Enrichment of antigen-specific antibodies for analytical and therapeutic uses

Abstract

The present invention relates to methods of using particles (e.g., microparticles, nanoparticles; magnetic, non-magnetic) comprising surfaces that include capture moieties as described herein to remove interferences as described herein or to enrich for biomarkers, particularly antibodies, prior to diagnostic testing or to isolate and use for prophylactic or therapeutic purposes. Described herein are methods for simple, efficient, and cost-effective conditioning of biological samples to manage 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 receiving them for diagnostic purposes.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 008,472, filed April 10, 2020, the entire contents of which are 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 surfaces that include capture moieties as described herein to isolate antigen-specific antibodies for subsequent analytical, prophylactic, or therapeutic use. [Background technology]

[0003] background Laboratory testing plays a critical role in health assessment, health care, and ultimately public health, affecting people at all stages of life. Nearly everyone will undergo one or more laboratory tests during their lifetime. An estimated 7-10 billion laboratory tests are performed each year in the United States alone, and laboratory tests influence approximately 70% of medical decisions.

[0004] Additionally, the Centers for Medicare & Medicaid Services (CMS) implemented the new Clinical Laboratory Fee Schedule (CLFS) required by the Protecting Access to Medicare Act (PAMA) on January 1, 2018, which is reducing reimbursement for laboratory testing. Even more importantly, laboratory results are accurate the first time, and troubleshooting is less effortful or time-consuming and does not impact laboratory workflow.

[0005] Interferences are substances present in patient samples that can alter the accuracy of diagnostic test results, for example, by interfering with antibody binding, or that can increase or decrease the assay signal through cross-linking, steric hindrance, or autoantibody mechanisms. Although immunoassays are known to be susceptible to interference, clinical laboratories can still report erroneous results if such results are not recognized and flagged by the instrument (analyzer) or laboratory, or if a physician does not notify the laboratory that a patient's results do not fit the clinical picture. Incorrect results can occur unexpectedly with any sample for which there is no practical means of identifying in advance such samples as potentially problematic. As a result of such interference, erroneous results can result in false-negative and false-positive test results, which can affect patient care and lead to unnecessary invasive, diagnostic, or therapeutic procedures, or to failed patient treatment.

[0006] Clinical laboratories face many sources of sample-specific interference, including sample type (i.e., plasma), carryover, freeze / thaw, stability, hemolysis, jaundice, lipemia, 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 troublesome and problematic because it can be difficult to detect and affect patient management.

[0007] Despite the complications caused by interferences, biomarker screening and diagnostic testing can be challenging due to, for example, their low presence or abundance in biological samples.

[0008] Thus, biomarkers found in the body can be used to detect, predict, or manage disease, but many are present in quantities too low to be detected using currently available commercially available tests. There is an unmet clinical need for new diagnostic technologies to improve test accuracy, measure hard-to-find biomarkers, 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 non-prescription health and beauty supplements. In vitro laboratory diagnostic tests that use the streptavidin-biotin binding mechanism are potentially affected by high circulating biotin concentrations. Biotin can be covalently bound to a variety of targets, from large antibodies to steroid hormones, with minimal impact on specific non-covalent binding with avidin, streptavidin, or neutravidin proteins. Therefore, biotin is frequently used in various forms of immunoassay detection systems.

[0010] Immunoassays are generally classified as either sandwich immunoassays (noncompetitive) or competitive inhibition immunoassays. Streptavidin-biotin binding is typically used to bind biotinylated antibodies in sandwich immunoassays or biotinylated antigens in competitive immunoassays to a streptavidin-coated surface during assay incubation. If the biological sample contains excess biotin, the biotin competes with the biotinylated antibody or antigen for binding to the streptavidin-coated surface, resulting in reduced capture of the biotinylated antibody or antigen. Excess biotin leads to erroneously low results in sandwich immunoassays because the assay signal is directly proportional to the analyte concentration. Excess biotin in competitive immunoassays leads to erroneously elevated results because the assay signal is inversely proportional to the analyte concentration.

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

[0012] Biotin in blood or other samples taken from patients with high biotin intake can cause falsely high or falsely low results in biotin-based immunoassays, depending on the assay design. False test results can lead to improper patient management as well as misdiagnosis.

[0013] Biotin interference thresholds vary widely between assays, even within a single platform. Tests with a biotin interference threshold below 51 ng / mL are considered high-risk tests, or weak immunoassays and competitive methods.

[0014] Biotin-based tests are also susceptible to anti-streptavidin interference, an interference mechanism associated with the use of streptavidin in test designs to capture biotin conjugated to antibodies, proteins, or antigens. Anti-streptavidin antibodies and proteins can significantly interfere with certain laboratory tests, causing erroneous test results. Similar to biotin interference, which can result in reduced test signal and false-low or false-high patient results depending on the assay design and format, anti-streptavidin interference also results in reduced test signal but via 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 be from the bacterium S. avidinii. Many people are exposed to these bacteria in their daily lives and may develop an immunological response to them.

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

[0016] Summary of the Invention Described herein are simple, efficient, and cost-effective methods for conditioning biological samples to manage 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 receiving them for diagnostic purposes. The methods described herein can also manage and mitigate sample-specific interferences resulting from biotin, which can arise 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] Also described herein are methods for enriching or increasing the concentration of biomarkers in biological samples. In particular, the biomarkers can be antigen-specific antibodies, e.g., 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 an antigenic fragment thereof, such as the receptor-binding domain (e.g., amino acids 331-524) 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 streptavidin-labeled beads, which can then serve as capture reagents for SARS-CoV-2 neutralizing antibodies. Typically, when both the N-terminal domain and the receptor-binding domain are used, the fragments are bound to separate beads, which are then mixed to serve as capture reagents.

[0018] In one aspect, provided herein are methods for isolating antigen-specific antibodies from a biological sample, the methods comprising: a) combining the sample with particles comprising a capture moiety to provide a mixture; and b) mixing the mixture to provide a particle complex to the antibody, thereby isolating the antibody from the biological sample. In some embodiments, the capture moiety is the spike protein of SARS-CoV-2. In some embodiments, the capture moiety is the S1 subunit of the spike protein of SARS-CoV-2, 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 can be blood, plasma, serum, or other antibody-containing biological fluid.

[0019] In some embodiments, the isolated antibodies are detected, quantified, or characterized in a serological assay.

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

[0021] In one aspect, provided herein is a method for removing interferences from a biological sample, the method comprising: a) combining the sample with particles comprising a capture moiety to provide a mixture; b) mixing the mixture to provide particle complexes to the interference; and c) removing or eliminating the particle complexes to provide a depletion solution, which can reduce or reduce the amount (e.g., mass, molarity, concentration) of the interference. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows a scheme for a validation and disqualification assay based on the removal (or depletion) of interferences from biological samples by the particles described herein.

[0023] [Figure 2] FIG. 2 shows a scheme of a depletion assay based on the removal (or depletion) of interferences from a biological sample by the lyophilized particles described herein.

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

[0025] [Figure 4] FIG. 4 is a graph showing biotin concentration over time after biotin intake.

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

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

[0028] [Figure 7] FIG. 7 is a graph showing biotin concentration over time after biotin intake.

[0029] [Figure 8] FIG. 8 is a graph showing biotin concentrations after ingestion of different biotin doses.

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

[0031] [Figure 10] FIG. 10 is a graph showing PTH concentrations.

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

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

[0034] [Figure 13] Figure 13 shows total SARS-CoV-2 neutralizing antibody levels in 37 PCR-positive patients with available serial samples. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention Described herein are methods of depleting or enriching a biological sample, comprising combining a particle described herein with a biological sample described herein.

[0036] In one aspect, provided herein is a method for isolating a biomarker from a biological sample, the method comprising: a) combining the sample with particles comprising a capture moiety to provide a mixture; and b) mixing the mixture to provide a particle complex with the biomarker, 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 a spike protein, such as the S1 subunit of the SARS-Cov-2 spike protein, 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 against 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. IgM, IgG, and IgA isotype antibodies that recognize either of these domains are all considered neutralizing. Therefore, it may be advantageous to capture and quantify both types of antibodies to accurately assess the degree of neutralizing antibodies in a biological sample. Similarly, when captured antibodies are used prophylactically or therapeutically, capturing and using both types can establish more robust passive immunity. For example, when antibodies that recognize both the receptor-binding domain and the N-terminal domain are used, variant viruses with mutations in one of these domains are less likely to escape neutralization than when antibodies that recognize only one of these domains are used.

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

[0040] In one aspect, provided herein is a method for removing interferences from a biological sample, the method comprising: a) combining the sample with particles comprising a capture moiety to provide a mixture; b) mixing the mixture to provide particle complexes to the interference; and c) removing or eliminating the particle complexes to provide a depletion solution, which can reduce or reduce the amount (e.g., mass, molality, concentration) of the interference.

[0041] In some embodiments, the method further comprises subjecting the depleted solution to characterization (eg, a diagnostic test).

[0042] In some embodiments, the particles are provided as a lyophilized product (eg, LyoSphere™ (BIOLYPH LLC)).

[0043] In one aspect, provided herein is a method for increasing the accuracy of a diagnostic test, the method comprising: a) combining a biological sample with particles comprising a capture moiety to provide a mixture; b) mixing the mixture to provide particle complexes to interferences; c) removing or eliminating the particle complexes to provide a depleted solution; and d) subjecting the depleted solution to a diagnostic test, thereby increasing the accuracy of the diagnostic test.

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

[0045] In some embodiments, the capture moiety is a human anti-animal antibody (eg, mouse IgG, sheep IgG, goat IgG, rabbit IgG, bovine IgG, porcine IgG, equine IgG). In some embodiments, the capture moiety is a heterophilic antibody (e.g., FR (Fc specific), Fab, F(ab)'2, polymerized IgG (type 1, 2a, 2b IgG and IgG fragments, serum components). In some embodiments, the capture moiety is an assay-specific binding agent (e.g., biotin, fluorescein, anti-fluorescein poly / Mab, anti-biotin poly / Mab, streptavidin, neutravidin). In some embodiments, the capture moiety is an assay-specific signal molecule (e.g., HRP, ALP, acridinium ester, isoluminol / luminol, ruthenium, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) / cyclic ABEI). In some embodiments, the capture moiety is an assay-specific blocker (e.g., BSA, fish skin gelatin, casein, ovalbumin, PVP, PVA). In some embodiments, the capture moiety is an assay-specific conjugate linker (e.g., LC, 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, neutravidin, avidin, polyA, polyDT, an aptamer, an antibody, Fab, F(ab')2, an antibody fragment, a recombinant protein, an enzyme, a protein, a biomolecule, or a polymer. In some embodiments, the capture moiety is biotin, fluorescein, PolyDT, PolyA, an antigen, or the like.

[0046] In some embodiments, the removing or excluding is a separation. In some embodiments, the separation comprises physical separation. In some embodiments, the separation comprises 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 built 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 96-well or 384-well microtiter plates. In some embodiments, the separation comprises chemical separation. In some embodiments, the removing or excluding comprises centrifugation at or above 1000 x g for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes to provide a pellet and supernatant, and removing the supernatant. In some embodiments, the removing or excluding comprises filtration (e.g., through a filter). In some embodiments, the filter has a porosity or molecular weight cutoff (MWCO) sufficiently smaller than the diameter of the particles (e.g., nanoparticles, microparticles). In some embodiments, filtering is by gravity, vacuum, or centrifuge. In some embodiments, removing or excluding comprises magnetization. In some embodiments, magnetization occurs using a strong magnet (e.g., a neodymium magnet) to provide a pellet and supernatant. In some embodiments, the magnet is within a centrifuge rotor. In some embodiments, the magnet is within a disposable pipette tip, cover, or sheath.

[0047] In one aspect, provided herein is a method for isolating a biomarker from a biological sample, the method comprising: a) combining the sample with particles comprising a capture moiety to provide a mixture; b) mixing the mixture to provide a particle complex comprising 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 comprising 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, e.g., the S1 subunit of the SARS-Cov-2 spike protein, or its receptor-binding domain and / or N-terminal domain. In some embodiments, the method for isolating a biomarker from a biological sample is performed before performing a diagnostic test on the biological sample.

[0048] In some embodiments, the wash reagent comprises a human immunoglobulin, e.g., IgG, IgA, and / or IgM, as a capture moiety. In some embodiments, the wash reagent comprises an animal immunoglobulin, e.g., rabbit, goat, or mouse IgG, as a capture moiety. In some embodiments, the wash reagent comprises BSA. It is desirable to use the same microparticles for the wash reagent(s) as for the biomarker capture reagent. In this way, one or more heterophilic interference(s) specific to the analyte antibody and assay reagents (e.g., streptavidin and the beads themselves) can be removed.

[0049] In one aspect, provided herein is a method of determining whether a biomarker is present in a biological sample, the method comprising: a) combining the sample with a capture moiety to provide a mixture; b) combining the mixture with particles comprising the capture moiety 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 the biomarker is present in the biological sample.

[0050] In one embodiment, provided herein is a method for determining whether a biomarker is present in a biological sample, the method comprising: a) combining the sample with particles comprising a capture moiety to provide a mixture; b) mixing the mixture to provide particle complexes against interferences; c) removing or eliminating the particle complexes to provide a depletion solution; d) combining the depletion solution with second particles comprising a second capture moiety to provide a second mixture; e) mixing the second mixture to provide second particle complexes comprising the 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 comprising the biomarker, thereby isolating the biomarker from the biological sample.

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

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

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

[0054] In one aspect, provided herein is a method of enriching the amount of biomarkers in a sample, the method comprising: a) adding particles comprising a capture moiety to a sample to provide a mixture; b) mixing the mixture to obtain particle complexes; c) separating the particle complexes 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 biomarkers from the pellet to provide an enriched sample, enriching the amount of biomarkers in the sample. In some embodiments, the method for enriching biomarkers 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 a tumor marker, e.g., p53. In some embodiments, the tumor antigen is a neoantigen. Some tumor antigens are expressed in a developmentally inappropriate manner, for example, in tissues or maturation stages where they would not normally be expressed at all, or at high levels as they are expressed. This can result in the production of antibodies that recognize the tumor antigen. Other tumor antigens involved in the carcinogenesis process may be mutated, making the tumor antigen immunogenic (neoantigen). Again, antibodies that recognize the altered tumor antigen can be produced. Detection of such autoantibodies can be useful for the detection and diagnosis of cancer, including early detection or changes in the malignant state, and is useful for selecting appropriate treatments. Such tumor antigens can be used as capture reagents for anti-tumor antigen autoantibodies.

[0056] Additional autoantibodies against tumor markers that may be useful for early cancer detection 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 amounts in response to low-circulating or low-abundance cancer proteins, making them promising biomarkers for early cancer detection.

[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), neurofilament light chain (NFL), truncated 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, or soluble Aβ precursor protein (sAPP). In some embodiments, the biomarker is an indicator of a sexually transmitted disease (STD). In some embodiments, the STD is chlamydia, gonorrhea, syphilis, trichomonas, HPV, herpes, hepatitis B, hepatitis C, or HIV. In some embodiments, the biomarker is an indicator of a bacterial infection. In some embodiments, the biomarker is a capture moiety for bacteria. In some embodiments, the biomarker is cleaved from the complex by a cleavage reagent. In some embodiments, the presence of the biomarker is determined by MALDI-MS. In some embodiments, the presence of the biomarker is determined by molecular diagnostics. In some embodiments, the presence of the biomarker is determined by immunoassay.

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

[0059] Referring to Figure 1, a scheme for a confirmatory and reject assay based on the removal (or depletion) of interferences from a biological sample by 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). Particles described herein, for example, particles comprising a surface containing free biotin and / or heterophile antibody capture moieties, are added to the STT to bind to and deplete sample interferences.

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

[0061] 3, a scheme for a depletion assay based on the removal (or depletion) of interferences from a biological sample by a magnetized pipette tip as described herein is shown. To remove an interference as described herein or a biomarker as described herein from a biological sample, a pipette tip containing a magnet is added to the biological sample.

[0062] Separation method The particles described herein can be added to a collection device, such as a primary 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 specimen collection tube or device, prior to the addition of a biological sample.

[0063] The particles described herein can also be added to a sample after collection of the sample in a collection device or after transfer of the sample from the primary collection 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 a liquid sample.

[0064] In some embodiments, the particles described herein are added to a collection device containing a biological sample, hi some embodiments, the particles described herein are added to the collection device prior to the addition of the biological sample.

[0065] In some embodiments, particularly those involving preparative rather than analytical applications, biological samples from multiple donors are pooled before adding particles. At least 10 liters can be processed at one time.

[0066] In one aspect, described herein is a device for releasing particles from a biological sample (i.e., a collection device described herein that includes a screw cap that activates a release mechanism, such as on a urine collection device. For example, the device is a tube with a screw cap that releases particles described herein upon closure of the screw cap.

[0067] In one aspect, described herein are devices that involve chemical release of particles into a container containing a biological sample (i.e., an encapsulated composition or a composition that dissolves into solution at a defined rate or time point). In some embodiments, the devices described herein are configured to delay the addition of the particles described herein, for example, to provide sample pretreatment prior to biomarker enrichment or isolation, or to perform diagnostic tests.

[0068] In some embodiments, samples described herein can be pretreated with chemicals, proteins, blockers, surfactants, or combinations thereof prior to adding the particles described herein, e.g., to adjust pH, deplete or compete sample-specific interferences, and / or manage matrix-specific challenges before adding, introducing, dispersing, or mixing the nanoparticles into the sample to improve the specificity and binding kinetics of the nanoparticles to their target biomarker(s). Delayed addition of nanoparticles to a sample after sample pretreatment can be physically controlled by adding nanoparticles to the sample after sample pretreatment. Nanoparticles can also be present in a sample during sample pretreatment if they are encapsulated, shielded, or protected by a chemical, polymer, or sugar shell, coating, or polymerization, such that the chemical, polymer, or sugar must dissolve before the nanoparticles can be released, added, dispersed, or mixed into the sample. Delayed release of nanoparticles can use chemistries known to those skilled in the art, such as those currently used in delayed drug release technology.

[0069] Preparative affinity separation is a commonly used form of column chromatography. Magnetic particle separation technology can avoid the column clogging problem that some samples can cause. In one example, magnetic particle technology allows for the processing of whole blood or cell-containing blood fractions. Magnetic particle separation technology can also be accomplished in a shorter time than typical column-based affinity separations. Another advantage of particle separation technology is that elution of captured ligands (biomarkers) can be achieved in a smaller volume, resulting in more concentrated molecules without further processing.

[0070] Magnetic separation of particles

[0071] In one aspect, provided herein are methods for removing interferences from biological samples (e.g., prior to diagnostic testing or prior to biomarker enrichment or isolation), or for isolating or separating magnetic particles (e.g., in a primary blood collection tube, in a custom specimen collection device, in a secondary transfer tube or custom sample device, or in a pooled sample). For example, a magnet-based device rapidly (less than 2 minutes; preferably less than 30 seconds) separates magnetic nanoparticles to the side(s) and / or bottom to form an essentially particle-free supernatant. The particle-free supernatant can then be aspirated, drained, or otherwise removed without disrupting 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] Device for magnetic separation of particles

[0073] Provided herein are devices comprising the particles described herein that can be used in the methods described herein to remove or deplete biomarkers, e.g., for diagnostic testing. In some embodiments, the devices comprise a physical mechanism that delays the combination of the particles described herein with a sample described herein. In some embodiments, the devices described herein comprise a time-release mechanism that delays the combination of the particles described herein with a sample described herein.

[0074] Magnetic tube holder. A custom magnetic tube holder, or a custom magnetic tube holder that can be removed from its stand and inserted inside a sample rack for subsequent diagnostic testing of particle-free supernatant. The custom magnetic tube holder can be designed to have a physical opening or clear / transparent plastic (if a magnet or magnet array is not present) in its design, so that the sample tube barcode can still be detected and read by the analyzer, or index tests such as lipemia, hemolysis, cell debris / clot detection, level detection, etc. can still be performed by the analyzer. The sample tube can be a custom sample tube designed with a notch or tongue and groove design to only fit into the custom magnetic tube holder in a specific position to ensure that the opening (space) or clear / transparent plastic of the magnetic tube holder allows the analyzer to see and read the barcode and / or perform index tests such as lipemia, hemolysis, cell debris / clot detection, level detection, etc.

[0075] In some embodiments, use of magnet(s) that can be attached to the sample rack via adhesive, Velcro®, or other methods. When sample tubes containing magnetic nanoparticles are inserted into the sample rack position(s) using the magnet(s), the magnetic nanoparticles rapidly segregate to the side(s) and / or bottom of the sample tube to form an essentially particle-free sample supernatant for diagnostic testing by a sample rack-specific test platform or analyzer.

[0076] The sample rack itself functions as a custom magnetic sample rack that is specific to a given analyzer (e.g., 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, every tube position in the rack has a number of magnets designed to rapidly separate magnetic nanoparticles to the side(s) and / or bottom of the sample tube, forming an essentially particle-free sample supernatant for diagnostic testing.

[0077] In one aspect, provided herein is a device (eg, a separation device) that includes a holder (eg, a test tube holder) for a rack of test tubes, wherein the holder includes a magnet.

[0078] Disposable pipette tip. In one embodiment, the device is a disposable pipette tip containing a custom magnet inserted into the interior of 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 be removed from the sample without disrupting the pellet containing the particles. The disposable tip containing the particles can be discarded if there is no need to measure or characterize what the particles captured (i.e., interference depletion), or it can be inserted into a new tube for particle isolation and characterization in a subsequent diagnostic test (i.e., enrichment). For example, the disposable tip with the particles can be inserted into a secondary transfer tube containing a buffer solution. When the magnet is removed from the tip or turned off (e.g., an electromagnet), the particles disperse freely in the buffer solution.

[0079] In one aspect, provided herein is a device comprising a disposable pipette tip, the tip comprising a magnet.

[0080] Methods for physical separation of particles

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

[0082] For example, magnetic nanoparticles specific for fibronectin and / or other coagulation factors, or clot components / components for subsequent capture or binding of "clots" (in serum), cellular debris (i.e., red blood cell membrane-specific), and / or cellular debris (in serum or plasma) can be integrated into the centrifuge rotor and / or tube holder to increase centrifugation speed and efficiency (shortening spin times to improve laboratory efficiency, workflow, and throughput). This combination of magnetic separation of RCFs or Gs and magnetic nanoparticle complexes (i.e., clots + magnetic beads, cellular 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 shortened to 2 minutes or less (preferably 1 minute or less) by combining centrifugation with magnetic separation / isolation of the magnetic nanoparticle-clot / cellular debris complexes.

[0083] Furthermore, if the nanoparticle or nanoparticles are also specific for one or more different sample interference mechanisms, such as 1, 5, 10, 20, 30, or more different interference mechanisms, these interferences, if present, will be captured by the nanoparticles and removed from the sample after physical separation by centrifugation, or removed from the sample by a combination of centrifugation and magnetic separation as described above.

[0084] These magnetic nanoparticles need not also have specificity for the clots or cellular debris that are 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, where one or more antibodies would be specific for clots and / or cellular debris, while other antibody(ies) and / or antigens would be specific for sample interferences. In this regard, the nanoparticles will specifically bind to both sample interferences and clots and / or cellular 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 cellular debris increases the clotting rate by specific binding by the magnetic nanoparticles, which then magnetically attract everything for magnetic separation and isolation. This bead-based pellet formed by the magnetic field and strength also accelerates clot formation based on the forced proximity of the clot or the clotting factors specifically captured by the nanoparticles and subsequent magnet.

[0086] Chemical separation of particles

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

[0088] In one embodiment, a method for chemical separation of particles is provided, the method 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, partitions the particles into phase A, and the nanoparticle-free sample partitions into phase B, whereupon phase B is tested. Agents for liquid-liquid phase separation (chemical phase separation) can be salts, soluble polymers, and detergents.

[0090] For example, liquid-liquid phase separation can be performed by adding a non-polar solvent, such as hexane, to a polar aqueous sample, and the particles will partition into the non-polar phase, leaving a nanoparticle-free aqueous phase for testing by the diagnostic tests described herein. In some embodiments, the separation methods described herein provide nanoparticles in an organic phase. In some embodiments, the separation methods described herein provide nanoparticles in an aqueous phase.

[0091] 1. A method for isolating particles in a biological sample, comprising: providing a non-polar solvent and an aqueous polar solvent to the particles and the biological sample to provide a non-polar solvent layer and a polar solvent layer; removing the non-polar solvent layer containing the non-polar solvent; and isolating the aqueous polar solvent containing the particles, thereby isolating the particles.

[0092] Sample collection can be adjusted or corrected by the addition and use of an internal standard, such as a deuterated internal standard for LC-MS / MS, before aspirating and discarding the non-polar phase.

[0093] In some embodiments, separation is physical separation that is used in combination with magnetic separation.For example, in one aspect, a device (for example, a magnetized centrifuge or a centrifugal separator that is equipped with a magnet, which helps separation by both gravity and the magnetic force of a magnet) is provided.In one aspect, the present specification provides a device for separating particles described herein, which comprises a magnet and a centrifugal separator.In some embodiments, the device significantly reduces the time of centrifugation.

[0094] Method for Removal of Interference Described herein are methods for removing or minimizing interferences, including depleting (e.g., mitigating, reducing, or managing) known pre-analytical and analytical sources of testing error (e.g., interference) due to other interfering substances, such as hemolysis, lipemia, jaundice, bilirubin, microfibrin clots, cellular debris, blood cells, fibrinogen, drugs, metabolites, supplements, herbal medicines, and multivitamins. In some embodiments, the methods described herein provide methods for removing 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 removing interference prior to diagnostic testing (e.g., diagnostic or biomarker testing, e.g., in clinical trials). In some embodiments, the methods for removing biomarkers described herein are used in clinical trials to improve the accuracy and reliability of diagnostic testing of biomarkers described herein. For example, the methods described herein can be used for patient selection or screening, e.g., for inclusion or exclusion criteria. In some embodiments, the methods described herein or removal or depletion can be used to identify outliers in clinical data or clinical trial results. For example, outliers in clinical data or clinical trial results include false positive or false negative identifications of the biomarkers described herein.

[0095] Depletion is defined as complete if a sufficient amount of interference is captured and / or removed for subsequent interference-free or reduced quantitative, semi-quantitative, or qualitative analysis. Depletion is defined as partial if a sufficient amount of interference(s) or interfering mechanism(s) is captured and / or removed for subsequent semi-quantitative or qualitative analysis, or if a sufficient amount of interference(s) or interfering mechanism(s) and internal standard(s) are captured for quantitative, semi-quantitative, or qualitative analysis by measurement methods that can use internal standards to adjust for recovery of the target analyte(s) or biomarker(s), such as LCMS and LC-MS / MS (i.e., deuterated internal standards) and HPLC (C14 or tritiated internal radioisotope internal standards).

[0096] Depletion does not mean 100% removal of interferences from the sample, but rather that residual interferences no longer lead to erroneous results. However, pre-treatment depletion of a sample can result in 100% removal of interferences when required for a specific assay or purpose, such as subsequent elution and analysis by LC-MS / MS, or for sample pre-analysis, 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 performed in less than 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 performed in less than 1 day.

[0098] interference The methods provided herein reduce, minimize, or eliminate interferences in biological samples, which are substances present in patient specimens that can alter the accuracy of diagnostic test results, for example, by interfering with antibody binding, or that can increase or decrease assay signals through cross-linking, steric hindrance, or autoantibody mechanisms. As used herein, "interference" refers to any endogenous or exogenous substance or combination of endogenous and / or exogenous substances in blood, plasma, serum, CFS, urine, stool, saliva, semen, amniotic fluid, or other bodily fluid or sample matrix such as immunoglobulins (IgG, IgM, IgA, IgE, IgD), proteins, antigens, lipids, triglycerides, cellular constituents, foreign substances, chemicals, drugs, drug metabolites, supplements, vitamins, herbal medicines, foreign bodies (viruses, bacteria (gram positive, gram negative), fungi, yeast), and any foreign body, food or waste products generated by food substances that may interfere with the test and result in erroneous test results by specific or nonspecific interactions with the test materials, formulations, biological and synthetic components, test design, and / or test format.The interference may be heterophilic or heterophile-like interference such as, but not limited to, autoantibodies, rheumatoid factors (RF), human anti-mouse antibodies (HAMA), human anti-animal antibodies (HAAA), e.g., goat, rabbit, sheep, bovine, murine, horse, pig, and donkey polyclonal and / or monoclonal antibodies, assay-specific interferences used in the test design or assay formulation, e.g., chemiluminescent substrates (luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, ruthenium, acridinium esters), fluorescent labels, e.g., fluorescein or other fluorophores and dyes, capture moieties (streptavidin, neutravidin, 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, PolyDT, PolyA, antigens, etc.), conjugation linkers (LC, LC-LC, PEO, PEOn), 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 pretreatment methods and devices for antibody-based diagnostic tests, non-antibody-based diagnostic tests, or subsequent analysis by mass spectrometry (i.e., HPLC, MS, LCMS, LC-MS / MS), radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), chemiluminescence immunoassays (CLIA), molecular diagnostics, lateral flow, point-of-care (PoC), CLIA and CLIA-exempt tests and devices.

[0099] In one aspect, provided herein is a method of removing interference (e.g., biotin) from a biological sample, the method comprising providing particles (which bind to inference) derivatized with a capture moiety. In some embodiments, the interference is biotin.

[0100] In another aspect, the sample can be pretreated with particles (e.g., nanoparticles, microparticles) 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 biomarker removal or enrichment Described herein are methods for enriching or increasing the concentration of biomarkers in a biological sample. "Enrichment" is defined as complete or partial particle capture and binding of target analyte(s) or biomarkers from a biological sample (e.g., human or animal serum, plasma, blood, whole blood, processed blood, urine, saliva, stool (liquid and solid), semen or seminal fluid, cells, tissues, biopsy material, DNA, RNA, or any liquid or solid) to the particles. In some embodiments, enrichment involves washing and concentrating the biological sample, for example, by washing and then isolating the biomarker-specific nanoparticles to allow for the removal or minimization of interferences prior to the biomarker characterization and measurement process.

[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 uses, or to enrich or increase the concentration of biomarkers prior to diagnostic testing, further purification, formulation or other uses.

[0104] After washing or isolating the biomarker-specific particles, the particles can be dispersed, reconstituted, or resuspended in a buffer solution, such as phosphate-buffered saline (i.e., PBS pH 7.2) or an LC-MS / MS-compatible buffer, prior to the characterization or measurement step. This means that the critical characterization or measurement step of the particle-enriched biomarkers is performed in a buffer system rather than in an animal or human matrix, which would introduce or cause matrix effects or bias between biomarkers measured in animal blood, plasma, serum, or urine compared to the same biomarkers measured in blood, plasma, serum, or urine using the same characterization, measurement, or testing method or system. Washing allows for the sample matrix, components, proteins, and cellular components, as well as associated interferences or matrix effects, to be washed away. Similarly, isolated biomarkers can be removed from the animal or human matrix and released into a formulation buffer for therapeutic or prophylactic use.

[0105] Enrichment is defined as complete if a sufficient amount of analyte(s) is captured for subsequent diagnostic testing, e.g., quantitative, semi-quantitative, or qualitative analysis; partial if a sufficient amount of analyte(s) or biomarker(s) is captured for subsequent semi-quantitative or qualitative analysis; or partial if a sufficient amount of target analyte(s) or biomarker(s) and internal standard(s) is captured for quantitative, semi-quantitative, or qualitative analysis by measurement methods that can use internal standards to adjust for recovery of the target analyte(s) or biomarker(s), such as LCMS and LC-MS / MS (i.e., deuterated internal standards) and HPLC (C or tritiated internal radioisotope internal standards). Enrichment is defined as preparative if a sufficient amount of the captured species is obtained for subsequent use in a prophylactic or therapeutic product.

[0106] Provided herein are methods for enriching a biomarker in a sample prior to a diagnostic test, the methods 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 the biomarker 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 an appropriate wash diluent to remove nonspecific material; and g) measuring the amount, mass, molality, concentration, or yield of the biomarker captured by the particles (e.g., nanoparticles, microparticles) using a qualitative, semi-quantitative, or quantitative diagnostic test specific for the biomarker. In some embodiments, the diluent comprises water (e.g., deionized water, water for injection, saline, aqueous buffer solution).

[0107] In some embodiments, the enrichment methods described herein include a washing step. The washing step removes interferences described herein and / or provides a washed, purified, or isolated biomarker of interest (e.g., a biomarker described herein). In some embodiments, the enrichment methods described herein reduce matrix or seed effects. In some embodiments, the enrichment methods described herein are used prior to a diagnostic test comparing two biological samples of different origins. In some embodiments, the enrichment methods described herein are used prior to a diagnostic test comparing an animal sample with a human sample. In some embodiments, the enrichment methods described herein are used prior to a diagnostic test comparing a serum sample with a plasma sample. In some embodiments, the enrichment methods described herein are used with samples that are highly viscous.

[0108] In some embodiments, the enrichment method comprises combining a first biological sample enriched in biomarkers with a second biological sample enriched in biomarkers.

[0109] Provided herein are methods for measuring the amount, mass, molality, concentration, or yield of a target biomarker captured and enriched by a particle (e.g., nanoparticle, microparticle), whereby the biomarker is cleaved by a cleavage reagent as described herein, for example, by changing the pH (e.g., increasing the pH with a base such as sodium bicarbonate, decreasing the pH with an acid such as acetic acid, trichloroacetic acid, sulfosalicylic acid, HCl, formic acid, etc., common 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 displacing agents, competitive elution (e.g., greater than 0.1 M counterligand or analog), 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, denaturing agents (e.g., 2–6 M guanidine HCl, 2–8 M urea, 1% deoxycholic acid, 1% SDS), organic solvents (e.g., alcohol, chloroform, ethanol, methanol, acetonitrile, hexane, DMSO, 10% dioxane, 50% ethylene glycol) They are eluted, dissociated, or liberated from particles (e.g., nanoparticles, microparticles) by disrupting binding interactions using elution strategies such as 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 agitation, sonication, and protein digesting 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 detection antibodies (e.g., AlexaFluor 488 anti-human IgG, AlexaFluor 555 anti-human IgM, AlexaFluor 647 anti-human IgA) or IgA, IgG, and / or IgM antibodies complexed with labeled detection antibodies from the capture beads. The magnetic beads are then ferromagnetically isolated, 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 fluorophore stability for subsequent detection by a fluorometer or fluorescence reader. This neutralization of the acidic elution pH can be important for improving assay accuracy and reproducibility.

[0111] Unless otherwise stated or implied from this disclosure, any embodiment described in connection with any particular method or composition described herein can be used in combination with any of the other embodiments described herein.

[0112] The methods and compositions of various embodiments can be used in combination with any suitable affinity or immunoassay known in the art, including, but not limited to, protein-protein affinity assays, protein-ligand affinity assays, nucleic acid affinity assays, indirect fluorescent antibody assays (IFAS), enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and enzyme immunoassays (EIAs), direct or indirect assays, competitive assays, sandwich assays, CLIA or CLIA-waived tests, LC-MS / MS, analytical assays, and the like.

[0113] A method for both depleting sample interferences and enriching biomarkers from the same sample prior to a diagnostic test, comprising: a) adding chemical and / or biological reagents, additives, or compositions to the sample to block or deplete sample-specific interferences before adding biomarker-specific particles (e.g., nanoparticles, microparticles) to the sample; b) pretreating or incubating the sample with the chemical and / or biological reagents, additives, or compositions before adding the biomarker-specific particles (e.g., nanoparticles, microparticles) to the sample; 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 or isolating the particles (e.g., nanoparticles, microparticles) from the sample and the chemical and / or biological reagents, additives, or compositions; and e) using a diagnostic test to characterize the biomarker(s) captured and enriched by the particles (e.g., nanoparticles, microparticles).

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

[0115] Biomarkers Described herein is a method for isolating or isolating or enriching biomarkers present in biological samples. As used herein, the term "biomarker" is defined as a characteristic biological or biologically derived indicator (e.g., a metabolite) of a process, event, or condition, such as aging or disease. Biomarkers can 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 (e.g., a substance present in a patient sample that can alter the accuracy of a diagnostic test result by interfering with antibody binding or that can increase or decrease the assay signal through cross-linking, steric hindrance, or autoantibody mechanisms) as described herein. In some embodiments, the biomarker is an antibody against an infectious disease antigen, e.g., a viral antigen. Antibodies against an infectious disease antigen can indicate exposure to and recovery from infection with an infectious disease pathogen. 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, e.g., the S1 subunit of the SARS-Cov-2 spike protein, or its receptor-binding domain and / or N-terminal domain."Interference" as used herein may be heterophilic or heterophile-like interference such as, but not limited to, autoantibodies, rheumatoid factors (RF), human anti-mouse antibodies (HAMA), human anti-animal antibodies (HAAA), e.g., goat, rabbit, sheep, bovine, murine, horse, pig, and donkey polyclonal and / or monoclonal antibodies, assay-specific interferences used in the test design or assay formulation, e.g., chemiluminescent substrates (luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, ruthenium, acridinium esters), fluorescent labels, e.g., fluorescein or other fluorophores and dyes, capture moieties (streptavidin, neutron capture, etc.), and the like. Avidin, avidin, polyA, polyDT, aptamers, antibodies, Fab, F(ab')2, antibody fragments, recombinant proteins, enzymes, proteins, biomolecules, polymers) and their binding partners (i.e., biotin, fluorescein, PolyDT, PolyA, antigens, etc.), conjugation linkers (LC, LC-LC, PEO, PEOn), 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® Commercially available blockers, blocking proteins, and polymer-based blocking reagents, such as X-100, triblock copolymers such as Pluronic® and Tetronic, and those from Surmodics and Scantibodies, are typically used in the design of sample pretreatment methods and devices for antibody-based diagnostic tests, non-antibody-based diagnostic tests, or subsequent analysis by mass spectrometry (i.e., HPLC, MS, LCMS, LC-MS / MS), radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), chemiluminescence immunoassays (CLIA), molecular diagnostics, lateral flow, point-of-care (PoC), CLIA, and CLIA-exempt tests and devices. In some embodiments, the biomarkers are found in the biological samples described herein.

[0116] Fibrinogen. Fibrinogen is converted to fibrin by thrombin during tissue and vascular injury, resulting in the subsequent formation of a fibrin-based clot. In some embodiments, the particles described herein (e.g., particle-derived antifibrinogen (e.g., mouse antifibrinogen)) used in the methods described herein bind to fibrinogen in whole blood, allowing for its separation (e.g., chemical separation). Particles that bind to the clot via fibrin can be isolated and removed from the serum after centrifugation for particle-free serum testing. In some embodiments, the biomarker is fibrinogen. In some embodiments, the methods described herein use particle-derived antifibrinogen, eliminating the need for centrifugation of the sample (e.g., blood sample).

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

[0118] In some embodiments, the methods described herein (e.g., enrichment methods) are used to isolate or enrich for the presence of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the following traumatic brain injury biomarkers selected from the group consisting of S100B, GFAP, NLF, NFH, gamma-enolase (NSE), alpha-II spectrin, UCH-L1, total tau, and phosphorylated tau.

[0119] Alzheimer's disease. In one embodiment, the biomarker is related to Alzheimer's disease. There are two biomarkers associated with the severity and magnitude 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 methods described herein (e.g., enrichment methods) are used to isolate or enrich for 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). 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 Diseases. In one embodiment, the biomarkers are for sexually transmitted diseases (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, and HIV 1 and 2. In some embodiments, the methods (e.g., enrichment methods) described herein are used to isolate or enrich for the presence of 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 HIV antibodies. In some embodiments, the biomarkers are in urine (e.g., chlamydia, gonorrhea, trichomonas). In some embodiments, the biomarkers are in blood, serum, or plasma (eg, syphilis, HPV, herpes 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 blood culture, which can take 24 to 48 hours before a positive result can be reflected in a confirmatory test, such as a molecular diagnostic. Given that time is critical to successfully treat a patient to prevent or manage sepsis, methods are described herein for rule-in / rule-out of 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). There are at least 30 biomarkers characteristic of bacterial infection. In some embodiments, the bacterial biomarker is selected from the group consisting of biomarkers for 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 a yeast pathogen (e.g., a yeast pathogen associated with a bloodstream pathogen).

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

[0123] In some embodiments, the gram-negative bacterium is Acinetobacter baumannii, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Enterobacteriaceae, Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, or Serratia marcescens.

[0124] In some embodiments, the yeast pathogen is Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, or Candida tropicalis.

[0125] In some embodiments, mass spectrometry is then performed. A cleavage agent (e.g., a reducing agent (e.g., DTT or TCEP)) is added to the bacteria-particle binding complex to cleave the linker (i.e., the linker that connects the particle to the surface capture moiety). The resulting bacteria are grown in culture or analyzed by MALDI-TOF mass spectrometry.

[0126] A method is envisioned in which a cleaving agent (e.g., a reducing agent (e.g., DTT or TCEP)) is added to the bacteria-particle bound complex to cleave the linker (i.e., the linker that conjugates the particle to the surface capture moiety). The resulting bacteria are grown in culture, analyzed by MALDI-TOF mass spectrometry, or analyzed by a molecular diagnostic 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 moiety on the magnetic particle. Whole blood, a blood fraction, plasma, or serum is mixed with the magnetic particles so that the antigen-specific antibody can bind to the capture moiety (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 moiety. This can be done on an analytical scale, and the antibody can be subjected to mass spectrometry (including, for example, LC-MS to separate multiple antibody species that may be present), Edman degradation, and other proteochemical analytical methods to determine the protein sequence of the antibody. The sequence information can be used to construct monoclonal antibodies (paratopes) with the same specificity(s). This can also be done on a preparative scale, and the enriched or isolated antigen-specific antibodies can be used in the clinic for treatment or prevention. In some embodiments, the antibody that recognizes a pathogen-specific 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 and / or receptor-binding domain.

[0128] Thyroid function. TSH concentrations are measured as part of thyroid function testing in patients suspected of thyroid hormone excess (hyperthyroidism) or deficiency (hypothyroidism). In some embodiments, the methods described herein are used to assess thyroid function. In some embodiments, the biomarker is an antigen (e.g., TSH). In some embodiments, the capture moiety is an autoantibody (e.g., free autoantibody, complexed autoantibody) with specificity for the antigen (e.g., TSH).

[0129] In some embodiments, the interference (which affects the measurement of thyroid stimulating hormone, free thyroxine, and free triiodothyronine) is macro-TSH, biotin, anti-streptavidin antibodies, anti-ruthenium antibodies, thyroid hormone autoantibodies, or heterophilic antibodies.

[0130] Cardiac function. In some embodiments, the methods described herein are used to evaluate cardiac function. Elevated levels of circulating troponin in the blood are biomarkers for cardiac disorders, such as myocardial infarction. Cardiac I and T are specific indicators of myocardial damage. Troponin subunits are also markers of cardiac health. Specifically, cTnI and cTnT are biomarkers for acute myocardial infarction (AMI), such as type 1 and type 2 myocardial infarction, unstable angina, postoperative myocardial trauma, and related diseases. In some embodiments, the biomarker is free cTnI, free cTnT, binary cTnI-TnC, or ternary cTnI-TnC-TnT. In some embodiments, the biomarker is an indicator of heart failure. In some embodiments, the biomarker is an indicator of stroke (e.g., 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 by reference in their entireties). In some embodiments, the biomarker is an indicator of fibrosis (e.g., as described in http: / / www.onlinejacc.org / content / 65 / 22 / 2449, which is incorporated by reference in its entirety). In some embodiments, the biomarker is for the diagnosis of acute coronary syndrome (ACS). In some embodiments, the biomarkers are for cardiac troponin (I, IC, ICT, T) and other cardiac troponin fragments, natriuretic peptides (BNP, ANP, CNP), N-terminal fragments (i.e., NT-proBNP, NT-proCNP), glycosylated, non-glycosylated, CRP, myoglobin, creatine kinase (CK), CK-MB, sST2, GDF-15, and galectin-3.

[0131] In some embodiments, precision and accuracy are enhanced by the ability to test large sample volumes (i.e., 1 mL, 10 mL, 100 mL, 1000 mL, etc.) to improve the accuracy of detection of very dilute or low concentration biomarker(s), as well as very small sample volumes (i.e., neonates, children, elderly individuals), which are typically not testable today or require sample dilution prior to testing (which compromises test sensitivity, precision, and accuracy). In some embodiments, the biological sample is 1 mL, 10 mL, 100 mL, 1000 mL, or more in volume. In some embodiments, the biological sample is 0.5 mL, 0.25 mL, 0.1 mL, 0.05 mL, or less in volume.

[0132] Also provided herein are methods or test methods that use particle sample pretreatment to aid in biomarker enrichment prior to diagnostic testing by enabling a washing step or particle isolation followed by selective release or elution of captured biomarker(s), or selective release or elution of capture moiety-biomarker complexes from the particles prior to a biomarker characterization step.

[0133] The use of a "cleavage reagent" or "release agent" that breaks the bond between the capture moiety on the particle surface and the biomarker, e.g., acidic or basic pH, high molarity salt, sugar, chemical displacer, detergent, surfactant, and / or chelator, or a combination thereof, leaving only the biomarker, without displacing or eluting the capture moiety. After washing or isolating the particles from the sample matrix using a magnet(s), the particles can subsequently be treated with an elution solution containing a release agent(s) to selectively release the biomarker and / or labeled detection reagent into solution. The particles can be rapidly (less than 2 minutes; ideally less than 30 seconds) isolated to the side(s) and / or bottom of the sample device (vial, test tube, etc.) to form an essentially particle-free sample supernatant. The particle-free supernatant can then be aspirated and dispensed into a separate transfer tube without disrupting the particle-containing pellet, or injected directly into an analytical system (i.e., LC-MS / MS or MALDI-TOF) for biomarker testing. In some embodiments, the supernatant containing the eluted components is transferred to a neutralization buffer to re-establish less harsh conditions (such as pH) and preserve the biomarkers and / or labels from degradation or denaturation by the elution solution. For example, the cleavage reagents or releasing agents described herein may be eluted using, for example, pH (e.g., increasing the pH with a base such as sodium bicarbonate, decreasing the pH with an acid such as acetic acid, trichloroacetic acid, sulfosalicylic acid, HCl, formic acid, or common pH elution buffers such as 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 displacement agents, competitive elution (e.g., greater than 0.1 M counterligand or analog), 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, etc.). Disrupt the binding interactions or cleavable bonds described herein between the particles described herein and the capture moieties described herein using elution strategies such as solvents (e.g., 2-6 M guanidine HCl, 2-8 M urea, 1% deoxycholic acid, 1% SDS), surfactants, detergents, concentrated inorganic salts, denaturants (e.g., 2-6 M guanidine HCl, 2-8 M 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 agitation, sonication, and protein digesting enzymes (pepsin, trypsin), as well as combinations thereof. In some embodiments, the supernatant containing the eluted components is transferred to a neutralization buffer to re-establish less harsh conditions (such as pH) and preserve the biomarkers and / or labels from degradation or denaturation by the elution solution.

[0134] Characterization Method Described herein are methods for depleting and / or enriching biomarkers for subsequent characterization or diagnostic testing. Characterization of a biomarker (e.g., an interference) described herein includes identification and / or quantification of the biomarker (e.g., an interference) described herein.

[0135] Characterization may include detection and / or quantification of biomarkers, such as antigen-specific antibodies. By binding antigen-specific antibodies to particles, they can be isolated from other specificities, released into a smaller volume than the original sample, and optionally concentrated. Typical diagnostic assays for specific antibodies detect them without first isolating them, for example, in relation to whole serum. This makes absolute quantification difficult. Antibody activity is often characterized as a titer based on how diluted the serum can be while still retaining activity. By capturing the species of interest and separating it from the rest of the immunoglobulins in the serum, simple protein assays can be used to quantify how much specific antibody is present. Furthermore, each isotype of interest can be quantified separately in parallel aliquots, or multiplexed in a single aliquot if conjugated to a distinct label, by comparing it to a standard curve generated using known amounts of bead-bound immunoglobulins using isotype-specific reagents. The standard curve can be for immunoglobulins in general or for specific isotypes, the latter of which can be generated separately or in a multiplexed format. IgM is typical of early responses, while IgG and IgA are typical of more mature and effective immune responses. 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 antibodies recognize 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 Described herein are particles for the isolation, depletion, and / or enrichment of biological samples. In some embodiments, the particles comprise a cleavable bond and a capture moiety (e.g., a particle surface functionalized to present one capture moiety). In some embodiments, the particles comprise a non-cleavable bond and a capture moiety (e.g., a particle surface functionalized to present one capture moiety). In some embodiments, the particles described herein comprise a capture moiety (e.g., a capture moiety with high specificity for a biomarker described herein). In some embodiments, the particles described herein (e.g., the surface of a particle described herein, the particle surface not bound to a capture moiety described herein) are inert (e.g., do not exhibit significant binding to a biomarker described herein). In some embodiments, the particles described herein can be used in the diagnostic tests described herein without further modification of the particle or the diagnostic test. In some embodiments, the particles described herein can be added to and removed from a sample without altering the sample (e.g., without adding or removing additional biomarkers (e.g., interferences)).

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

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

[0139] In some embodiments, the particle surface comprises an organic polymer or an organic copolymer, and the organic polymer or organic copolymer is hydrophobic. In some embodiments, the particle (e.g., nanoparticle, microparticle) surface comprises, but is not limited to, ceramic, glass, polymer, copolymer, metal, latex, silica, colloidal metal such as gold, silver, or alloy, organic polymer or copolymer such as a material selected from the group consisting of polystyrene, derivatized polystyrene, poly(divinylbenzene), styrene-acylate copolymer, styrene-butadiene copolymer, styrene-divinylbenzene copolymer, poly(styrene-oxyethylene), polymethyl methacrylate, polymethacrylate, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, N,N'-methylenebisacrylamide, polyolefin, polyethylene, polypropylene, polyvinyl chloride, polyacrylonitrile, polysulfone, poly(ether sulfone), pyrolyzable materials, block copolymers, and copolymers thereof, silicone or silica, methylol melamine, biodegradable polymer such as dextran or poly(ethylene glycol)-dextran (PEG-DEX), or a combination thereof.

[0140] As used herein, "blocker" refers to a protein, polymer, surfactant, detergent, or combination thereof. In some embodiments, binding of a capture moiety on a particle (e.g., nanoparticle, microparticle) described herein is blocked with 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, and F68, Triton® X-100, polysorbate 20 (Tween®-20), Tween® The blocker may be selected from the group consisting of non-ionic detergents such as 80 (non-ionic), zwitterionic detergents such as CHAPS, ionic detergents such as sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl detergent, sugars such as sucrose, and commercially available blockers such as heterophilic blocking reagent (Scantibodies), MAK33 (Roche Diagnostics), immunoglobulin inhibiting reagent (IIR) (Bioreclamation), Heteroblock (Omega Biologicals), Blockmaster (JSR), TRU Block (Meridian Life Sciences), and StabilCoat® & StabilGuard® (Surmodics). In some embodiments, the blocker is bound (e.g., covalently or non-covalently) to the particles described herein. In some embodiments, the blocker is not bound (e.g., covalently or non-covalently) to the particles described herein.

[0141] Cleavable bond. In one embodiment, the capture moiety is bound to the biomarker by a cleavable bond as described herein. The cleavable bond can be a covalent or non-covalent bond. Examples of non-covalent bonds include affinity, ionic, van der Waals (e.g., dipole / dipole or London forces), hydrogen bonds (e.g., between polynucleotide duplexes), and hydrophobic interactions. When the association is non-covalent, the association between the entities is preferably specific. Non-limiting examples of specific non-covalent associations include the binding interaction between biotin and a biotin-binding protein, such as avidin, captavidin, SA, neutravidin, a fragment of SA, a fragment of avidin, a fragment of neutravidin, or a mixture thereof; the binding of a biotinylated Fab, a biotinylated immunoglobulin or a fragment thereof, a biotinylated small molecule (e.g., a hormone or receptor ligand), a biotinylated polynucleotide, a biotinylated macromolecule (e.g., a protein or natural or synthetic polymer) to a biotin-binding protein, such as avidin, SA, neutravidin, a fragment of SA, a fragment of avidin, a fragment of neutravidin, or a mixture thereof; the binding of a substrate to its enzyme; the binding of a glycoprotein to a lectin specific for the glycoprotein; the binding of a ligand to a receptor specific for the ligand; the binding of an antibody to an antigen against which the antibody is raised; and the formation of a duplex between a polynucleotide and a complementary or substantially complementary polynucleotide.

[0142] A cleavable bond, such as a disulfide bond (RSSR), is used to immobilize or attach the capture moiety (i.e., antibody or antibody fragment, e.g., SH-Fab) to the particle. After washing or isolating the particles from the sample matrix, the particles can subsequently be treated with a solution containing a reducing agent, such as TCEP or DTT, to cleave the disulfide bond and release the capture moiety-biomarker complex into solution. The particles can be rapidly isolated (in less than 2 minutes; ideally, in less than 30 seconds) to the side(s) and / or bottom of the sample device (vial, test tube, etc.) to form an essentially particle-free sample supernatant. The particle-free supernatant can then be aspirated without disrupting the particle-containing pellet and dispensed into a separate transfer tube, or directly injected into an analytical system (i.e., LC-MS / MS or MALDI-TOF, or molecular diagnostics such as FilmArray blood culture identification panels) for testing the capture moiety-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 captavidin, avidin, and biotin.

[0145] Capture moiety. Provided herein are particles comprising a capture moiety that binds to an interference described herein or a biomarker described herein. As referred to herein, a "capture moiety" is selected from the group consisting of an antibody, a binding fragment of an antibody, an IgG, IgM, IgA, IgE, or IgD receptor, a receptor ligand, a hormone, a hormone receptor, an enzyme, an enzyme substrate, a single-stranded oligonucleotide, a single-stranded polynucleotide, a double-stranded oligonucleotide, a double-stranded polynucleotide, an antigen, a peptide, a polymer, an aptamer, and a protein.

[0146] In some embodiments, the capture moiety is a protein. The protein can be, for example, a monomer, a dimer, a multimer, or a fusion protein. In certain embodiments, the protein comprises at least one of albumin, e.g., an antibody, an antibody fragment, BSA, ovalbumin, a fragment of BSA, a fragment of ovalbumin, mouse IgG, polymerized mouse IgG, antibody fragments of mouse IgG (Fc, Fab, F(ab')2) and different subclasses (IgG1, IgG2a, IgG2b, IgG3, IgE, IgD) targeting HAMA and RF interference mechanisms, purified animal polyclonal antibodies (i.e., bovine, goat, mouse, rabbit, sheep) targeting HAAA interference, streptavidin targeting MASI interference, ALP, HRP, BSA (conjugated to isoluminol, ruthenium, acridinium), or a mixture thereof. In some embodiments, the capture moiety is a structural or surface-exposed antigen of a pathogen, e.g., a bacterium or virus. In some embodiments, the capture moiety 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 moiety is a human anti-animal antibody (eg, mouse IgG, sheep IgG, goat IgG, rabbit IgG, bovine IgG, porcine IgG, equine IgG). In some embodiments, the capture moiety is a heterophilic antibody (e.g., FR (Fc specific), Fab, F(ab)'2, polymerized IgG (type 1, 2a, 2b IgG and IgG fragments, serum components). In some embodiments, the capture moiety is an assay-specific binding agent (e.g., biotin, fluorescein, anti-fluorescein poly / Mab, anti-biotin poly / Mab, streptavidin, neutravidin). In some embodiments, the capture moiety is an assay-specific signal molecule (e.g., HRP, ALP, acridinium ester, isoluminol / luminol, ruthenium, ABEI / cyclic ABEI). In some embodiments, the capture moiety is an assay-specific blocker (e.g., BSA, fish skin gelatin, casein, ovalbumin, PVP, PVA). In some embodiments, the capture moiety is an assay-specific conjugate linker (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, neutravidin, avidin, polyA, polyDT, aptamer, antibody, Fab, F(ab'), antibody fragment, recombinant protein, enzyme, protein, biomolecule, polymer, or molecularly imprinted polymer. In some embodiments, the capture moiety is biotin, fluorescein, PolyDT, PolyA, antigen, etc.

[0148] In some embodiments, the capture moiety binds to biotin (eg, avidin, streptavidin, neutravidin, CaptAvidin, anti-biotin antibodies, antibody fragments, apmers, molecularly imprinted polymers, etc.).

[0149] Some embodiments provide a binding surface with two or more different capture moieties.

[0150] Generation of Capture Moieties. In one aspect, a method for producing a capture moiety is provided, comprising producing or generating a complex-specific or conformation-specific antibody against a free autoantibody or an autoantibody complex. Free autoantibodies are autoantibodies that are not yet complexed to their antigen target. Complexed autoantibodies are autoantibodies that have formed a complex with their antigen target.

[0151] In one aspect, a method for producing a capture moiety is provided, comprising producing or generating 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, complex-specific or conformation-specific antibodies can be made into an autoantibody complex, such as MTSH, which can be purified from human serum and used as a capture moiety. In this manner, the produced antibodies will have specificity only for hIgG or hIgM complexes with TSH. MTSH can be purified based on technology and published methods or by those skilled in the art of protein biochemistry and purification. In some embodiments, patients with autoimmune diseases with the highest likelihood of autoantibody assay interference are used to produce or generate autoantibodies. See, e.g., HyTest SES assay for BNP, WO 2014114780, WO 2016113719 and WO 2016113720, the references of which are cited in their entireties.

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

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

[0154] In some embodiments, the autoantibody is a complexed autoantibody (e.g., MTSH). In some embodiments, the capture moieties described herein are antibodies generated with specificity for the complexed autoantibody or with confirmatory specificity for hIgG and / or hIgM already bound to its antigen target, such as MTSH.

[0155] The following is a non-limiting list of substances that can function as one member of a binding pair consisting of an analyte-binding agent (capture moiety) and an analyte, or alternatively as the other member, depending on the application for which the affinity assay is designed. Such substances can be used, for example, as capture moieties (analyte-binding agents) or to generate capture moieties 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 as analytes in a sample. In certain embodiments, analyte-binding capture moieties can be used to detect these substances as analytes in a sample. Alternatively, the substances disclosed herein can be used according to various embodiments to associate with a solid support surface and capture molecules that interact with them (e.g., antibodies or fragments thereof specific for the listed substances, binding proteins, or enzymes).

[0156] A non-limiting list of substances that can function as one member of a binding pair consisting of an analyte binding agent (capture moiety) and an analyte, or alternatively as the other member, 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-1 RA, TNF-α, IFN-γ, GM-CSF; isoforms of PSA (prostate-specific antigen) such as PSA, pPSA, and BPSA; PSA, non-α1-antichymotrypsin-complexed PSA, α1-antichymotrypsin-complexed PSA; prostate 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, B-type 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; antithyroglobulin; IgE, IgG, IgG1, IgG2, IgG3, IgG4, B. anthracis protective antigen, B. anthracis lethal factor, B. anthracis spore antigen, F. tularensis LPS, S. aureus enterotoxin B, Y. pestis capsular F1 antigen, 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, HCVAb, anti-p53, histamine; neopterin; s-VCAM-1, serotonin, sFas, sFas ligand, sGM-CSFR, s1CAM-1, thymidine kinase, IgE, EPO, intrinsic factor Ab, haptoglobulin, anticardiolipin, anti-dsDNA, anti-Ro, Ro, anti-La, anti-SM, SM, anti-nRNP, anti-histone, anti-Scl-70, Scl-70, antinuclear antibody, anticentromere antibody, SS-A, SS-B, Sm, U1-RNP, Jo-1, CK, CK-MB, CRP, ischemia-modified albumin, HDL, LDL, oxLDL, VLDL, troponin T, troponin I, troponin C, microalbumin, amylase, ALP, ALT, AST, GGT, IgA, IgG, prealbumin, antistreptolysin, chlamydia, CMV IgG, Toxo-IgG, Toxo-IgM, Apolipoprotein A, Apolipoprotein B, C3, C4, Properdin factor B, Albumin, α1-Acid glycoprotein, α1-Antitrypsin, α1-Microglobulin, α2-Macroglobulin, Antistreptolysin 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 subclasses, immunoglobulin M, immunoglobulin M (CSF), kappa light chain, lambda light chain, lipoprotein(a), microalbumin, prealbumin, properdin factor B, rheumatoid factor, ferritin, transferrin, transferrin (urine), rubella IgG, thyroglobulin Phosphoantibody, Toxoplasma IgM, Toxoplasma IgG, IGF-I, IGF-binding protein (IGFBP)-3, hepsin, pim-1 kinase, E-cadherin, 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, HIVp24, HIV 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, the 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 for which the affinity assay is designed, suitable materials that may function as one or alternatively as the other member of a binding pair consisting of an analyte-binding agent (capture moiety) and an analyte and that can be used in the presently disclosed embodiments also include moieties, such as antibodies or fragments thereof, specific for any of the WHO International Biological Reference Preparations maintained, 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 lists materials known in the art, and which is incorporated herein by reference).

[0158] A partial list of such appropriate international reference standards, identified in parentheses after the substance by its 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 cerebral CJD control and human cerebral sporadic CJD preparation 1 and human cerebral sporadic CJD preparation 2 and human cerebral variant CJD (none; WHO TRS ECBS Report No. 926, 53.sup.rd, respectively). Report cited, brain homogenate), human serum complement components C1q, C4, C5, factor B, and full complement CH50 (W1032), human serum immunoglobulin E (75 / 502), human serum immunoglobulin G, A, and M (67 / 86), human serum proteins 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 of 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, and X (99 / 826), human blood coagulation factor II and X concentrate (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 homozygote, FVL heterozygote (03 / 254, 03 / 260, 03 / 248), human blood coagulation factor VII concentrate (97 / 592), human blood coagulation factor VIIa concentrate (89 / 688), human antisyphilis serum (HS), human anti-tetanus immunoglobulin (TE-3), human antithrombin concentrate (96 / 520), human plasma antithrombin (93 / 768), human antithyroglobulin 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 serum types 1, 2, and 3 (66 / 202), human anti-rabies immunoglobulin (RAI), human anti-rubella immunoglobulin (RUBI-1-94), anti-smooth muscle serum (W1062), human anti-double-stranded DNASerum (Wo / 80), human anti-E complete blood typing 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), human These include alpha-fetoprotein (AFP), ancrod (74 / 581), human anti-A blood typing serum (W1001), human anti-B blood typing serum (W1002), human anti-C complete blood typing serum (W1004), anti-D (anti-Rh0) complete blood typing reagent (99 / 836), human anti-D (anti-Rh0) incomplete blood typing serum (W1006), and human anti-D immunoglobulin (01 / 572).

[0159] Other examples of suitable substances that may function as one member of a binding pair consisting of an analyte-binding agent (capture moiety) and an analyte, or alternatively as the other member, depending on the application for which the affinity assay is designed, include compounds that can be used as haptens to generate antibodies capable of recognizing the compounds, including, but not limited to, hormones including, but not limited to, progesterone, estrogen and testosterone, progestins, corticosteroids and dehydroepisterone, and any salt, ester or ether of any non-protein / non-polypeptide antigen listed as an International Reference Standard by the WHO. A partial list of such suitable 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 Catalogue of International Biological Reference Preparations (available on the WHO website, e.g., at http: / / www.who.int / bloodproducts / ref_materials / , updated June 30, 2005), which is incorporated herein by reference. The methods and compositions described herein can include the above-mentioned WHO reference standards or mixtures containing the reference standards.

[0160] Other examples of substances that can function as one or the other member of a binding pair consisting of an analyte binder (capture moiety) and an analyte, depending on the application for which the affinity assay is designed, include drugs of abuse. Drugs of abuse include, for example, the following list of drugs and their metabolites (e.g., metabolites present in blood, urine, and other biological materials), as well as any salts, esters, or ethers thereof: heroin, morphine, hydromorphone, codeine, oxycodone, hydrocodone, fentanyl, Demerol, methadone, Darvon, Stadol, Talwin, Paregoric, Buprenex; stimulants such as amphetamine, methamphetamine; methylamphetamine, ethylamphetamine, methylphenidate, ephedrine, pseudoephedrine, ephedra, mahango, etc. huang), methylenedioxyamphetamine (MDS), phentermine, phenylpropanolamine; amiphenazole, bemigride, benzphetamine, bromethane, chlorphentermine, clopropamide, crotethamide, diethylpropion, dimethylamphetamine, doxapram, etamivan, fencamfamin, meclofenoxate, methylphenidate, nikethamide, pemoline, pentetrazole, phendimetrazine, phenmetrazine, phentermine, phenylpropanolamine, picrotoxin, pipradol, prolintane, strychnine, synephrine, phencyclidine and angel dust PCP, ketamine, etc. analogs thereof; for example, barbiturates, glutethimide, methaqualone, and depressants such as meprobamate, methohexital, thiamyl, thiopental, amobarbital, pentobarbital, secobarbital, butalbital, butabarbital, talbutal, and aprobarbital, phenobarbital, and mephobarbital; benzodiazapenes such as estazolam, flurazepam, temazepam, triazolam, midazolam, alprazolam, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, prazepam, quazepam, clonazepam, and flunitrazepam;These include GBH drugs such as gamma-hydroxybutyrate and gamma-butyrolactone; glutethimide, methaqualone, meprobamate, carisoprodol, zolpidem, and zaleplon; cannabinoid drugs such as tetrahydracannabinol and analogs; cocaine and 3-4 methylenedioxymethamphetamine (MDMA); and hallucinogens such as mescaline and LSD. [Example]

[0161] Example 1: Biotin interference depletion after high-dose biotin intake Endogenous (non-spiked) biotin samples were collected serially. Baseline serum samples were obtained from five apparently healthy adult volunteers (four males, one female) via antecubital vein blood collection in BD brand Vacutainer™ 10 mL red-top tubes. Each volunteer then received a 20 mg dose of biotin (4 x 5 mg, Finest Nutrition Biotin 5000 mcg Strawberry, Quick Dissolve, product number 938508, sold by Walgreens). Serum samples were obtained 1, 3, 6, 8, and 24 hours after biotin ingestion. Blood was allowed to clot at room temperature for 1 hour and centrifuged at 2,000 rpm for 15 minutes in a Beckman Allegra 6R benchtop centrifuge. For each time point, serum samples from each volunteer were pooled, mixed for 15 minutes at room temperature, 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 serially collected samples using a free biotin ELISA. Biotin serum samples were tested using the Immundiagnostik IDK® Biotin ELISA Kit (Part No. K8141, Lot No. 180906, measuring range 48.1-1100 pg / mL). Samples exceeding the kit's measuring range were diluted with the kit's sample dilution buffer. Samples collected 1, 3, 6, and 8 hours after biotin ingestion were assumed to be in the range of 50,000-500,000 pg / mL and were diluted 1:1000. Samples collected 24 hours after biotin ingestion were assumed to be near or below 20,000 pg / mL and were diluted 1:20. When samples were tested according to the ELISA kit protocol, biotin concentrations were still significantly elevated 8 hours (60–107 ng / mL; n = 5) and 24 hours (24–32 ng / mL; n = 4) after 20 mg of biotin ingestion (Figure 4 and Table 1). [Table 1]

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

[0164] In the first study, increasing amounts (mg) of VERAPREP Biotin Reagent were added to different aliquots of the same high-biotin (370 ng / mL) endogenous serum sample to determine the amount of reagent required to deplete 100% of the free biotin in the sample. 20 μL of VERAPREP Biotin Reagent (230 nm diameter, 32 μg streptavidin per mg beads) was added to each 200 μL aliquot of serum sample collected 1 hour after 20 mg biotin uptake, mixed by gentle inversion for 10 minutes at room temperature, 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 No. K8141, Lot No. 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, a 200 μL sample, and only 0.39 mg of reagent (Figure 5).

[0165] In a second study, increasing amounts (mg) of two different VERAPREP Biotin reagents were added 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 mg of beads) or 20 μL of 550 nm VERAPREP Biotin reagent (4 μg streptavidin per mg of beads) was added to each 200 μL aliquot of serum collected 1 hour after 50 mg biotin uptake, mixed by gentle inversion for 10 minutes at room temperature, and magnetically separated for 10 minutes using a Dexter LifeSep 1.5S magnet. 175 μL of serum supernatant was carefully aspirated and measured using an Immundiagnostik IDK® Biotin ELISA kit (part number K8141, lot number 180906), and 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 0.75 mg of reagent, while the 550 nm VERAPREP Biotin reagent depleted only 89% of free biotin using 1.86 mg of reagent. 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 biotin binding capacity per mg of beads, and / or by adding increasing concentrations or amounts (mg) of VERAPREP Biotin reagent, resulting in smaller bead diameters and increased 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 apparently healthy adults aged 25–46 years, one 65-year-old with type 2 diabetes) had fasting serum samples collected at baseline via antecubital vein sampling in BD brand Vacutainer™ 10 mL red-top tubes. Each volunteer then received over-the-counter (OTC) biotin at a dose 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 ingestion. For the 100 and 200 mg doses, serum samples were collected 1, 6, and 24 hours after biotin ingestion. Blood was allowed to clot at room temperature for 1 hour and then centrifuged at 2,000 rpm for 15 minutes in a Beckman Allegra 6R tabletop centrifuge. For each time point and biotin dose, serum samples from each volunteer were pooled, mixed for 15 minutes at room temperature, aliquoted into 1.2 mL aliquots in 2 mL cryovials, and frozen at -80°C. All samples were sent for LC-MS / MS biotin measurement at the University of Washington, Department of Laboratory Medicine, 1959 NE Pacific Street, Seattle, WA 98195. At the 20 mg dose, biotin levels were highest at 1 hour [96–179 ng / mL]; at 6 hours, all five volunteers still had serum biotin levels above 15 ng / mL [17–35]; at 8 hours, four of the five volunteers had serum biotin levels above 15 ng / mL [16–28]; and at 24 hours, volunteer 1, a known type 2 diabetic, had a biotin level above 15 ng / mL

[18] (Figure 7).

[0167] For the 100 and 200 mg doses, biotin levels were highest at 1 hour, ranging from 294 to 459 ng / mL for the 100 mg dose and 610 to 861 ng / mL for the 200 mg dose. At 6 hours, volunteers receiving either 20 mg or 100 mg biotin had serum biotin levels above 15 ng / mL, ranging from 17 to 35 ng / mL for the 20 mg dose and 95 to 347 ng / mL for the 200 mg dose. At 24 hours, two volunteers receiving 100 mg biotin, both with known diabetes, had biotin levels above 15 ng / mL: Volunteer 1 (84 ng / mL) and Volunteer 6 (54 ng / mL) (Figure 8).

[0168] Four samples were selected with high endogenous biotin levels [294-861 ng / mL] as measured 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 1 hour after biotin ingestion had PTH results below 1.57 pg / mL, or below the lower 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 below 500 ng / mL by LC-MS / MS and were pretreated with 0.5 mg of reagent, and samples 2 and 3 had biotin levels above 500 ng / mL by LC-MS / MS and were pretreated with 1.5 mg of reagent using the following protocol. 1. Remove the VeraPrep Biotin reagent vial from storage and vortex at medium speed for a minimum of 10 seconds to mix well and resuspend the reagent. 2. Insert the reagent vial into the foam vial holder. 3. Insert an empty 2 ml microtube (SARSEDT Order Number 72.694) into the LifeSep® 1.5S magnet until the collar of the tube touches the magnetic frame. 4. Dispense 200 μL (0.5 mg) or 600 μL (1.5 mg) of well-mixed reagent into an empty tube and separate the reagent on a magnet for >30 seconds to form a reagent pellet. 5. Without disturbing the reagent pellet, carefully aspirate and discard all storage buffer supernatant (approximately 200 µL or approximately 600 µL). 6. Dispense 400 μL of well-mixed serum or plasma sample into the tube containing the reagent pellet. 7. Screw the cap back onto the tube, remove the tube from the magnet, and vortex at medium speed for a minimum of 10 seconds to mix well 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 collar of the tube contacts the magnet frame. 10. Magnetically separate the reagents for >4 minutes to form a reagent pellet. 11. Carefully aspirate the sample supernatant without disturbing the reagent pellet and dispense the sample into a 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 specimen is ready for testing.

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

[0171] While samples taken 1 hour after biotin ingestion had high levels of biotin interference (294–861 ng / mL) per LC-MS / MS and undetectable PTH values ​​(<1.57 pg / mL) by PTH Intact ELISA, samples taken 1 hour after biotin ingestion pretreated with VeraPrep Biotin had physiologically normal biotin values ​​(<1.1 ng / mL) per Biotin ELISA kit and normal PTH values ​​(26.7–52.0 pg / mL) by PTH Intact ELISA (Figures 9 and 10). Comparing the PTH values ​​before VeraPrep Biotin sample treatment to baseline PTH values ​​resulted in a recovery of 95–113% (mean recovery of 105%) (Figure 10). The significant difference in test results after VeraPrep biotin sample pretreatment, or the significant increase in PTH values ​​in this PTH Intact ELISA sandwich immunoassay, confirms that biotin interference was clinically significant in all four samples tested.

[0172] Example 3: Low Abundance Biomarker Enrichment 550 nm superparamagnetic nanoparticles coated with streptavidin and subsequently coated with biotinylated anti-TSH antibody (VERAPREP Concentrate TSH Reagent) or biotinylated anti-PTH monoclonal antibody (VERAPREP Concentrate PTH Reagent) were used to enrich very low levels of biomarkers in 40 mL of PBS at either 0.0195 μIU TSH / mL or 0.497 pg PTH / mL).

[0173] In the first study, VERAPREP Concentrate TSH reagent was prepared by coating 550 nm VERAPREP Biotin with a biotinylated anti-TSH capture antibody. 0.08 mL of TSH antigen (10 μIU / mL ELISA calibrator) was diluted to 0.0195 μIU / mL with 41 mL of PBS buffer, which is lower than the functional sensitivity (<0.054 μIU / mL) of the DRG TSH Ultrasensitive ELISA (Part No. EIA-1790, Lot No. RN 58849). 1 mL was saved as a baseline sample (before enrichment). The 40 mL sample was processed using the VERAPREP Concentrate TSH protocol to generate a 1.0 mL enriched sample for subsequent TSH ELISA testing.

[0174] Dilute 80 μL of the 10 μIU / mL TSH standard to 0.0195 μIU / mL in 41.0 mL of PBS and save 1.0 mL as the baseline sample (before enrichment). Dilute 1.80 μL of 10 μIU / mL TSH standard to 0.80 μIU / mL in 1.0 mL of VERAPREP Cleave as a control. 2. Add 40 mL of 0.0195 μIU / mL TSH in PBS to a 50 mL Falcon tube 3. Add VERAPREP Concentrate TSH and mix. 4. Incubate at room temperature for 60 minutes while mixing. 5. Magnetically separate VERAPREP Concentrate TSH using Dexter LifeSep® 50SX for 60 minutes 6. Decant 40 mL of PBS and discard into waste. 7. Add 4.0 mL of PBS Wash Buffer and mix. 8. Magnetically separate VERAPREP Concentrate TSH in 4 mL of PBS Wash Buffer using Dexter LifeSep® 50SX for 30 minutes. Decant 9.4 mL of PBS and discard into waste Add 10.1 mL of PBS Wash Buffer and mix. 11. Transfer 1 mL of VERAPREP Concentrate TSH to a 1.75 mL conical bottom snap cap vial. 12. Magnetically separate VERAPREP Concentrate TSH in 1 mL PBS Wash Buffer using Dexter LifeSep® 1.5S for 10 minutes. 13. Aspirate 1 mL of PBS and discard into waste Add 14.1 mL of VERAPREP Cleave and mix. 15. Magnetically separate VERAPREP Concentrate TSH in 1 mL of VERAPREP Cleave using Dexter LifeSep® 1.5S for 10 minutes. 16.1 mL of the supernatant (enriched sample) is aspirated and saved to 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 in 1 mL of VERAPREP Cleave buffer as a control. The baseline sample, enriched sample, and control were tested by DRG TSH Ultrasensitive ELISA, and the percent TSH recovery of the enriched sample was calculated as [enriched sample result] / [control result] × 100%. As expected, the diluted TSH baseline sample was undetectable by Ultrasensitive ELISA, reading 0.00 μIU / mL. Using only 0.80 mg of reagent, VERAPREP Concentrate TSH successfully enriched diluted TSH from undetectable to 0.73 μIU / mL (Table 2). Compared to the control, this was a 98.6% recovery; however, there may have been a matrix effect of the VERAPREP Cleave buffer in the TSH ELISA that suppressed the assay signal (Table 3). [Table 2] [Table 3]

[0176] In the second study, VERAPREP Concentrate PTH reagent was prepared by coating 550 nm of VERAPREP Biotin with 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, below the functional sensitivity (<1.56 pg / mL) of the DRG PTH (parathyroid) Intact ELISA (Part No. EIA-3645, Lot No. 2896), and 1 mL was saved as a baseline sample (before enrichment). The 40 mL sample was processed using the VERAPREP Concentrate PTH protocol to generate a 1.0 mL enriched sample for subsequent PTH ELISA testing. Dilute 21 µL of the 1.971 pg / mL PTH standard solution to 0.497 pg / mL in 41.0 mL with PBS, and save 1.0 mL as the baseline sample (before enrichment). As a control, 21 μL of 2.971 pg / mL PTH standard solution is diluted to 20.4 pg / mL by volume to 1.0 mL with VERAPREP Cleave. 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. Magnetically separate VERAPREP Concentrate PTH using Dexter LifeSep® 50SX for 15 minutes. 7. Decant 40 mL of PBS and discard into waste 8. Add 4.0 mL of PBS Wash Buffer and mix. 9. Magnetically separate VERAPREP Concentrate PTH in 4 mL of PBS Wash Buffer using Dexter LifeSep® 50SX for 10 minutes. Decant 10.4 mL of PBS and discard into waste 11. Add 1 mL of PBS Wash Buffer and mix. Transfer 12.1 mL of VERAPREP Concentrate PTH to a 1.75 mL conical-bottom snap-cap vial. 13. Magnetically separate VERAPREP Concentrate PTH in 1 mL of PBS Wash Buffer using Dexter LifeSep® 1.5S for 10 minutes. 14. Aspirate 1 mL of PBS and discard into waste Add 15.1 mL of VERAPREP Cleave and mix. 16. Magnetically separate VERAPREP Concentrate PTH in 1 mL of VERAPREP Cleave using Dexter LifeSep® 1.5S for 10 minutes. 17.1 mL of the supernatant (enriched sample) is aspirated and saved to 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 in 1 mL with VERAPREP Cleave buffer as a control. The baseline sample, enriched sample, and control were tested by DRG PTH (parathyroid) Intact ELISA, and the PTH % recovery of the enriched sample 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 an enhanced 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 was a 109% recovery (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, we describe a mass spectrometry sample preparation protocol for enriching low-abundance biomarkers and spiked internal standards (ISTDs) from large 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 ISTD from the same sample using multiple superparamagnetic nanoparticle populations mixed or pooled together, each population coated with a different capture moiety. 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 high-sensitivity C-reactive protein. 1. Collect the patient's urine (using standard urine collection protocols such as urine collection cups) 2. Mixing the collected urine sample 3. Add 40 mL of urine to a 50 mL Falcon tube 4. Add deuterated internal standards for enriched biomarkers and mix 5. Add VERAPREP Condition and mix. 6. Add VERAPREP Concentrate and mix. 7. Incubate: Biomarker + Deuterated Internal Standard Capture with VERAPREP Concentrate 8. Magnetically separate VERAPREP Concentrate in 40 mL urine using Dexter LifeSep® 50SX. 9. Aspirate the urine and discard it in the waste Add 10.4 mL of PBS Wash Buffer and mix. 11. Magnetically separate VERAPREP Concentrate in 4 mL of PBS Wash Buffer using Dexter LifeSep® 50SX. 12. Aspirate the urine and discard it in the waste 13. Repeat step 12 two more times (2X) Add 14.1 mL of VERAPREP Cleave and mix (mass spectrometry compatible buffer). 15. Magnetically separate VERAPREP Concentrate in 1 mL of VERAPREP Cleave using Dexter LifeSep® 1.5S. 16. Aspirate and test a 1 mL sample of the supernatant by LC-MS 17. Final biomarker concentrations determined based on 1) a 40 mL urine sample size, 2) LC-MS quantification of biomarkers, and 3) adjusting reported biomarker values ​​based on deuterated internal standard recovery.

[0179] Selective release or cleavage of the captured and enriched biomarker(s) can be achieved by a change in pH (acidic pH, e.g., glycine pH 2.5 elution followed by neutralization, or alkaline pH 10.0 or above), using cleavable linkers such as disulfide bonds cleaved with reducing agents such as TCEP or DTT, or by using competitive elution such as a molar excess of D-biotin and monomeric avidin, or a molar excess of sugars and concanavalin A competing for binding sites 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 separately quantifies IgG, IgA, and IgM. As performed below, serum antibodies were assayed, but this procedure can also be performed with an oral saline rinse to assay saliva antibodies. In the assay, the sample is first washed to remove potential heterophilic interference, antibodies are captured on beads (microparticles), reacted with a 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. The generalized procedure for the assay includes the following:

[0181] 1. Prime the plate washer with washing / blocking buffer a. First sonicate in water for 30 minutes.

[0182] 2. Prepare all reagents a. All beads should be mixed and on a rocker to be homogenous for use.

[0183] 3. Plate Conditioning—To prevent nonspecific binding of, e.g., immunoglobulins, to the well surfaces, a pair of clear, round-bottom, 96-well microtiter plates are washed with a wash 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 serves as the "wash" plate, and the other as the "capture" plate.

[0184] 4. Pre-analytical sample cleanup to remove heterophilic interference. a. Use sample directly from refrigerated storage without centrifugation mixing. Avoid pipetting any lipids directly. b. Add 60 μL of each sample to the blocked round bottom plate in the "clean" plate layout. c. Once all samples have been added, use a multichannel pipette to add 140 μL of clean beads (a mixture of 36 μg of rabbit IgG-biotin-streptavidin beads and 4 μg of human IgG-biotin-streptavidin beads to capture and remove heterophilic interference specific to rabbit IgG, human IgG, streptavidin, and / or the beads themselves) to all wells containing 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 with shaking in a plate washer. i. Orbital shaking on fast setting, 425 cpm e. Place on magnet for 4 minutes. i. For example, Alpaqua Catalyst 96, part number A000550.

[0185] 5. Neutralizing antibody capture for detection and multiplexed quantification of IgM, IgG, and IgA specific for SARS-CoV-2 S1-RBD or S1-NTD. a. Using a multichannel pipette, transfer 50 μL of washed sample to a blocked capture plate (clear flat bottom). b. Use a multichannel pipette to 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 human immunoglobulins IgA, IgG, and / or IgM specific for the RBD or NTD) to each well. i. Add the same volume of capture beads to empty wells to serve as capture bead blanks. ii. SARS-CoV-2 spike glycoprotein (S1) RBD (His-tagged, produced in HEK293; The Native Antigen Company, Part No. REC31882). iii. SARS-CoV-2 spike NTD (His-tagged, produced in HEK293; The Native Antigen Company, Part No. REC31905). c. Add 60 μL of triplicate calibrator beads to six wells of the plate, each containing a different calibrator level (see below). i. Place plate on magnet while adding triplicate calibrator beads, "rinsing" tip between each calibrator dispense. d. Incubate at 37°C for 30 minutes with shaking in a plate reader. i. Orbital shaking on fast setting, 425 cpm. e. Wash three times in the plate washer. i. 2 minute initial / shaking step.

[0186] 6. Multiple labeling of captured antibodies. a. Add 200 μL of triple conjugate per well. i. The same set of 8 tips on a multichannel pipette can be used to add the conjugate to all wells across the plate. ii. Once all wells have received the conjugate, return to pipetting up and down 5 times to thoroughly mix the wells (use a multichannel pipette but prepare a new tip for each row). iii. Triple conjugate: 0.002 mg / ml polyclonal rabbit anti-human IgM conjugated to AlexaFluor-488, 0.002 mg / ml polyclonal rabbit anti-human IgG conjugated to AlexaFluor-555, and 0.002 mg / ml polyclonal rabbit anti-human IgA conjugated to AlexaFluor-647 in conjugation buffer (0.1% BSA in TTA). b. Incubate at 37°C for 30 minutes with shaking in a plate reader. i. Orbital shaking on fast setting, 425 cpm. c. Wash three times in the plate washer. i. 2 minute initial / shaking step.

[0187] 7. Elution of Antibody-Conjugate Complexes from Beads a. Pre-fill wells on a clear bottom black plate (unblocked) with 35.5 Ml of Neutralization 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. The same set of 8 tips on a multichannel pipette can be used to add the conjugate to all wells across the plate. ii. Once all wells have been filled with elution buffer, return to pipetting up and down 5 times to thoroughly mix the wells (use a multichannel pipette but prepare a new tip for each row). d. Place on magnet for 2 minutes. e. Using a multichannel pipette, transfer 200 uL from each well to a black, clear, flat-bottom reading plate (from step 7a).

[0188] 8. Read fluorescence on a plate reader.

[0189] The triplex calibrator beads are assembled from the following four components: 1.6 μm magnetic beads covalently modified with streptavidin and reacting with biotinylated human IgA (affinity purification), 1.6 μm magnetic beads covalently modified with streptavidin and reacting with biotinylated human IgG (affinity purification), 1.6 μm magnetic beads covalently modified with streptavidin and reacting with biotinylated human IgM (affinity purification), 1.6 μm magnetic beads (carboxyl) quenched with Tris (TRis beads), Each was stored at 10 mg / ml in a calibrator bead stock solution of 0.1% BSA in TTA.

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

[0191] Following essentially the protocol described above, 146 serum samples collected before December 2018 were tested for the presence of SARS-CoV-2 neutralizing antibodies. Because this was well before the virus emerged in the human population, the samples were expected to be negative, and this is what was found (see Table 6). [Table 6]

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

[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 specimens 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 Emergency Use Authorization sensitivity requirement of 90%. Some samples that showed false-negative results on the initial assay showed positive results on subsequently collected samples (Figure 12). Many patients with two or three serial samples showed similar levels of antibodies over time, although some patients showed rapidly increasing or decreasing antibody levels (Figure 13).

[0195] Example 6. Cleaning and Capture of Biomarkers from Saline Oral Rinse To clean 1 mL (1000 μL) of saline oral rinse sample (5 mL 0.9% NaCl rinse for 25 seconds, gargle for 5 seconds, and expectorate into a collection tube = 5 mL saline + saliva sample, or saliva-based sample in saline), add either 2x or 4x cleaning beads with the following composition:

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

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

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

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

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

[0201] The results of the assay using 10x cleaning beads and 10x capture beads for three patients who received one dose (of the usual two) of a SARS-CoV-2 mRNA vaccine and three unvaccinated subjects are shown in Table 8. These data were collected only 8 days after administration and demonstrate that the assay can detect (and quantify) SARS-CoV-2 neutralizing antibody responses even at this early time point. (People are generally not considered "fully vaccinated" until two weeks after the second dose of a vaccine.) [Table 8]

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

[0203] Abbreviation 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 antibodies or immunoglobulins IgM immunoglobulin M HRP horseradish peroxidase LC-MS / MS Liquid Chromatography Tandem Mass Spectrometry LDT Laboratory Developed Test Mab monoclonal antibody MASI production assay-specific interference MFG IVD Manufacturer PMP superparamagnetic particles PBCT primary blood collection tube RF Rheumatoid Factor RLU Relative Light Units or Assay Response Signal RUO Research Use Only SAv Streptavidin STT secondary transfer pipe TAT Turnaround Time WF Workflow

[0204] definition As used herein, "sample" or "biological sample" refers to any human or animal serum, plasma (i.e., EDTA, lithium heparin, sodium citrate), blood, whole blood, processed blood, urine, saliva, stool (liquid and solid), semen or seminal fluid, amniotic fluid, cerebrospinal fluid, cells, tissue, biopsy material, DNA, RNA, or any fluid, dissolved solid, or processed solid material being tested for diagnosis, prognosis, screening, risk assessment, risk stratification, and monitoring, such as therapeutic drug monitoring. 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 comprises a biomarker present in low abundance in the sample.

[0205] In some embodiments, the sample is collected in any collection tube or device such as a primary blood collection tube (PBCT), secondary transfer tube (SST), blood collection bag, 24-hour (24-hour) urine collection device, Vericor tube, nanotainer, saliva collection tube, blood spot filter paper, or stool and semen collection tubes or devices, light green top or green top plasma separator tubes (PST) containing sodium heparin, lithium heparin, or ammonium heparin, 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 to collect serum in glass (no additives) or plastic tubes (with clot activators), red top tubes for chemistry to collect serum in glass (no additives) or plastic tubes (with clot activators), EDTA K2, EDTA K3, liquid EDTA solution (i.e., 8%), or EDTA for testing plasma in molecular diagnostics and viral load detection. 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 are obtained in the absence of anticoagulant), yellow-top tubes containing ACD solution A or ACD solution B, wisteria-purple top (serum, no additives, or sodium heparin), white-top tubes for any use or diagnostic test type, or any color or tube type without additives or any additives or combinations thereof for collecting blood.

[0206] In some embodiments, the sample is a difficult sample type, such as urine, 24-hour urine, saliva, and stool, or the biomarker of interest may be diluted or difficult to measure. For example, biological samples may be difficult for patient populations (e.g., neonatal, pediatric, geriatric, pregnant, tumor, autoimmune disease). For example, some biomarkers are too dilute or too low in concentration, for example, in the circulation or urine, to be reliably detected and accurately and precisely measured by existing POCT analyzers and central laboratory analyzers. In some embodiments, the difficult sample is cerebrospinal fluid (CSF).

[0207] As used herein, a "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 specimen collection tube or device prior to the addition of a sample.

[0208] PBCT and secondary transfer tubes (SST) include any commercially available standard or custom collection tubes (with or without gel separators) from companies such as Becton Dickinson (BD), Greiner, VWR, and Sigma Aldrich; glass tubes; plastic tubes; light green-top or green-top plasma separator tubes (PST) containing sodium heparin, lithium heparin, or ammonium heparin; light blue-top tubes containing sodium citrate (i.e., 3.2% or 3.8%) or citrate, theophylline, adenosine, and dipyridamole (CTAD); red-top tubes for serology or immunohematology for collecting serum in glass (no additives) or plastic tubes (with clot activators); red-top tubes for chemistry for collecting serum in glass (no additives) or plastic tubes (with clot activators); and EDTA K2, EDTA K3, liquid EDTA solution (i.e., 8%), or EDTA for testing plasma in molecular diagnostics and viral load detection. 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 (in the absence of anticoagulant, serum samples are obtained), yellow top tubes containing ACD solution A or ACD solution B, wisteria purple top (serum, no additives or sodium heparin), white top tubes for any use or diagnostic test type without additives or any additives or combinations thereof for drawing blood, or can be any color or tube type.

[0209] As used herein, a "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 a liquid sample. As referred to herein, a "diagnostic test" includes, but is not limited to, any antibody-based diagnostic test, non-antibody-based diagnostic test, 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-exempt tests and devices, research use only (RUO) tests, in vitro diagnostic (IVD) tests, laboratory-developed tests (LDTs), companion diagnostics, and sample pretreatment methods or devices for subsequent analysis by mass spectrometry (i.e., HPLC, MS, LCMS, LC-MS / MS) for diagnosis, prognosis, screening, risk assessment, risk stratification, and therapeutic drug monitoring, etc. In some embodiments, diagnostic tests include 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, radioimmunoassays (RIAs), enzyme-linked immunoassays (ELISAs), chemiluminescent immunoassays (CLIAs), CLIA and CLIA rule-out tests, and any diagnostic test used for diagnosis, prognosis, screening, risk assessment, risk stratification, treatment monitoring, and therapeutic drug monitoring.

[0210] As used herein, a pathogen is a bacterium, virus, or other microorganism 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 mismatched blood transfusions), or the body's own proteins (in the case of autoimmune diseases).

[0212] Finally, while aspects of the present specification have been 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. Accordingly, it should be understood that the disclosed subject matter is in no way limited to the particular methods, protocols, and / or reagents, etc., described herein. Accordingly, various modifications or variations of the disclosed subject matter or alternative configurations can be made in accordance with the teachings herein without departing from the spirit of the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Therefore, the present invention is not limited to that precisely as shown and described.

[0213] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect such variations to be utilized by those skilled in the art, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of all possible variations of the above-described embodiments is encompassed by the present invention unless otherwise indicated herein or clearly contradicted by context.

[0214] The grouping of alternative embodiments, elements, or steps of the 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. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the group as modified and thus fulfill all Markush group descriptions used in the appended claims.

[0215] Unless otherwise specified, all numbers expressing properties, items, quantities, parameters, characteristics, terms, etc. used in the specification and claims should be understood to be modified in all instances by the term "about." As used herein, the term "about" means that the so-qualified property, item, quantity, parameter, characteristic, or term encompasses a range of 10% above and below the value of the stated property, item, quantity, parameter, characteristic, or term. Thus, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical designation should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each individual value of the numerical range is incorporated herein as if it were individually recited herein.

[0216] As used in the context of describing the present invention (particularly in the context of the claims below), the terms "a," "an," "the," and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better clarify the invention and does not limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0217] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of." When used in a claim, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim, whether added at the time of filing or by amendment. The transitional term "consisting essentially of" limits the scope of the claim to specific materials or steps, as well as those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are essentially or explicitly described and enabled herein.

[0218] All patents, patent publications, and other publications referenced and identified herein are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

Claims

1. 1. A method for isolating an antigen-specific antibody from a biological sample, comprising: a) combining said biological sample with first particles comprising a capture moiety for an interference to provide a mixture; b) mixing the mixture to provide a first particle complex to the interference; c) removing or eliminating the first particle complex to provide a depletion solution; d) combining the depleted solution with second particles comprising a capture moiety for the antigen-specific antibody to provide a mixture; e) mixing the mixture resulting from step d to provide a second particle complex to the antigen-specific antibody; f) isolating the second particle complex from the biological sample. thereby isolating said antigen-specific antibodies from said biological sample; wherein the antigen-specific antibody is a pathogen-specific antibody; the pathogen is SARS-CoV-2, The method, wherein the capture moieties for interference include human and non-human animal immunoglobulins.

2. The method of claim 1 , further comprising dissociating the antigen-specific antibodies from the second particles.

3. The method of claim 1 , wherein dissociating comprises cleaving or eluting the antigen-specific antibodies from the second particles.

4. The method of claim 3, further comprising subjecting the dissociated antigen-specific antibodies to characterization.

5. The method of claim 4 , wherein the characterization comprises forming a complex with an anti-immunoglobulin antibody conjugated to a detectable label.

6. The method of claim 5 , wherein the detectable label is a fluorescent label.

7. The method of any one of claims 4 to 6, further comprising comparing the signal associated with the antigen-specific antibody to a standard curve of immunoglobulins.

8. The method of any one of claims 5 to 7, wherein claim 7 is directly or indirectly dependent on claim 5 or 6, wherein said anti-immunoglobulin antibody is not isotype specific.

9. The method of any one of claims 5 to 7, wherein claim 7 is directly or indirectly dependent on claim 5 or 6, wherein said anti-immunoglobulin antibody is isotype-specific.

10. 10. The method of claim 9, wherein the isotype-specific anti-immunoglobulin antibodies comprise at least two of anti-IgA, anti-IgG, and anti-IgM, each conjugated to a different label.

11. The method of claim 1, wherein the capture moiety for the interference comprises streptavidin.

12. The method of claim 1 , wherein the first and / or second particles are provided as a lyophilized product.

13. A method according to any one of claims 1 to 3 and 11 to 12 for use in producing enriched antibodies.

14. The method of any one of claims 1 to 13, wherein the capture moiety for the interference is the spike protein of SARS-CoV-2.

15. 15. The method of claim 14, wherein the spike protein is the S1 subunit, or its receptor binding domain and / or N-terminal domain.

16. The method of any one of claims 1 to 15, wherein the antigen-specific antibody is a human antibody.

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