic detergents such as sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarcosyl surfactants, sugars such as sucrose, heterophilic blocking reagents (Scantibodies), MAK33 (Roche Diagnostics), immunoglobulin inhibitors (IIR) (Bioreclamation), Heteroblock (Omega Biologicals), Blockmaster (J The blockers are selected from the group consisting of commercially available blockers such as SR), TRU Block (Meridian Life Sciences), and StabilCoat® & StabilGuard® (Surmodics). In some embodiments, the blockers are bound (e.g., covalently, non-covalently) to the particles described herein. In some embodiments, the blockers are not bound (e.g., covalently, non-covalently) to the particles described herein.

[0141] Cleavable bonds. In one embodiment, the capture portion binds to a biomarker by a cleavable bond as described herein. The cleavable bond may be covalent or non-covalent. Examples of non-covalent bonds include affinity, ionicity, van der Waals (e.g., dipole / dipole or London force), hydrogen bonds (e.g., between polynucleotide double strands), and hydrophobic interactions. If the association 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, such as avidin, captavidin, SA, neutraavidin, fragments of SA, fragments of avidin, fragments of neutraavidin, or mixtures thereof; binding of biotinylated Fab, biotinylated immunoglobulins or fragments thereof, biotinylated small molecules (e.g., ligands for hormones or receptors), biotinylated polynucleotides, biotinylated macromolecules (e.g., proteins or natural or synthetic polymers) to biotin-binding proteins such as avidin, SA, neutraavidin, fragments of SA, fragments of avidin, fragments of neutraavidin, or mixtures thereof; binding of substrates to their 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 complementary or substantially complementary polynucleotides; and so on.

[0142] A cleavable bond, such as a disulfide bond (RSSR), is used to immobilize or bind the capture portion (i.e., antibody or antibody fragment, e.g., SH-Fab) to the particle. After washing or isolating the particle from the sample matrix, the particle 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. The particle can be rapidly isolated (less than 2 minutes; ideally less than 30 seconds) to the side(s) and / or bottom of the sample device (vial, test tube, etc.) to form a sample supernatant that is essentially particle-free. The particle-free supernatant can then be aspirated without destroying the pellet containing the particle and dispensed into a separate transfer tube, or injected directly into an analytical system (i.e., LC-MS / MS or MALDI-TOF, or molecular diagnostics such as a FilmArray blood culture identification panel) to test the capture portion-biomarker complex.

[0143] In some embodiments, the cleavable bond is a disulfide bond (RSSR).

[0144] In some embodiments, the cleavable bond is a non-covalent bond between streptavidin or captoavidin, avidin, and biotin.

[0145] Capture portion. A capture portion that binds to an interference as described herein, or a particle containing a biomarker as described herein, is provided herein. Where referred to herein, “capture portion” is selected from the group consisting of antibodies, antibody binding fragments, IgG, IgM, IgA, IgE, IgD receptors, receptor ligands, hormones, hormone receptors, enzymes, enzyme substrates, single-stranded oligonucleotides, single-stranded polynucleotides, double-stranded oligonucleotides, double-stranded polynucleotides, antigens, peptides, polymers, aptamers, and proteins.

[0146] In some embodiments, the capture portion is a protein. The protein may be, for example, a monomer, dimer, polymer, or fusion protein. In certain embodiments, the protein includes at least one of the following: albumin, e.g., antibodies, antibody fragments, BSA, ovalbumin, BSA fragments, ovalbumin fragments, mouse IgG, polymerized mouse IgG, antibody fragments of mouse IgG targeting HAMA and RF interference mechanisms (Fc, Fab, F(ab')2) and different subclasses (IgG1, IgG2a, IgG2b, IgG3, IgE, IgD), purified animal polyclonal antibodies targeting HAAA interference (i.e., bovine, goat, mouse, rabbit, sheep), streptavidin targeting MASI interference, ALP, HRP, BSA (conjugated with isolminol, ruthenium, acridinium), or mixtures thereof. In some embodiments, the capture portion is a pathogen, e.g., a structural or surface-exposed antigen of a bacterium or virus. In some embodiments, the capture portion is a viral structural protein, such as the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the spike protein is the S1 subunit, or its receptor-binding domain and / or N-terminal domain.

[0147] In some embodiments, the capture portion is a human anti-animal antibody (e.g., mouse IgG, sheep IgG, goat IgG, rabbit IgG, bovine IgG, pig IgG, horse IgG). In some embodiments, the capture portion is heterophile antibodies (e.g., FR(Fc-specific), Fab, F(ab)'2, polymerized IgG (types 1, 2a, 2b IgG and IgG fragments, serum components). In some embodiments, the capture portion is assay-specific conjugates (e.g., biotin, fluorescein, anti-fluorescein poly / Mab, anti-biotin poly / Mab, streptavidin, neutraavidin). In some embodiments, the capture portion is assay-specific signaling molecules (e.g., HRP, ALP, acridinium ester, isoluminol / luminol, ruthenium, ABEI / cyclic ABEI). In some embodiments, the capture portion is assay-specific blockers (e.g., BSA, fish skin gelatin, casein, ovalbumin, PVP, PVA). In some embodiments, the capture portion is assay-specific conjugate linkers (e.g., LC, LC-LC, PEO4, PEO16). In some embodiments, the capture moiety is an antigen autoantibody (e.g., free T3, free T4). In some embodiments, the capture moiety is a protein autoantibody (e.g., MTSH, TnI, TnT, non-cardiac TnT (skeletal muscle disease)). In some embodiments, the capture moiety is a chemiluminescent substrate (e.g., luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, ruthenium, acridinium esters) or a fluorescent label (e.g., fluorescein or other fluorophores and dyes). In some embodiments, the capture moiety is streptavidin, neutraavidin, avidin, polyA, polyDT, aptamer, antibody, Fab, F(ab')2, antibody fragment, recombinant protein, enzyme, protein, biomolecule, polymer, or molecularly imprinted polymer. In some embodiments, the capture moiety is biotin, fluorescein, Po1yDT, PolyA, antigen, etc.

[0148] In some embodiments, the capture portion binds to biotin (e.g., avidin, streptavidin, neutraavidin, CaptAvidin, anti-biotin antibodies, antibody fragments, apmers, molecular imprinted polymers, etc.).

[0149] Some embodiments provide a bonding surface having two or more different capture portions.

[0150] Generation of a capture portion. In one embodiment, a method for producing a capture portion, comprising the production of a complex-specific or conformation-specific antibody against a free autoantibody or autoantibody complex. A method including the production of such antibodies is provided. Free autoantibodies are autoantibodies that have not yet been complexed with their antigenic targets. Complexed autoantibodies are autoantibodies that have formed complexes with their antigenic targets.

[0151] In one embodiment, a method is provided for preparing a capture portion, comprising the production or generation of a complex-specific or conformation-specific antibody against an autoantibody complex such as MTSH. In some embodiments, the autoantibody is triiodothyronine (T3) or thyroxine (T4). In some embodiments, the autoantibody complex is MTSH. For example, a complex-specific or conformation-specific antibody can be made into an autoantibody complex such as MTSH, which can be purified from human serum and used as a capture portion. In this way, the antibody produced will have specificity only for the hIgG or hIgM complex with TSH. MTSH can be purified based on the art and published methods, or by those skilled in protein biochemistry and purification. In some embodiments, a patient with an autoimmune disease with the highest accuracy of autoantibody assay interference is used to produce or generate the autoantibody. For example, see the HyTest SES assays for BNP, International Publication No. 2014114780, International Publication No. 2016113719, and International Publication No. 2016113720, which cite the entire reference.

[0152] Thyroid-specific autoantibodies. For example, in one embodiment, the autoantibody is an anti-thyroid autoantibody (e.g., anti-thyroid peroxidase antibody, thyrotropin receptor antibody, thyroglobulin antibody). An anti-thyroid autoantibody is an autoantibody that targets one or more components on the thyroid gland.

[0153] In some embodiments, the autoantibody is a free autoantibody (e.g., thyrotropin (TSH)).

[0154] In some embodiments, the autoantibody is a complexed autoantibody (e.g., MTSH). In some embodiments, the capture portion described herein is an antibody produced to have specificity for the complexed autoantibody or confirmation specificity for hIgG and / or hIgM already bound to its antigenic target, such as MTSH.

[0155] The following is a non-limiting list of substances that may function as one or the other member of a binding pair consisting of an analyte binder (capture portion) and an analyte, depending on the application for which the affinity assay is designed. Such substances can, for example, be used as a capture portion (analyte binder), or can be used to generate a capture portion that can be used in various embodiments (e.g., by using them as haptens / antigens to generate specific antibodies). Affinity assays, including immunoassays, can be designed according to various embodiments to detect the presence and / or levels of such substances that are analytes in a sample. In certain embodiments, these substances can be detected as analytes in a sample using an analyte-binding capture portion. Alternatively, the substances disclosed herein can be used according to various embodiments to capture molecules (e.g., antibodies or fragments thereof that associate with and interact with a solid support surface, such as the listed substances, binding proteins, or enzymes).

[0156] An unrestricted list of substances that may function as one or the other member of a binding pair consisting of an analyte binder (capture portion) and the analyte includes: inducible nitric oxide synthase (iNOS), CA19-9, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-t, IL-5, IL-7, IL-10, IL-12, IL-13, sIL-2R, sIL-4R, sIL-6R, SIV core antigen, IL-1RA, TNF- α, IFN-γ, GM-CSF; PSA (prostate-specific antigen) isoforms such as pPSA, BPSA, etc., in PSA, non-α1-antichymotrypsin complexed PSA, α1-antichymotrypsin complexed PSA, prostatic kallikreins such as hK2, hK4, and hK15, ek-rhK2, Ala-rhK2, TWT-rhK2, Xa-rhK2, HWT-rhK2, and other kallikreins; HIV-1 p24; ferritin, L-ferritin, troponin I, BNP, leptin, digoxin, myoglobin, type B natriuretic peptide or brain natriuretic peptide (BNP), NT-proBNP, 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 gonadotropin (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 capsule F1 Antigens, insulin, alpha-fetoprotein (e.g., AFP 300), carcinoembryonic antigen (CEA), CA15.3 antigen, CA19.9 antigen, CA125 antigen, HAV Ab, HAV Igm, HBc Ab, HBc Igm, HIV1 / 2, HBsAg, HBsAb, HCV Ab, anti-p53, histamine; neopterin; s-VCAM-1, serotonin, sFas, sFas ligand, sGM-CSFR, s1CAM-1, thymidine kinase, IgE, EPO, endogenous 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 modified albumin, HDL, LDL, oxLDL, VLDL, troponin T, troponin I, troponin C, microalbumin, amylase, ALP, ALT, AST, GGT, IgA, IgG, prealbumin, anti-streptrycin, Chlamydia, CMV IgG, ToxoIgG, ToxoIgM, Apolipoprotein A, Apolipoprotein B, C3, C4, Propergin Factor B, Albumin, α1-Acid Glycoprotein, α1-Antitrypsin, α1-Microglobulin, α2-Macroglobulin, Anti-Streptoricin O, Antithrombin-III, Apolipoprotein Al, Apolipoprotein B, β2-Microglobulin, Ceruloplasmin, Complement C3, Complement C4, C-Reactive Protein, DNase B, ferritin, free kappa light chain, free lambda light chain, haptoglobin, immunoglobulin A, immunoglobulin A (CSF), immunoglobulin E, immunoglobulin G, immunoglobulin G (CSF), immunoglobulin G (urine), immunoglobulin G subclass, immunoglobulin M, immunoglobulin M (CSF), kappa light chain, lambda light chain, lipoprotein (a), microalbumin, prealbumin, propagin 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-cadherein, EZH2, and α-methylacyl-CoA racemase, TGF- Beta, 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 Contains gp41 / 120, HCV 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. dificille toxins A & B, H. pylori, HSV-1, and HSV-2.

[0157] Depending on the application in which the affinity assay is designed, it may function as one member or the other member of a binding pair consisting of the analyte binder (capture portion) and the analyte. Suitable materials that can be used in the currently disclosed embodiments include, for example, portions specific to any of the WHO International Biological Reference Preparations held, characterized, and / or distributed by the WHO International Laboratories for Biological Standards (available at http: / / www.who.int / bloodproducts / re_materials, updated June 30, 2005, which enumerates materials well known in the art, and this list is incorporated herein by reference), such as antibodies or fragments thereof.

[0158] The partial list of such appropriate international reference standards, identified in parentheses after the substance by the WHO code, 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; respectively, WHO TRS ECBS Report No. 926, 53.sup.rd The following substances are cited in the report: brain homogenate, human serum complement components C1q, C4, C5, factor B, and full-function 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 (10Set 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 concentrates (98 / 590), human carcinoembryonic antigen (73 / 601), human C-reactive protein (85 / 506), recombinant human ferritin (94 / 572), apolipoprotein B (SP3-07), beta-2-microglobulin (B2M), human beta-thromboglobulin (83 / 501), human blood coagulation factor IX concentrate (96 / 854), human blood coagulation factor IXa concentrate (97 / 562), human blood coagulation factor V Leiden, human gDNA samples: FV wild-type, FVL homozygous, FVL heterozygous (03 / 254, 03 / 260, 03 / 248), human coagulation factor VII concentrate (97 / 592), human coagulation factor VIIa concentrate (89 / 688), human anti-syphilis serum (HS), human anti-tetanus immunoglobulin (TE-3), human antithrombin 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-beta serum (G038-501-572), human anti-measles serum (66 / 202), anti-nuclear ribonucleoprotein serum (W1063), anti-nuclear factor (homogeneous) 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 group testing serum (W1005), Human anti-echinococcus serum (ECHS), Human anti-hepatitis A immunoglobulin (97 / 646), 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 α serum (B037-501-572), Hi This includes alfafetoprotein (AFP), ancrod (74 / 581), human anti-A blood group testing serum (W1001), human anti-B blood group testing serum (W1002), human anti-C complete blood group testing serum (W1004), anti-D (anti-Rh0) complete blood group testing reagent (99 / 836), human anti-D (anti-Rh0) incomplete blood group testing serum (W1006), and human anti-D immunoglobulin (01 / 572).

[0159] Depending on the application for which the affinity assay is designed, other examples of suitable substances that may function as one or the other member of a binding pair consisting of the analyte conjugate (capture portion) and the analyte include compounds that can be used as haptens to produce antibodies capable of recognizing the compound, including but not limited to hormones such as progesterone, estrogen and testosterone, progestin, corticosteroid and dehydroepisterone, as well as any salts, esters or ethers of any non-protein / non-polypeptide antigens listed by the WHO as international reference standards. A partial list of such appropriate international reference standards, identified by the WHO code in parentheses after the substance, includes vitamin B12 (WHO 81.563), folate (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., page updated June 30, 2005, http: / / www.who.int / bloodproducts / ref_materials / ), which are incorporated herein by reference. The methods and compositions described herein may include the aforementioned WHO reference standards or mixtures containing the reference standards.

[0160] Depending on the application for which the affinity assay is designed, other examples of substances that may function as one or the other member of a binding pair consisting of the analyte binder (capture portion) and the analyte include abuse drugs. Abuse drugs include, for example, the following drugs and their metabolites (e.g., metabolites present in blood, urine, and other biological substances), as well as any of their salts, esters, or ethers: heroin, morphine, hydromorphone, codeine, oxycodone, hydrocodone, fentanyl, demerol, methadone, darvon, stadol, talwin, palegoric, buprenex; stimulants such as amphetamine and methamphetamine; methylamphetamine, ethylamphetamine, methylphenidate, ephedrine, pseudoephedrine, ephedra, ma huang, methylenedioxyamphetamine (MDS), phentermine Phenylpropanolamine; Amifenazole, Bemigride, Be Amphetamine, Bromatan, Chlorphentermine, Clopropamide, Clotetamide, Diethylpropion, Dimethylamphetamine, Doxapram, Etamiban, Fencamfamine, Meclofenoxate, Methylphenidate, Niketamide, Pemoline, Pentetrazole, Fendimethrazine, Fenmetrazine, Phentermine, Phenylepropion Nolamines, picrotoxin, piperadol, prolintan, strychnine, synephrine, phencyclidine, and Angel Dust PCP, ketamine and its analogs; for example, barbiturates, glutethimide, methacarone, and meprobamate, methhexital, thiamil, thiopental, amobarbital, pentobarbital, secobarbital, butarbital, butabarbital, talbutal, and aprobal Suppressants such as phenobarbital, mefobarbital, and ester; benzodiazapenes such as estazolam, flurazepam, temazepam, triazolam, midazolam, alprazolam, chlordiazepoxide, clorazepate, diazepam, harazepam, lorazepam, oxazepam, prazepam, quazepam, clonazepam, and flunitrazepam; and GBH drugs such as gamma hydroxyl butyrate and gamma butyrolactone. ;Glutethimide, methacarone, meprobamate, carisoprodol, zolpidem, zaleplon;cannabinoid drugs such as tetrahydracannabinol and its analogues;cocaine, 3-4 methylenedioxymethamphetamine (MDMA);halogens such as mescaline and LSD, for example. [Examples]

[0161] Example 1: Biotin interference depletion after high-dose biotin intake Endogenous (non-spike) biotin samples were collected sequentially. Baseline serum samples were obtained from five seemingly healthy adult volunteers (4 males, 1 female) by precubital vein collection in a BD brand Vacutainer® 10 mL red-top tube. Each volunteer then ingested a 20 mg dose of biotin (4 × 5 mg, Finest Nutrition Biotin 5000 mcg Strawberry, Quick Dissolve, trademark 938508, sold by Walgreens). Serum samples were obtained 1, 3, 6, 8, and 24 hours after biotin ingestion. Blood was allowed to coagulate at room temperature for 1 hour and centrifuged at 2,000 rpm for 15 minutes using a Beckman Allegra 6R benchtop centrifuge. For each time point, serum samples from each volunteer were pooled, mixed at room temperature for 15 minutes, aliquoted into 1.2 mL aliquots in 2 mL cryovials, and frozen at -80°C.

[0162] Biotin metabolism was determined by measuring biotin levels in sequentially collected samples using free biotin ELISA. Biotin serum samples were tested with the Immundiagnostik IDK® Biotin ELISA kit (part number K8141, lot number 180906, measurement range 48.1–1100 pg / mL). Samples exceeding the kit's measurement range were diluted with the kit's sample dilution buffer. Samples collected 1, 3, 6, and 8 hours after biotin intake were diluted to 1:1000, assuming they were in the range of 50,000–500,000 pg / mL. Samples collected 24 hours after biotin intake were diluted to 1:20, assuming they were around or below 20,000 pg / mL. When the samples were tested according to the ELISA kit protocol, biotin concentrations remained significantly elevated 8 hours (60–107 ng / mL; n=5) and 24 hours (24–32 ng / mL; n=4) after 20 mg of biotin intake (Figure 4 and Table 1). [Table 1]

[0163] High levels of endogenous biotin (370 or 550 ng / mL) were depleted from serum samples continuously collected using superparamagnetic nanoparticles coated with streptavidin (VERAPREP Biotin reagent). This was achieved by adding 200 μL of serum to a 1.5 mL microcentrifuge tube, adding 20 μL of VERAPREP Biotin reagent, gently mixing / shaking the sample for 10 minutes, magnetically separating the VERAPREP Biotin reagent for 10 minutes using Dexter LifeSep® 1.5S, carefully aspirating the serum to avoid damage to the magnetic particles, and testing the serum sample with free biotin ELISA.

[0164] In the first study, the amount of VERAPREP Biotin reagent required to deplete 100% of the free biotin in the sample was determined by adding gradually increasing doses (mg) of VERAPREP Biotin reagent to different aliquots of the same high-biotin (370 ng / mL) endogenous serum sample. 20 μL of VERAPREP Biotin reagent (230 nm diameter, 32 μg streptavidin per 1 mg bead) was added to each 200 μL aliquot of serum sample collected 1 hour after 20 mg biotin intake. The samples were mixed by gently inverting them at room temperature for 10 minutes and then magnetically separated for 10 minutes using a Dexter LifeSep 1.5S magnet. 175 μL of serum supernatant was carefully aspirated and measured using the Immundiagnostik IDK® Biotin ELISA kit (part number K8141, lot number 180906). Samples exceeding the kit's measurement range were diluted with the kit's sample dilution buffer. The 230 nm VERAPREP Biotin reagent successfully depleted 100% of free biotin using a simple 20-minute process, 200 μL of sample, and only 0.39 mg of reagent (Figure 5).

[0165] In the second study, two different VERAPREP Biotin reagents were added in gradually increasing doses (mg) to different aliquots of the same high-biotin (550 ng / mL) serum sample to determine the amount of each reagent required to deplete 100% of the free biotin in the sample. 20 μL of 230 nm VERAPREP Biotin reagent (32 μg streptavidin per 1 mg bead) or 20 μL of 550 nm VERAPREP Biotin reagent (4 μg streptavidin per 1 mg bead) were added to each 200 μL aliquot of serum collected 1 hour after 50 mg biotin intake, mixed by gentle inversion at room temperature for 10 minutes, and magnetically separated for 10 minutes using a Dexter LifeSep 1.5S magnet. 175 μL of serum supernatant was carefully aspirated and measured using the Immundiagnostik IDK® Biotin ELISA kit (part number K8141, lot number 180906). Samples exceeding the kit's measurement range were diluted with the kit's sample dilution buffer. The 230 nm VERAPREP Biotin reagent was tested using only a simple 20-minute process, 200 μL of sample, and 0.75 mg of reagent. While 0% of free biotin was successfully depleted, the 550nm VERAPREP Biotin reagent, using 1.86 mg of the reagent, depleted only 89% of free biotin. These results demonstrate that the binding capacity and binding efficiency of the VERAPREP Biotin reagent can be improved by increasing the amount of streptavidin and the biotin binding capacity per 1 mg of beads, and / or by increasing the concentration or amount (mg) of VERAPREP Biotin reagent added, thereby reducing the bead diameter and increasing the surface area per unit mass (Figure 6).

[0166] Example 2. Biotin interference depletion using an optimized sample pretreatment reagent to bind and deplete high concentrations of free biotin in serum samples. Six volunteers (five clearly healthy adults aged 25–46 years, and one 65-year-old with type 2 diabetes) had fasting serum samples collected at baseline by precubital vein sampling in BD brand Vacutainer® 10 mL red-top tubes. Each volunteer then took over-the-counter (OTC) biotin in doses of 20 mg, 100 mg, or 200 mg. For the 20 mg dose, serum samples were obtained 1, 3, 6, 8, and 24 hours after biotin intake. For the 100 and 200 mg doses, serum samples were collected 1, 6, and 24 hours after biotin intake. Blood was allowed to clot at room temperature for 1 hour and centrifuged at 2,000 rpm for 15 minutes using a Beckman Allegra 6R benchtop centrifuge. Serum samples from each volunteer were pooled for each time point and biotin dose, mixed at room temperature for 15 minutes, aliquoted into 1.2 mL aliquots in 2 mL cryovials, and frozen at -80°C. All samples were sent to the Department of Laboratory Medicine, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195 for LC-MS / MS biotin analysis. At a dose of 20 mg, biotin levels were highest at 1 hour [96–179 ng / mL], all five volunteers still had serum biotin levels above 15 ng / mL [17–35] at 6 hours, four of the five volunteers had serum biotin levels above 15 ng / mL [16–28] at 8 hours, and at 24 hours, volunteer 1, who had known type 2 diabetes, had a biotin level above 15 ng / mL

[18] (Figure 7).

[0167] For 100 and 200 mg doses, biotin levels were highest at 1 hour for the 100 mg dose (294–459 ng / mL) and for the 200 mg dose (610–861 ng / mL). At 6 hours, volunteers who took 20 mg or 100 mg of biotin had serum biotin levels greater than 15 ng / mL, at 17–35 ng / mL for the 20 mg dose and 95–347 ng / mL for the 200 mg dose. At 24 hours, two volunteers who took 100 mg of biotin had biotin levels greater than 15 ng / mL; Volunteer 1, who was known to have diabetes, had a level of 84 ng / mL, and Volunteer 6 had a level of 54 ng / mL (Figure 8).

[0168] Four samples with high endogenous biotin levels [294–861 ng / mL] were selected by LC-MS / MS (Figure 9). Baseline serum samples were tested by PTH Intact ELISA (DRG PTH Intact ELISA, part number EIA-3645), and PTH values ​​ranged from 28.1 to 50.3 pg / mL. All samples taken one hour after biotin ingestion showed PTH results of less than 1.57 pg / mL or below the limit of detection (LLD) (Figure 10).

[0169] All four samples were pretreated with optimized 550 nm superparamagnetic nanoparticles coated with streptavidin (VeraPrep Biotin). Samples 1 and 4 had biotin levels less than 500 ng / mL as determined by LC-MS / MS and were pretreated with 0.5 mg of reagent, while samples 2 and 3 had biotin levels greater than 500 ng / mL as determined by LC-MS / MS and were pretreated with 1.5 mg of reagent using the following protocol. Ta. 1. Remove the VeraPrep Biotin reagent vial from storage, vortex it at medium speed for at least 10 seconds to mix thoroughly, and resuspend the reagent. 2. Insert the reagent vial into the foam vial holder. 3. Insert an empty 2ml microtube (SARSEDT Order Number 72.694) into the LifeSep® 1.5S magnet until the tube's collar touches the magnetic frame. Dispense 4,200 μL (0.5 mg) or 600 μL (1.5 mg) of well-mixed reagent into an empty tube and separate the reagent on the magnet for more than 30 seconds to form a reagent pellet. 5. Carefully aspirate and discard all of the stored buffer supernatant (approximately 200 μL or 600 μL) without disturbing the reagent pellet. Dispense 6,400 μL of well-mixed serum or plasma sample into a tube containing the reagent pellet. 7. Tighten the cap of the tube, remove the tube from the magnet, and vortex it at a medium speed for at least 10 seconds to mix thoroughly and resuspend the reagent in the sample. 8. Place the tube on a laboratory mixer at medium speed and incubate at room temperature for 10 minutes. 9. Loosen the screw cap and insert the tube into the magnet until the tube collar touches the magnetic frame. 10. Separate the reagents magnetically for more than 4 minutes to form a reagent pellet. 11. Carefully aspirate the sample supernatant without disturbing the reagent pellet and dispense the sample into the transfer tube for testing. Note: If this step is performed carefully, all of the sample supernatant (approximately 400 μL) can be aspirated. If any of the reagents are accidentally aspirated, simply return the sample / reagent mixture to the tube and return to step 10. 12. The sample is ready for testing.

[0170] To verify biotin interference removal, VeraPrep Biotin-pretreated samples were tested using the Immundiagnostik IDK® Biotin ELISA kit (part number K8141, measurement range 48.1–1,100 ng / L). Biotin concentrations were in the range of 0.2–1.0 ng / mL or within normal plasma levels (200–1,200 ng / L) (Figure 9). Immediately after VeraPrep Biotin pretreatment of samples 1–4, PTH levels were measured using PTH Intact ELISA, and PTH levels were in the range of 26.7–52.0 pg / mL (Figure 10).

[0171] Samples taken one hour after biotin ingestion showed high levels of biotin interference per LC-MS / MS (294–861 ng / mL) and undetectable PTH levels by PTH Intact ELISA (<1.57 pg / mL). However, samples taken one hour after biotin ingestion pretreated with VeraPrep Biotin showed physiologically normal biotin levels per Biotin ELISA kit (<1.1 ng / mL) and normal PTH levels by PTH Intact ELISA (26.7–52.0 pg / mL) (Figures 9 and 10). When comparing PTH levels before VeraPrep biotin sample treatment with baseline PTH levels, the results showed a recovery of 95–113% (mean recovery rate of 105%) (Figure 10). The significant difference in test results after VeraPrep biotin sample pretreatment, or the significant increase in PTH levels in this PTH Intact ELISA sandwich immunoassay, supports the clinical significance of biotin interference in all four samples tested.

[0172] Example 3: Enrichment of low-abundance biomarkers Using 550 nm superparamagnetic nanoparticles coated with streptavidin and then with biotinylated anti-TSH antibody (VERAPREP Concentrate TSH reagent) or biotinylated anti-PTH monoclonal antibody (VERAPREP Concentrate PTH reagent), 0.0195 μIU of TSH / mL or 0.0195 μIU was measured. Very low levels of any of the biomarkers (497 pg PTH / mL) were enriched in 40 mL of PBS.

[0173] In the first study, VERAPREP Concentrate was obtained by coating 550 nm VERAPREP Biotin with a biotinylated anti-TSH capture antibody. TSH reagents were prepared. 0.08 mL of TSH antigen (10 μIU / mL ELISA calibrator) was diluted to 0.0195 μIU / mL in 41 mL of PBS buffer, which is lower than the functional sensitivity (less than 0.054 μIU / mL) of the DRG TSH Ultrasensitive ELISA (part number EIA-1790, lot number RN 58849). 1 mL was stored as the baseline sample (before enrichment). 40 mL of the sample was processed using the VERAPREP Concentrate TSH protocol to prepare a 1.0 mL enriched sample for subsequent TSH ELISA testing.

[0174] Dilute 80 μL of 10 μIU / mL TSH standard solution to 0.0195 μIU / mL in 41.0 mL of PBS, and store 1.0 mL as the baseline sample (before enrichment). Dilute 1.80 μL of 10 μIU / mL TSH standard solution to 0.80 μIU / mL in 1.0 mL of VERAPREP Cleave as a control. 2. Add 40 mL of PBS containing 0.0195 μIU / mL of TSH to a 50 mL Falcon tube. 3. Add VERAPREP Concentrate TSH and mix. 4. Incubate at room temperature for 60 minutes while mixing. 5. Use Dexter LifeSep(registered trademark) 50SX with VERAPREP The concentrated TSH is magnetically separated for 60 minutes. Decant 6.40 mL of PBS and dispose of it as waste. Add 7.4.0 mL of PBS Wash Buffer and mix. 8. Magnetically separate VERAPREP Concentrate TSH in 4 mL of PBS Wash Buffer for 30 minutes using Dexter LifeSep® 50SX. Decant 9.4 mL of PBS and dispose of it as waste. Add 10.1 mL of PBS Wash Buffer and mix. Transfer 11.1 mL of VERAPREP Concentrate TSH to a 1.75 mL cone-bottom snap-cap vial. 12. Use Dexter LifeSep® 1.5S to magnetically separate VERAPREP Concentrate TSH in 1 mL of PBS Wash Buffer for 10 minutes. Aspirate 13.1 mL of PBS and discard it as waste. Add 14.1 mL of VERAPREP Cleave and mix. 15. Using Dexter LifeSep® 1.5S, VERAPREP Concentrate TSH in 1 mL of VERAPREP Cleave is magnetically separated for 10 minutes. Aspirate 16.1 mL of supernatant (enriched sample) and store it. Test the control, baseline, and enriched samples.

[0175] 0.08 mL of TSH antigen (10 μIU / mL ELISA calibrator) was also diluted to 0.800 μIU / mL by adding 1 mL of VERAPREP Cleave buffer as a control. The baseline sample, enriched sample, and control were tested using DRG TSH Ultrasensitive ELISA, and the TSH% recovery rate of the enriched sample was calculated as [enriched sample result] / [control result] × 100%. As expected, the diluted TSH baseline sample was undetectable by the Ultrasensitive ELISA, with a reading of 0.00 μIU / mL. Using only 0.80 mg of reagent, VERAPREP EP Concentrate TSH successfully enriched TSH from undetectable to diluted 0.73 μIU / mL (Table 2). Compared to the control, this represented a 98.6% recovery rate, but VERAPREP suppressed the assay signal in TSH ELISA. There may have been a matrix effect from the Cleave buffer (Table 3). [Table 2] [Table 3]

[0176] In the second study, the VERAPREP Concentrate PTH reagent was prepared by coating 550 nm VERAPREP Biotin with a biotinylated anti-PTH capture antibody. 0.021 mL of PTH antigen (971 pg / mL ELISA calibrator) was diluted to 0.497 pg / mL in 41 mL of PBS buffer at a concentration less than the functional sensitivity (<1.56 pg / mL) of the DRG PTH (parathyroid) Intact ELISA (part number EIA-3645, lot number 2896), and 1 mL was stored as the baseline sample (before enrichment). 40 mL of the sample was processed using the VERAPREP Concentrate PTH protocol to prepare a 1.0 mL enriched sample for subsequent PTH ELISA testing. Dilute 21 μL of 1.971 pg / mL PTH standard solution to 0.497 pg / mL by adding 41.0 mL of PBS, and store 1.0 mL as the baseline sample (before enrichment). A 21 μL PTH standard solution at 2.971 pg / mL was used as a control and diluted to 1.0 mL using a VERAPREP Cleave to a concentration of 20.4 pg / mL. 3. Add 40 mL of 0.497 pg / mL PTH in PBS to a 50 mL Falcon tube. 4. Add VERAPREP Concentrate PTH and mix. 5. Incubate at room temperature for 30 minutes while mixing. 6. Use Dexter LifeSep(registered trademark) 50SX with VERAPREP The concentrated PTH is magnetically isolated for 15 minutes. Decant 7.40 mL of PBS and dispose of it as waste. Add 8.4.0 mL of PBS Wash Buffer and mix. 9. Use Dexter LifeSep® 50SX to magnetically separate VERAPREP Concentrate PTH in 4 mL of PBS Wash Buffer for 10 minutes. Decant 10.4 mL of PBS and dispose of it as waste. Add 11.1 mL of PBS Wash Buffer and mix. Transfer 12.1 mL of VERAPREP Concentrate PTH to a 1.75 mL cone-bottom snap-cap vial. 13. Use Dexter LifeSep® 1.5S to magnetically separate VERAPREP Concentrate PTH in 1 mL of PBS Wash Buffer for 10 minutes. Aspirate 14.1 mL of PBS and discard it as waste. Add 15.1 mL of VERAPREP Cleave and mix. 16. Use Dexter LifeSep® 1.5S to magnetically separate VERAPREP Concentrate PTH from 1 mL of VERAPREP Cleave for 10 minutes. Aspirate 17.1 mL of supernatant (enriched sample) and store it. Test the control, baseline, and enriched samples.

[0177] 0.021 mL of PTH antigen (971 pg / mL ELISA calibrator) was also diluted to 20.4 pg / mL by adding 1 mL of VERAPREP Cleave buffer as a control. Baseline, enriched, and control samples were tested by DRG PTH (parathyroid) Intact ELISA, and the PTH% recovery rate for enriched samples was calculated as [enriched sample result] / [control result] × 100%. The diluted PTH baseline sample read 13.5 pg / mL due to the matrix effect of VERAPREP Cleave buffer in the ELISA assay. This matrix effect resulted in enhancement of the assay signal. Using only 0.80 mg of reagent, VERAPREP Concentrate PTH successfully enriched diluted PTH to 42.3 pg / mL (Table 4). Compared to the control, this represented a 109% recovery rate (Table 5). [Table 4] [Table 5]

[0178] Example 4: Enrichment of low-abundance biomarkers from urine for subsequent analysis by mass spectrometry (LC-MS / MS or MALDI-MS). Below is a mass spectrometry sample preparation protocol for enriching low-abundance biomarkers and spiked internal standards (ISTDs) from large volumes of urine samples using superparamagnetic nanoparticles coated with biomarker-specific capture moieties. The exact same protocol can also be used to multiplex and enrich more than one biomarker and corresponding spiked ISTDs from the same sample, using multiple different superparamagnetic nanoparticle populations, each population coated with a different capture moiety, either mixed or pooled together. Enrichment and characterization of two or more biomarkers facilitates the use of algorithms for clinical diagnosis and / or prognosis of diseases that are not possible with the characterization of a single biomarker. For example, for the diagnosis of obstructive sleep apnea (OSA) from urine, the VERAPREP Concentrate reagent may contain four different antibodies to capture and enrich kallikrein-1, uromodulin, urocortin-3, and orosomucoid-1, or seven different antibodies to capture and enrich kallikrein-1, uromodulin, urocortin-3, and orosomucoid-1, IL-6, IL-10, and highly sensitive C-reactive proteins. 1. Collect the patient's urine (using a standard urine collection protocol, such as a urine collection cup). 2. Mix the urine samples. Add 3.40 mL of urine to a 50 mL Falcon tube. 4. Add and mix the deuterated internal standard for the enriched biomarker. 5. Add VERAPREP Condition and mix. 6. Add VERAPREP Concentrate and mix. 7. Incubation: Deuterated internal standard capture using biomarker + VERAPREP Concentrate 8. VERAPREP Concentrate is magnetically separated from 40 mL of urine using Dexter LifeSep(registered trademark) 50SX. 9. Aspirate the urine and dispose of it as waste. Add 10.4 mL of PBS Wash Buffer and mix. 11. Use Dexter LifeSep® 50SX to magnetically separate VERAPREP Concentrate in 4 mL of PBS Wash Buffer. 12. Aspirate the urine and dispose of it as waste. 13. Repeat step 12 two more times (2X). Add 14.1 mL of VERAPREP Cleave and mix (mass spectrometry-compatible buffer). 15. Use Dexter LifeSep® 1.5S to magnetically separate VERAPREP Concentrate from 1 mL of VERAPREP Cleave. 16. Aspirate 1 mL of supernatant sample using LC-MS and test it. 17.1) Final biomarker concentration determined based on adjusting the reported biomarker values ​​based on 40 mL urine sample size, 2) LC-MS quantification of biomarkers, and 3) deuterated internal standard recovery rate.

[0179] Selective release or cleavage of one or more captured and enriched biomarkers can be achieved by a change in pH (acidic pH, e.g., elution of glycine at pH 2.5 followed by neutralization, or alkaline pH 10.0 or higher) using a cleavable linker such as a disulfide bond cleaved with a reducing agent such as TCEP or DTT, or by using competitive elution such as molar excess D-biotin and monomeric avidin, or molar excess sugar and concanavalin A competing for a binding site on concanavalin A.

[0180] Example 5. SARS-CoV-2 neutralizing antibody assay This assay isolates and quantifies SARS-CoV-2 neutralizing antibodies that recognize both the receptor-binding domain and the N-terminal domain. It quantifies IgG, IgA, and IgM separately. Although serum antibodies were assayed as described below, this procedure can also be performed with oral saline rinse to assay salivary antibodies. In the assay, the sample is first washed to remove potential heterophilic interference, the antibodies are captured on beads (microparticles), reacted with the detection reagent, the captured antibodies (still bound by the detection reagent) are eluted from the beads, and the supernatant is transferred for quantification. The assay can be performed manually or automatically. A generalized procedure for the assay includes the following:

[0181] 1. Prime the plate washer with washing / blocking buffer. a. First, treat the water with sound waves for 30 minutes.

[0182] 2. Prepare all reagents. a. All beads should be mixed to be homogeneous for use and placed in a locker.

[0183] 3. Plate Conditioning - To prevent nonspecific binding of, for example, immunoglobulins to the well surface, wash a pair of clear round-bottom 96-well microtiter plates with a washing buffer of 0.023% (w / v) Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), triblock copolymer) in TTA (Tris-buffered saline, 0.05% Tween® 20 (polysorbate 20), 0.05% azide, pH 7.4). One round-bottom plate will function as the "wash" plate and the other as the "capture" plate.

[0184] 4. Pre-analysis sample cleaning to eliminate heterophilic interference. a. Use samples directly from refrigerated storage without mixing by centrifugation. Avoid direct pipetting of any lipids. b. Add 60 μL of each sample from the "clean" plate layout to the blocked round-bottom plate. c. After adding all samples, use a multichannel pipette to add 140 μL of clean beads (a mixture of 36 μg rabbit IgG-biotin-streptavidin beads and 4 μg human IgG-biotin-streptavidin beads to capture and remove heterophilic interference specific to rabbit IgG, human IgG, streptavidin, and / or the beads themselves) to each well containing the samples. i. Note: The 1.6 micron superparamagnetic streptavidin beads used in the base, clean beads are the exact same base 1.6 micron superparamagnetic streptavidin beads used in the capture beads. In this way, any heterophilic interference specific to the base streptavidin beads is removed from the sample before testing the clean sample with the capture beads. d. Incubate at 37°C for 15 minutes while shaking in a plate washer. i. Orbital oscillation at fast settings, 425 cpm e. Place on a magnet for 4 minutes. i. For example, Alpaqua Catalyst 96, part number A000550.

[0185] 5. Capture of neutralizing antibodies for detection and multiplex quantification of IgM, IgG, and IgA specific to SARS-CoV-2 S1-RBD or S1-NTD. a. Using a multichannel pipette, transfer 50 μL of the washed sample to a blocked capture plate (transparent, flat-bottomed). b. Using a multichannel pipette, add 150 μL of capture beads (a mixture of 30 μg of SARS-CoV-2 S1-RBD-biotin-streptavidin beads and 10 μg of SARS-CoV-2 S1-NTD-biotin-streptavidin beads used to capture RBD or NTD-specific human immunoglobulins IgA, IgG, and / or IgM) to each well. i. Add the same volume of trapping beads to an empty well to use it as a trapping bead blank. ii. SARS-CoV-2 spike glycoprotein (S1) RBD (with His tag, prepared with HEK293; The Native Antigen Company, Part.No.REC31882). iii. SARS-CoV-2 Spike NTD (with His tag, manufactured with HEK293; The Native Antigen Company, Part.No.REC31905). c. Add 60 μL of triple calibrator beads to each of the six wells of the plate, each containing a different calibrator level (see below). i. Add the triple calibrator beads and set the plate on the magnet, and each calibrator Rinse the tip when dispensing with a blister. d. Incubate at 37°C for 30 minutes while shaking with a plate reader. i. Orbital oscillation at a fast setting, 425 cpm. e. Wash three times with a plate washer. i. Initial / shaking process for 2 minutes.

[0186] 6. Multiple labeling of the captured antibody. a. Add 200 μL of triple conjugate per well. i. Using the same set of 8 tips on a multichannel pipette, the conjugate can be added to all wells in the entire plate. ii. Once all wells have been filled with conjugate, return the pipette and pipette up and down five times to thoroughly mix the wells (use a multichannel pipette, but prepare a new tip for each column). iii. The triple conjugate consists of 0.002 mg / ml of polyclonal rabbit anti-human IgM conjugated with AlexaFluor-488, 0.002 mg / ml of polyclonal rabbit anti-human IgG conjugated with AlexaFluor-555, and 0.002 mg / ml of polyclonal rabbit anti-human IgA conjugated with AlexaFluor-647 in a conjugate buffer (0.1% BSA in TTA). b. Incubate at 37°C for 30 minutes while shaking with a plate reader. i. Orbital oscillation at a fast setting, 425 cpm. c. Wash three times with a plate washer. i. Initial / shaking process for 2 minutes.

[0187] 7. Elution of antibody-conjugate complex from beads a. Pre-fill the wells on a black plate with a clear bottom (unblocked) with 35.5 ml of neutralizing buffer (300 mM Tris pH 10.0). b. Remove the capture plate from the plate washer. c. Add 220 μL of elution buffer (100 mM glycine, pH 2.5). i. Using the same set of 8 tips on a multichannel pipette, the conjugate can be added to all wells in the entire plate. ii. Once all wells have been filled with elution buffer, return the pipette to its original position and pipette up and down five times to thoroughly mix the wells (use a multichannel pipette, but prepare a new tip for each column). d. Place on a magnet for 2 minutes. e. Using a multichannel pipette, transfer 200 μL from each well to the black, clear, flat-bottomed reading plate (from step 7a).

[0188] 8. Read the fluorescence using a plate reader.

[0189] The triplex calibrator bead is assembled from the following four components: • 1.6 μm magnetic beads (affinity purified) that are covalently modified with streptavidin and react with biotinylated human IgA. • 1.6 μm magnetic beads (affinity purified) that are covalently modified with streptavidin and react with biotinylated human IgG. • 1.6 μm magnetic beads (affinity purified) that are covalently modified with streptavidin and react with biotinylated human IgM. • 1.6 μm magnetic beads (carboxyl) (TRis beads) quenched with Tris, The solutions stored at 10 mg / ml were calibrator bead storage solutions containing 0.1% BSA in TTA.

[0190] The beads were diluted to 1.00 mg / ml with the calibrator bead storage solution, and the IgA, IgG, and IgM beads were pooled separately with the Tris beads in the following ratios: 100:0, 75:25, 50:50, 25:75, 10:90, and 0:100, respectively. Then, for each non-zero Ig calibration level, the IgA, IgG, and IgM mixtures were pooled in a 1:1:1 ratio to prepare a set of triple calibrator beads. The triple calibrator beads were used at a final concentration of 0.3 mg / ml in the calibrator bead storage solution. The calibration curve is shown in Figure 11.

[0191] Essentially following the protocol described above, 146 serum samples collected prior to December 2018 were tested for the presence of SARS-CoV-2 neutralizing antibodies. Since these samples were collected well before the virus appeared in human populations, they were expected to be negative, and indeed, this was the case (see Table 6). [Table 6]

[0192] "Diagnostic routine" refers to serum samples remaining after routine diagnostic testing, where the donor's health status is unknown. "Donor" refers to samples preserved from blood donations from healthy donors. These results demonstrate that this assay meets the FDA emergency use authorization specificity requirement of 95%. ru.

[0193] This assay was also used to test 122 samples from 63 symptomatic patients with PCR-confirmed SARS-CoV-2 infection. These samples included one or more consecutive samples collected from the date of symptom onset. The results are shown in Table 7. [Table 7]

[0194] These results demonstrate that this assay meets the FDA's 90% sensitivity requirement for emergency use authorization. Several samples that showed false-negative results in the initial assay later showed positive results for samples collected (Figure 12). Many patients with two or three consecutive samples showed similar levels of antibodies over time, but some patients showed rapidly increasing or decreasing antibody levels (Figure 13).

[0195] Example 6. Cleaning and capture of biomarkers from saline mouthwash. To purify the oral rinse sample in 1 mL (1000 μL) of saline solution (5 mL of 0.9% NaCl rinsed for 25 seconds, followed by gargling for 5 seconds and spitting into a collection tube = 5 mL of saline solution + saliva sample, or a saliva-based sample in saline solution), add either 2 × or 4 × purifying beads having the following composition.

[0196] For 2 x clean beads per test: 25ug rabbit IgG beads 25ug BSA beads 10 ug of purified human IgA beads 10ug purified human IgG beads 10ug purified human IgM beads

[0197] For 4 x clean beads per test: 50ug Rabbit IgG Beads 50ug BSA beads 20 ug of purified human IgA beads 20ug purified human IgG beads 20ug purified human IgM beads

[0198] To capture SARS-CoV-2 neutralizing antibodies from a 1 mL sample of purified saline mouth rinse, 5× or 10× capture beads are added, and this amount of RBD and NTD capture beads is used to enrich and capture total neutralizing immunoglobulins from the purified saline mouth rinse sample.

[0199] For 5 × capture beads per test: 150ug RBD beads 50ug NTD beads

[0200] For 10 x capture beads per test: 300ug RBD beads 100ug NTD beads

[0201] Table 8 shows the results of assays using 10× clean beads and 10× capture beads in three patients who received one dose (of the usual two) of the SARS-CoV-2 mRNA vaccine and three unvaccinated subjects. These data were collected as early as 8 days post-administration, demonstrating that even at this early stage, the assay can detect (and quantify) the SARS-CoV-2 neutralizing antibody response. (In general, individuals are not considered "fully vaccinated" until two weeks after the second dose of the vaccine.) [Table 8]

[0202] As can be seen from the figure, patient 2 has levels of SARS-CoV-2-specific antibodies of all three isotypes that exceed the background levels, and all three vaccinated patients have levels of SARS-CoV-2-specific IgG that exceed the background level.

[0203] Abbreviations ABEI N-(4-aminobutyl)-N-ethylisoluminol ALP Alkaline phosphatase BSA Bovine serum albumin Fab Fragment antibody binding Fc Fragment, crystallizable HAAA Human anti-animal antibody HAMA Human anti-mouse antibody HASA Human anti-sheep antibody IFU Instructions for use IgG Antibody or immunoglobulin IgM Immunoglobulin M HRP Horseradish peroxidase LC-MS / MS Liquid chromatography tandem mass spectrometry LDT Laboratory-developed test Mab Monoclonal antibody MASI Manufacturing assay-specific interference MFG IVD manufacturer PMP Superparamagnetic microparticles PBCT Primary blood collection tube RF Rheumatoid factor RLU Relative light unit or assay response signal RUO For research use only SAv Streptavidin STT Secondary transfer tube TAT Turnaround time WF Workflow

[0204] Definitions As used herein, “sample” or “biological sample” means serum, plasma (i.e., EDTA, lithium heparin, sodium citrate), blood, whole blood, processed blood, urine, saliva, feces (liquid and solid), semen or semen, amniotic fluid, cerebrospinal fluid, cells, tissues, biopsy material, DNA, RNA, or any fluid, dissolved solid, or processed solid material being tested for monitoring such as diagnosis, prognosis, screening, risk assessment, risk stratification, and therapeutic drug monitoring of any human or animal. In some embodiments, the sample is a large volume sample. In some embodiments, the sample comprises multiple samples (e.g., two or more samples from the same or different subjects). In some embodiments, the sample contains biomarkers present in low abundance in the sample.

[0205] In some embodiments, the sample is a primary blood collection tube (PBCT), a secondary transfer tube (SST), a blood collection bag, a 24-hour urine collection device, a vericore tube, a nanotainer, a saliva collection tube, a blood spot filter paper, or any collection tube or device such as feces and semen, a light green top or green top plasma separator tube (PST) containing sodium heparin, lithium heparin, or ammonium heparin, a light blue top tube containing sodium citrate (i.e., 3.2% or 3.8%) or citrate, theophylline, adenosine, dipyridamole (CTAD), a red top tube for serology or immunohematology for collecting serum in a glass (without additives) or plastic tube (containing a clot activator), a red top tube for chemistry for collecting serum in a glass (without additives) or plastic tube (containing a clot activator), a purple lavender top tube containing EDTA K2, EDTA K3, a liquid EDTA solution (i.e., 8%), or an EDTA K2 / gel tube for testing plasma in molecular diagnostics and viral load detection, a pink top tube for blood bank EDTA, a gray top tube containing potassium oxalate and sodium fluoride, sodium fluoride / EDTA, or sodium fluoride (when there is no anticoagulant, a serum sample is obtained), a yellow top tube containing ACD solution A or ACD solution B, a vine purple top (serum, without additives or sodium heparin), a white top tube for any application or diagnostic test type that does not contain an additive or any additive or combination thereof for blood collection, or is collected in any color or tube type.

[0206] In some embodiments, the sample is a difficult sample type such as urine, 24-hour urine, saliva, and feces, or the biomarker of interest is diluted or the measurement is This can be difficult. For example, biological samples can be difficult for patient populations (e.g., neonates, children, the elderly, pregnant women, tumors, autoimmune diseases). For example, some biomarkers are too dilute or too low in concentration, for instance, in circulation or urine, to be reliably detected and accurately measured by existing POCT analyzers and central laboratory analyzers. In some embodiments, a difficult sample is cerebrospinal fluid (CSF).

[0207] As used herein, “collection device” may be a primary blood collection tube (PBCT), a 24-hour urine collection device, a urine collection device, a saliva collection tube, a stool collection device, a semen collection device, a blood collection bag, or any sample collection tube or device before the addition of the sample.

[0208] PBCTs and secondary transfer tubes (SSTs) include any commercially available standard or custom collection tubes (with or without gel separators), glass tubes, plastic tubes, light green top or green top plasma separator tubes (PSTs) containing sodium heparin, lithium heparin, or ammonium heparin from companies such as Becton Dickinson (BD), Greiner, VWR, and Sigma Aldrich; light blue top tubes containing sodium citrate (i.e., 3.2% or 3.8%) or citrate, theophylline, adenosine, dipyridamole (CTAD); red top tubes for serology or immunohematology for collecting serum in glass (no additives) or plastic tubes (containing coagulation activators); red top tubes for chemistry for collecting serum in glass (no additives) or plastic tubes (containing coagulation activators); 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 (serum samples can be obtained if no anticoagulant is present), yellow top tubes containing ACD solution A or ACD solution B, wisteria purple top tubes (serum, no additives, or sodium heparin), white top tubes for any use or diagnostic test type without additives for blood collection or any combination thereof, or any color or tube type.

[0209] As used herein, “storage device” or “transfer device” refers to a device that receives the sample and / or other components received by the collection device. The storage or transfer device may be a plastic or glass tube, vial, bottle, beaker, flask, bag (e.g., blood collection bag, can, microtiter plate, ELISA plate, 96-well plate, 384-well plate, 1536-well plate), cuvette, reaction module, reservoir, or any container suitable for holding, storing or processing liquid samples. Where used herein, “diagnostic testing” includes, but is not limited to, any antibody-based diagnostic testing, non-antibody-based diagnostic testing, immunoextraction (IE) and solid-phase extraction (SPE), radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), chemiluminescence immunoassay (CLIA), molecular diagnostics, lateral flow (LF), point of care (PoC), direct-to-consumer (DTC), CLIA and CLIA exclusion testing and devices, research use only (RUO) testing, in vitro diagnostic (IVD) testing, laboratory development testing (LDT), companion diagnostics, and sample preparation methods or devices for subsequent analysis by mass spectrometry (i.e., HPLC, MS, LCMS, LC-MS / MS) for diagnostic, prognosis, screening, risk assessment, risk stratification, and therapeutic drug monitoring. In some embodiments, the diagnostic test includes short turnaround time (STAT) diagnostic tests, outpatient tests, lateral flow tests, point-of-care (PoC) tests, molecular diagnostic tests, HPLC, MS, LCMS, LC-MS / MS, radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), and chemiluminescent immunoassay (CLIA). This includes CLIA and CLIA exclusion studies, as well as any diagnostic studies used for diagnosis, prognosis, screening, risk assessment, risk stratification, treatment monitoring, and therapeutic drug monitoring.

[0210] As used herein, pathogens are bacteria, viruses, or other microorganisms that can cause disease.

[0211] Serology is the scientific study of serum and other body fluids. In practice, the term usually refers to the diagnostic identification of antibodies in serum. Such antibodies are typically formed in response to infection (against a given microorganism), other foreign proteins (e.g., in response to inconsistent blood transfusions), or the body's own proteins (in the case of autoimmune diseases).

[0212] Finally, while aspects of this specification are emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily recognize that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific methods, protocols, and / or reagents, etc., described herein. Accordingly, various modifications, alterations, or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of this specification. Finally, the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the invention as defined solely by the claims. Therefore, the invention is not limited to what is precisely illustrated and described.

[0213] Specific embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the foregoing description. The inventors expect that those skilled in the art will appropriately use such variations, and they intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter enumerated in the appended claims, as permitted by applicable law. Furthermore, unless otherwise indicated herein, or unless clearly inconsistent with the context, any combination of all possible variations of the above embodiments is incorporated into the Invention.

[0214] Any grouping of alternative embodiments, elements, or processes of the present invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of such inclusion or removal, this specification shall be deemed to include the modified groups and thus satisfy the description of all Markush groups used in the appended claims.

[0215] Unless otherwise specified, all figures representing properties, items, quantities, parameters, characteristics, terms, etc., used herein and in the claims should be understood to be modified in all cases by the term “approximately.” Where used herein, “approximately” means that the thus limited property, item, quantity, parameter, characteristic, or term encompasses a range of plus or minus 10 percent of the stated value of the property, item, quantity, parameter, characteristic, or term. Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are variable approximations. Each numerical representation should be interpreted, at least in light of the number of significant figures reported, by applying common rounding techniques, not as an attempt to limit the application of the doctrine of equivalents to the claims. Although the numerical ranges and values ​​representing the broad scope of the invention are approximations, the numerical ranges and values ​​shown in specific examples are reported as accurately as possible. However, any numerical range or value inherently contains certain errors that inevitably arise from the standard deviation observed in the respective test measurements. The enumeration of numerical ranges of values ​​in this specification is intended simply as a simplified way of referring individually to each distinct numerical value that falls within that range. Unless otherwise indicated herein, the individual values ​​of a numerical range are incorporated herein as if they were individually enumerated herein.

[0216] In the context describing the present invention (particularly in the context of the following claims), the terms “a,” “an,” “the,” and similar reference subjects should be construed to encompass both singular and plural unless otherwise specifically indicated herein or unless clearly inconsistent with the context. All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless clearly inconsistent with the context. The use of any examples or illustrative language provided herein (e.g., “etc.”) is intended merely to better illustrate the present invention and not to limit the scope of the claimed invention. No language herein should be construed to indicate an unclaimed element essential to the practice of the present invention.

[0217] Certain embodiments disclosed herein may be further limited in the claims by using the terms "consisting of" or "essentially consisting of." Where used in the claims, the transitional term "consisting of," whether added at filing or by amendment, excludes elements, processes, or components not specified in the claims. The transitional term "consisting essentially of" limits the scope of the claims to specific materials or processes, as well as those that do not substantially affect the basic and novel features. Embodiments of the invention as thus claimed are essentially or expressly described and enabled herein.

[0218] All patents, patent publications, and other publications referenced and identified herein are explicitly incorporated herein by whole by reference for the purpose of describing and disclosing, for example, compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are provided only for their disclosures prior to the filing date of this application. Nothing in this regard should be construed as an acknowledgment by the inventors that they do not have prior rights to such disclosures by prior art or for any other reason. All statements or expressions relating to the dates or contents of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or contents of these documents. The present invention provides, for example, the following items: (Item 1) A method for isolating antigen-specific antibodies from a biological sample, a) To provide a mixture by combining the sample with a first particle containing a capture portion for the antigen-specific antibody, b) Mixing the mixture and providing the particle complex to the biomarker, c) Separating the particles from the biological sample. A method comprising isolating the antibody from the biological sample. (Item 2) The method according to item 1, further comprising dissociating the antigen-specific antibody from the particles. (Item 3) The method according to item 1, wherein dissociation comprises cleavage or elution of the antibody from the first particle. (Item 4) The method according to item 3, further comprising subjecting the released antibody to characterization. (Item 5) The method according to item 4, wherein the characterization involves forming a complex with an anti-immunoglobulin antibody conjugated to a detectable label. (Item 6) The method according to item 5, wherein the detectable label is a fluorescent label. (Item 7) The method according to any one of Items 4 to 6, further comprising comparing the signal related to the antigen-specific antibody with a standard curve of immunoglobulins. (Item 8) The method according to any one of Items 5 to 7, wherein the anti-immunoglobulin antibody is not isotype-specific. (Item 9) The method according to any one of Items 5 to 7, wherein the anti-immunoglobulin antibody is isotype-specific. (Item 10) The method according to Item 9, wherein the isotype-specific anti-immunoglobulin antibody comprises at least two of anti-IgA, anti-IgG, and anti-IgM, each conjugated to a different label. (Item 11) Further comprising a pretreatment, wherein the pretreatment a) providing a mixture by combining the biological sample with a second particle comprising a capture portion for interference, b) mixing the mixture to provide a second particle complex for the interference, c) removing or excluding the second particle complex to provide a depletion solution, the method according to Item 1. (Item 12) The method according to Item 11, wherein the capture portion comprises human and / or non-human animal immunoglobulins. (Item 13) The method according to Item 11 or 12, wherein the capture portion comprises streptavidin. (Item 14) The method according to Item 1 or 11, wherein the first and / or second particles are provided as lyophilized products. (Item 15) An enriched antibody produced by the method according to any one of Items 1 to 3 or 11 to 14. (Item 16) The method according to any one of Items 1 to 15, wherein the antigen-specific antibody is a pathogen-specific antibody. (Item 17) The method described in any one of item 16, wherein the pathogen is SARS-CoV-2. (Item 18) The method according to item 17, wherein the capture portion is the spike protein of SARS-CoV-2. (Item 19) The method according to item 18, wherein the spike protein is the S1 subunit, or its receptor-binding domain and / or N-terminal domain. (Item 20) The method according to any one of items 1 to 15, wherein the antigen-specific antibody is an autoantibody. (Item 21) The method according to any one of item 20, wherein the antigen-specific antibody is a tumor antigen-specific autoantibody. (Item 22) The method according to any one of items 1 to 15, wherein the antigen-specific antibody is against an exogenous protein. (Item 23) The method according to any one of items 1 to 22, wherein the antigen-specific antibody is a human antibody.

Claims

[Claim 1] The invention described in the specification.

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