Method for detecting biomarkers

Abstract

To provide methods for using multiple kinds of particles comprising surfaces each independently comprising a capture moiety to isolate and characterize biomarkers (e.g., for obstructive sleep apnea).SOLUTION: A method for removing biomarkers from a biological sample comprises: a) combining the sample with multiple kinds of particles to provide a mixture, where each particle independently comprises a capture moiety (i.e., a type or species of capture moiety), to provide a mixture; b) mixing the mixture to provide one or more particle complexes with the biomarkers; and c) removing or isolating the particle complexes to provide a depleted solution; thereby removing biomarkers from the biological sample.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority based on U.S. Provisional Patent Application No. 62 / 711,415, filed on Jul. 27, 2018, and U.S. Provisional Patent Application No. 62 / 747,017, filed on Oct. 17, 2018. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] (Technical Field) The present invention relates to a method of using a plurality of types of particles (e.g., microparticles, nanoparticles; magnetic, non - magnetic) each independently comprising a capture site as described herein to isolate and characterize biomarkers (e.g., for obstructive sleep apnea).

Background Art

[0003] Clinical testing plays an important role in health assessment, healthcare, and ultimately public health, affecting people at all life stages. Most people will have a clinical test one or more times in their lives. In the United States alone, an estimated 70 - 10 billion clinical tests are performed each year, and the results of clinical tests influence approximately 70% of medical decisions.

[0004] Furthermore, since the implementation of the new Clinical Laboratory Fee Schedule (CLFS) required by the Medicare Access and CHIP Reauthorization Act (PAMA) at the Centers for Medicare & Medicaid Services (CMS) in January 2018, PAMA has been reducing reimbursement for clinical tests. It is even more important that lab results are accurate the first time, troubleshooting efforts are reduced, or time is shortened, without affecting the lab workflow.

[0005] An interfering substance is a substance present in a patient's sample that can, for example, change the correct value of the result of a diagnostic test by interfering with antibody binding, or can increase or decrease an assay signal by cross-linking, steric hindrance, or the mechanism of autoantibodies. While it is known that immunoassays are susceptible to interference, if incorrect results are not recognized in the device (analyzer) or laboratory, there is no warning signal, or the physician does not communicate to the laboratory that the patient's results do not match the clinical picture, the clinical laboratory may still report incorrect results. Without practical means to pre-identify such samples that can cause problems, inaccurate results may unexpectedly occur with any sample. As a result of such interference, incorrect results can lead to false-negative and false-positive test results, affecting the treatment of the patient, leading to unnecessary invasive, diagnostic, or therapeutic measures, or the failure of the patient's treatment.

[0006] Despite the problems arising from interference, screening for biomarkers and diagnostic tests can be difficult, for example, due to low abundance or abundance ratio in biological samples.

[0007] Urine and / or blood biomarkers for obstructive sleep apnea (OSA) are used in children and adults to facilitate diagnosis and for cost-effective treatment of the disease. The overall purpose of urine and / or blood biomarkers is to address and measure the characteristics of the optimal biomarker for OSA. OSA is a highly prevalent disorder in children and adults, associated with an increased risk of cardiovascular disease, diabetes, and other chronic diseases. Unfortunately, up to 90% of OSA patients are undiagnosed and not receiving appropriate treatment for the disease. While much is known about the pathophysiology and consequences of OSA, the molecular mechanisms of OSA have not yet been fully defined. However, advances in proteomics-based technologies have facilitated the discovery of new biomarkers as potential diagnostic and therapeutic targets for many diseases, including OSA.

[0008] Epidemiological studies have shown that OSA affects 6 - 13% of the adult population and 1 - 4% of the pediatric population. The prevalence of OSA is more than 50% higher in patients with heart disease or metabolic disorders than in the general population. Untreated OSA worsens the quality of life and results in long - term consequences including cardiovascular disease, hypertension, diabetes, obesity, stroke, depression, and various metabolic disorders. In children, OSA causes cognitive and behavioral disorders and may be misdiagnosed as attention - deficit disorder (ADD).

[0009] Currently, a definitive diagnosis of OSA requires an overnight sleep study by laboratory - based polysomnography (PSG). However, sleep studies are time - consuming, expensive, inaccessible, and inconvenient for the diagnosis of OSA in children and adults. While home - based unattended studies using portable sleep monitors are being promoted, 56% of home - based OSA diagnoses still require confirmation by a PSG test in the laboratory. There is a need to identify patients with OSA early by simple and inexpensive screening or diagnostic tests such as 1) discovery and simplified analysis of potential OSA biomarkers in known OSA - positive patient samples, and 2) combining multiple biomarkers into a panel to achieve the desired sensitivity and specificity for accurate diagnosis of OSA and access the effectiveness of OSA treatment.

[0010] The development of an obstructive sleep apnea (OSA) diagnostic test solution that can accurately, precisely, and highly sensitively measure low - concentration biomarkers is required to correctly confirm / rule out OSA and improve patient outcomes. OSA, which can cause obesity, hypertension, heart disease, diabetes, behavioral problems, and learning problems and may be misdiagnosed as attention - deficit disorder (ADD) in children, may be present in 43 million Americans. The CDC estimates the prevalence of pediatric OSA in the United States to be approximately 1 million. OSA is currently one of the most under - diagnosed conditions, and its diagnosis customarily requires an expensive and inconvenient sleep test (polysomnography). In the case of children, parents are often not proactive, and children may not be able to tolerate the sleep test.

[0011] Thus, there is a clinical need for a simple, inexpensive, automatable, and effective solution for removing or minimizing sample interfering substances and concentrating biomarker concentrations prior to diagnostic testing without affecting the workflow and required time in the testing laboratory. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0012] Disclosed herein is, for example, a simple, effective, and cost-effective method for detecting one or more biomarkers in a biological sample, for managing and reducing known sample-specific interferences that can lead to false test results and increased risk to patient safety, such as heterophile antibodies in patients being treated with or receiving monoclonal mouse antibodies for diagnostic purposes. The methods described herein can also manage and reduce sample-specific interferences caused by biotin, which can be derived from over-the-counter (OTC) supplements, multivitamins, and botanical extracts consumed by consumers for health, beauty, and weight loss, or for therapeutic purposes, such as the treatment of multiple sclerosis.

[0013] In one aspect, provided is a method for removing a biomarker from a biological sample, comprising: a) combining the sample with a plurality of types of particles, each particle independently comprising a capture site (i.e., type or kind of capture site), to provide a mixture; b) mixing the mixture to provide one or more particle complexes with the biomarker; and c) removing or isolating the particle complexes to provide a depleted solution, thereby removing the biomarker from the biological sample.

[0014] In one aspect, provided is a method for isolating a biomarker from a biological sample, comprising: a) combining the sample with a plurality of types of particles, each particle independently comprising a capture site (i.e., a type or kind of capture site), to provide a mixture; b) mixing the mixture to provide one or more particle complexes with the biomarker; and c) removing or isolating the particle complexes to provide a depleted solution and an enriched isolate, thereby isolating the biomarker from the biological sample.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Described herein is a method for isolating or enriching one or more biomarkers of obstructive sleep apnea (OSA) in a biological sample, comprising combining a plurality of types of particles as described herein with a biological sample as described herein.

[0016] In one aspect, described herein is a method for removing a biomarker of OSA from a biological sample, comprising: a) combining the sample with a plurality of types of particles, each particle independently comprising a capture site (i.e., a type or kind of capture site), to provide a mixture; b) mixing the mixture to provide one or more particle complexes with the biomarker; and c) removing or isolating the particle complexes to provide a depleted solution, thereby removing the biomarker from the biological sample.

[0017] In one aspect, described herein is a method for isolating a biomarker from a biological sample, the method comprising: a) combining the sample with a plurality of particles, each particle independently comprising a capture site (i.e., a type or kind of capture site), to provide a mixture; b) mixing the mixture to provide one or more particle complexes with the biomarker; and c) removing or isolating the particle complexes to provide a depleted solution and a concentrated isolate, thereby isolating the biomarker from the biological sample. In some embodiments, the plurality of particles includes a plurality of capture sites (e.g., a plurality of particles each independently covalently or non-covalently bound to a plurality of capture sites).

[0018] In some embodiments, a conditioning agent is added to the biological sample prior to combining the sample with the plurality of particles. In some embodiments, the conditioning agent is a pH adjuster, a molarity adjuster, an interference blocker, or a releasing or liberating agent.

[0019] In some embodiments, the plurality of particle types includes a first particle having a first capture site. In some embodiments, the plurality of particle types includes a second particle having a second capture site. In some embodiments, the plurality of particle types includes a third particle having a third capture site. In some embodiments, the plurality of particle types includes a fourth particle having a fourth capture site. In some embodiments, the plurality of particle types includes a fifth particle having a fifth capture site. In some embodiments, the plurality of particle types includes a sixth particle having a sixth capture site. In some embodiments, the plurality of particle types includes a seventh particle having a seventh capture site. In some embodiments, the plurality of particle types includes an eighth particle having an eighth capture site. In some embodiments, the plurality of particle types includes a ninth particle having a ninth capture site. In some embodiments, the plurality of particle types includes a tenth particle having a tenth capture site. In some embodiments, the method includes a method of removing or isolating a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth biomarker from a biological sample.

[0020] In some embodiments, the method further includes adding a cleavage reagent or release agent to the mixture to provide an enriched isolate.

[0021] In some embodiments, the method further includes performing a diagnostic test on the biomarker (e.g., after the removal or isolation method described herein). In some embodiments, the diagnostic test simultaneously detects the presence or absence of two or more biomarkers.

[0022] In some embodiments, the first particle differs from the second particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the third particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the fourth particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the fifth particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the sixth particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the seventh particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the eighth particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the ninth particle in size, shape, chemical properties, color, or other properties. In some embodiments, the first particle differs from the tenth particle in size, shape, chemical properties, color, or other properties. In some embodiments, the properties are selectivity, affinity, or binding to a biomarker described herein.

[0023] In some embodiments, the size is 50 to 1000 nm in diameter, for example, 50 to 500 nm in diameter, 50 to 300 nm in diameter, 50 to 100 nm in diameter, 200 to 600 nm in diameter, 400 to 600 nm in diameter, 100 to 500 nm in diameter. In some embodiments, the size is 1 to 3 microns in diameter. In some embodiments, the particle is 5 to 100 nm in diameter.

[0024] In some embodiments, the first particle population is present at a higher concentration than the second particle population. In some embodiments, the first particle population is present at a higher concentration than the third particle population. In some embodiments, the first particle population is present at a higher concentration than the fourth particle population. In some embodiments, the ratio of the first particle to the second particle is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10. The concentration of the particles is provided in units of mass per unit volume (e.g., mg / mL, g / L) or % solids (w / v). For example, 0.50% w / v is equivalent to 5 mg / mL or 5 g / L.

[0025] In some embodiments, the first particle is present at a first concentration. In some embodiments, the second particle is present at a second concentration. In some embodiments, the third particle is present at a third concentration. In some embodiments, the fourth particle is present at a fourth concentration. In some embodiments, the fifth particle is present at a fifth concentration. In some embodiments, the sixth particle is present at a sixth concentration. In some embodiments, the seventh particle is present at a seventh concentration. In some embodiments, the eighth particle is present at an eighth concentration. In some embodiments, the ninth particle is present at a ninth concentration. In some embodiments, the tenth particle is present at a tenth concentration.

[0026] In some embodiments, the first particle is a control particle (e.g., a particle containing a label or an indicator (e.g., a label or an indicator of a known amount, concentration)). In some embodiments, the label or indicator provides a measurement of the concentration or volume of the sample. In some embodiments, the label or indicator provides an indicator of the lot number or batch number of the sample. In some embodiments, the label or indicator provides a measurement of the yield or particle recovery rate.

[0027] In some embodiments, the biomarker is biotin, HAMA, RF, heterophile, or anti-SAv. In some embodiments, the biomarker is an indicator of bacterial infection. In some embodiments, the biomarker is a bacterial capture site.

[0028] In some embodiments, removal or isolation of the particle complex involves cleavage, elution, or selective release of the capture site-biomarker complex. In some embodiments, the capture site (e.g., the capture site of the capture site-biomarker complex) includes (or is pre-labeled with) a signal detection molecule for measurement in the test system. Suitable signal detection molecules include, but are not limited to, HRP, ALP, acridinium ester, isoluminol / luminol, ruthenium, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) / cyclic ABEI, or fluorescein.

[0029] In some embodiments, prior to the combinatorial step a) of the method for removing or isolating the biomarker described herein, the sample is pre-treated to remove or deplete interfering substances. In some embodiments, removal or depletion of interfering substances involves (i) combining the sample with particles comprising a capture site that is not specific for the biomarker to provide a mixture, (ii) mixing the mixture to provide a particle complex with the interfering substance, and (iii) removing the particle complex, or removing it completely, to provide a depleted solution. In some embodiments, the removal, or complete removal, of step (iii) is performed magnetically, physically, or chemically.

[0030] In some embodiments, the capture moiety is a human anti-animal antibody (e.g., mouse IgG, sheep IgG, goat IgG, rabbit IgG, bovine IgG, porcine IgG, equine IgG). In some embodiments, the capture moiety is a heterophile antibody (e.g., FR (Fc-specific)) Fab, F(ab)’2, polymeric IgG (IgG types 1, 2a, 2b 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 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 binding 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 ester), or a fluorescent label (fluorescein or other fluorescent dye molecules, and staining agents). In some embodiments, the capture moiety is streptavidin, neutravidin, avidin, polyA, polyDT, aptamer, antibody, Fab, F(ab)’2, antibody fragment, recombinant protein, enzyme, protein, biomolecule, polymer, or molecular imprint. In some embodiments, the capture moiety is biotin, fluorescein, polyDT, polyA, antigen, etc.

[0031] In one aspect, described herein is a kit comprising multiple types of particles and an instruction manual. In some embodiments, the kit includes multiple types of particles, a collection tube, and an instruction manual. In some embodiments, the kit includes multiple types of particles, a magnet, a collection tube, and an instruction manual.

[0032] (Multiple applications of the method)

[0033] Described herein is the multiple application of the method described herein. For example, in one embodiment, two or more types of particles (e.g., particles as described herein) are used to bind or complex with one or more biomarkers in a biological sample. In some embodiments, the first particle includes a first capture site that binds to or removes a first biomarker (e.g., a biomarker as described herein), the second particle includes a second capture site that binds to or removes a second biomarker (e.g., a biomarker as described herein), and when the first and second particles are combined with a biological sample (e.g., a biological sample as described herein), the first biomarker and the second biomarker bind or are removed from the biological sample.

[0034] In some embodiments, the first particle has physical properties (e.g., size, color) different from those of the second particle.

[0035] For example, a kit for multiplex applications of the methods described herein includes 1) blue beads coated with anti-human IgG, 2) red beads coated with anti-human IgM, and 3) black beads coated with biotin-PEG. The detection reagent is a mixture of these three different types of beads for qualitative visual detection. The beads can also be labeled with unique fluorescent molecules having different fluorescence / excitation lights for multiplex detection when read using a fluorometer or a fluorescence plate reader, and semi-quantitative or quantitative results can be obtained. In addition to the use of color beads, nanoparticles can also be labeled for analytical (fluorescence, UV / vis, chemiluminescence, electrochemiluminescence, etc.) detection.

[0036] The beads may be non-magnetic stained beads such as latex, nanobeads (i.e., blue, red, black, green, yellow, purple, white), stained magnetic beads (i.e., yellow, red, green), or unstained magnetic beads (brown), the bead surface of which is functionalized for covalent immobilization of antibodies, proteins, antigens, antibody fragments, aptamers, oligonucleotides (PolyA, PolyDT), molecular imprinted polymers, etc.

[0037] The microarray characterization kit includes one or more different interference targets or analytes immobilized (i.e., covalently bound, affinity interaction) to each well / spot that is specific for at least one interference mechanism. When an undiluted sample or a diluted sample is added to each well / spot, a sample-specific interfering substance (only if present in the sample) will interact with the immobilized interference target or analyte (e.g., biomarker, e.g., interfering substance) only if a sample-specific interfering substance is present. For example, if the sample contains a human anti-goat IgG interfering substance, human IgG will specifically bind to the goat IgG immobilized in a specific well / spot. As another example, if the sample contains human anti-streptavidin IgM, human IgM will specifically bind to the streptavidin immobilized in a specific well / spot. As another example, if free biotin is present in the sample, it will specifically bind to streptavidin, neutravidin, avidin, or anti-biotin IgG immobilized in a specific well / spot. As another example, if the sample contains two or more different interfering substances such as human anti-sheep IgG and human anti-ruthenium IgM, when the sample is added to the well / spot containing sheep IgG immobilized in the well / spot, only human anti-sheep IgG will specifically bind to this well / spot and human anti-ruthenium IgG will not bind. When the same sample is added to another well / spot containing ruthenium immobilized in the well / spot, only human anti-ruthenium IgM will specifically bind to this well / spot and human anti-sheep IgG will not bind.

[0038] After incubating the well / spot containing the sample, the well / spot was washed so that only human IgG, human IgM, and / or biotin interfering substances in the sample remained in the predetermined well / spot only if the sample was specific for the interference target immobilized in the predetermined well / spot. If there was no sample-specific interfering substance for the predetermined interference target immobilized in the well / spot, no detectable amount of human IgG, human IgM, or biotin would be present in the well / spot after washing.

[0039] For the characterization of potential sample interferents, a mixture of these three different beads is added to each well or reaction spot. When human IgG and / or human IgM bind to the well / spot via specific binding interactions with their interference targets, only the blue and / or red beads bind to the predetermined spot, and the black beads do not bind as they are specific only to biotin. If the amount of free biotin in the sample is less than the biotin-binding capacity or the threshold of free biotin, only the black beads bind to the well / reaction via immobilized streptavidin, neutravidin, avidin, or anti-biotin IgG. If the free biotin in the sample exceeds the biotin-binding capacity or the threshold of free biotin, all biotin-binding sites are occupied or saturated, and none of the black beads bind.

[0040] As a control, three or more wells / spots immobilized with human IgG, human IgM, or anti-biotin protein (streptavidin, neutravidin, avidin, or anti-biotin IgG) can be used. A mixture of three beads (blue, red, and black) is added to a predetermined control well / spot, and only the color beads specific to human IgG (blue beads), human IgM (red), or anti-biotin protein (black) bind to the control well / spot, and the other two-color beads do not bind. For example, when human IgM is immobilized in the control well / spot and a mixture of three-color beads is added, only the red beads (red beads coated with anti-human IgM) bind, and the blue or black beads do not bind. As another example, when anti-biotin protein is immobilized in the control well / spot and a mixture of three-color beads is added, only the black beads (black beads coated with biotin-PEG) bind, and the blue or red beads do not bind. However, if beads of the wrong color bind to the control well / spot, or if beads of two or more colors bind to the control well / spot, the control fails. The control passes only if beads of the correct color bind to the correct control well / spot.

[0041] However, in addition to human IgG (immunoglobulin G) and IgM (immunoglobulin M), there are three additional types of human antibodies: human IgA (immunoglobulin A), human IgD (immunoglobulin D), and human IgE (immunoglobulin E). Since there are five types of human antibodies, additional color beads (N = 6 colors) in the detection reagent mixture may be considered to detect these potential interference mechanisms. For example, the kit includes: 1) blue beads coated with anti-human IgG, 2) red beads coated with anti-human IgM, 3) yellow beads coated with anti-human IgA, 4) green beads coated with anti-human IgE, 5) brown beads coated with anti-human IgD, and 6) black beads coated with biotin-PEG.

[0042] As another example, yellow beads co-coated with anti-human IgA, anti-human IgE, and anti-human IgD, or each lot / batch is coated with a single anti-human antibody, and all batches are pooled or blended as a single batch of a yellow bed containing three different lots of yellow beads specific for human IgA, IgE, or IgD, etc. Monochromatic beads for detecting two or more human antibody types can be used. For example, additional color beads (N = 4 colors) in the detection reagent mixture may be used to detect those potential interference mechanisms. 1) blue beads coated with anti-human IgG, 2) red beads coated with anti-human IgM, 3) yellow beads coated with anti-human IgA, anti-human IgE, anti-human IgD, or one lot is coated with anti-human IgA, the second lot is coated with anti-human IgE, and the third lot is coated with anti-human IgD, a batch / pool of three different lots of yellow beads, 4) black beads coated with biotin-PEG.

[0043] IgM is divalent (having two Fabs and capable of binding 2 moles of antigen per mole of IgG1), and IgM is decavalent (having 10 Fabs and capable of binding 10 moles of antigen per mole of IgM). Therefore, when the same interference target immobilized on the surface of magnetic nanoparticles is also immobilized on a microarray spot or a microtiter plate well, it may be possible to use a magnetic nanoparticle depletion reagent as a characterization label in an assay interference characterization kit. For example, if the magnetic nanoparticle depletion reagent is coated with sheep IgG and is added to a sample containing human anti-sheep antibody (HASA) interfering substances as a sample pretreatment to deplete the interfering substances before the test, human anti-sheep IgG and / or human anti-sheep IgM will bind to the sheep IgG immobilized on the surface of the magnetic nanoparticles. After the incubation of the sample pretreatment is completed, the magnetic nanoparticles can be separated from the sample using magnetic, physical, or chemical separation methods and washed to remove unbound substances from the sample. The washed magnetic nanoparticles can be placed in water or buffer and added to the wells or spots of an assay interference characterization kit. If sheep IgG is immobilized on the surface of the well / spot, any divalent (human IgG) and / or decavalent (human IgM) can bind to the sheep IgG on the surface of the magnetic nanoparticles and also capture / bind to the sheep IgG immobilized on the well / spot to form a specific sandwich complex. [Magnetic nanoparticles]-(Sheep IgG)-[Human anti-sheep IgG / IgM]-(Sheep IgG)-[Microarray spot or microtiter plate well] After washing the well / spot, if brown color (i.e., magnetic nanoparticles) remains = positive result, or human anti-sheep antibody is detected in the sample. If the well / spot is transparent or colorless after washing = negative result, or human anti-sheep antibody is not detected in the sample.

[0044] (Method of separation) The particles described in this specification can be added to a collection device such as a primary blood collection tube, 24-hour urine collection device, urine collection device, saliva collection tube, feces collection device, semen collection device, blood collection bag, or any sample collection tube or apparatus, etc., before adding the biological sample.

[0045] The particles described in this specification can also be added to the sample after collecting the sample in the collection device or after transferring the sample from the first 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.

[0046] In some embodiments, the particles described in this specification are added to a collection device containing a biological sample. In some embodiments, the particles described in this specification are added to the collection device before adding the biological sample.

[0047] In one aspect, described herein is a device (i.e., a screw cap that triggers a release mechanism) for releasing particles, including a collection device described herein that contains a biological sample, such as a urine collection device. For example, the device is a tube having a screw cap that releases the particles described in this specification when the screw cap is closed.

[0048] In one aspect, described herein is a device containing a chemical substance for releasing particles into a container containing a biological sample (i.e., an encapsulated composition or a composition that dissolves in a solution at a defined rate or time point). In some embodiments, the device described herein is configured to delay the addition of the particles described in this specification, for example, to provide pre-treatment of the sample before a diagnostic test.

[0049] In some embodiments, the samples described herein can be pretreated with a chemical, a protein, a blocker, a surfactant, or a combination thereof to address matrix-specific issues, such as adjusting the pH, depleting or competing for sample-specific interferents, and / or improving the specificity and binding rate of the nanoparticles to the target biomarker, before adding the nanoparticles described herein to the sample, introducing, dispersing, or mixing them. The delay in adding the nanoparticles to the sample after pretreatment of the sample can be physically controlled by adding the nanoparticles to the sample after pretreatment of the sample. The nanoparticles can also be present in the sample during pretreatment of the sample if the nanoparticles are encapsulated, shielded, or protected by a chemical, a polymer, or a sugar shell, coating, or polymerization such that the nanoparticles need to be dissolved in a chemical, a polymer, or a sugar before the nanoparticles are released, added, dispersed, or mixed in the sample. Delayed release of the nanoparticles can be achieved using chemicals known to those of skill in the art, such as those used today in delayed drug release technology.

[0050] Method for magnetic separation of particles

[0051] In one aspect, provided herein is a method for removing interfering substances from a biological sample (e.g., prior to a diagnostic test), or for isolating or separating magnetic particles (e.g., within a primary blood collection tube, a custom sample collection device, a secondary transfer tube, or a custom sample device). For example, a magnet-based device will rapidly (in less than 2 minutes, preferably less than 30 seconds) isolate magnetic nanoparticles to the side and / or bottom to form a supernatant essentially free of particles. The particle-free supernatant can be continuously aspirated without disrupting the pellet containing the particles and dispensed into a separate transfer tube for a diagnostic test. In some embodiments, the pellet is separated or subjected to a diagnostic test. In some embodiments, the magnet for magnetic separation is a multi-magnet device having 2 to 12 magnets in a rack designed to hold 1 to 12 sample preparation tubes on a large pipette device. Examples of such pipette devices include, but are not limited to, those manufactured by Hamilton or Tecan. In some embodiments, the magnet for magnetic separation is a multi-magnet device having 96 or 384 magnets designed to magnetize a 96-well or 384-well microtiter plate.

[0052] Method for physical separation of particles

[0053] In one aspect, provided herein is a method for removing the particles described herein by a physical force (e.g., gravity). In some embodiments, the particles described herein are separated, isolated, or removed from a biological sample by a physical force (e.g., by centrifugation). In some embodiments, the method is used prior to the application of the diagnostic test methods described herein, e.g., in a primary blood collection tube, a custom sample collection tube, a secondary transfer tube, or a custom sample device. In some embodiments, the method for removing particles is filtration.

[0054] For example, magnetic nanoparticles specific to fibronectin and / or other thrombotic factors, or cell debris (i.e., specific to erythrocyte membranes) for the capture or binding of subsequent "thrombus" (in serum) after turning off thrombus components / constituents, and / or magnetic nanoparticles for the capture or binding of cell debris (in serum or plasma) increase the centrifugation speed and efficiency (shorter spin times to improve laboratory efficiency, workflow, and throughput) through the integration of a strong magnet or magnetic technology in a centrifuge rotor and / or tube holder. The combination of the relative centrifugal force (RCF) or G of centrifugation and the magnetic separation of magnetic nanoparticle complexes (thrombus + magnetic beads, cell debris + magnetic beads) enables much faster and efficient separation and supernatant formation on the side or bottom of the sample tube, clarifying the sample for subsequent analysis. For example, in most laboratories, this centrifugation step takes more than 4 minutes and can be shortened to less than 2 minutes (preferably less than 1 minute) by combining centrifugation with the magnetic isolation / separation of magnetic nanoparticle-thrombus / cell debris complexes.

[0055] Furthermore, if the nanoparticles or the majority of magnetic nanoparticles are specific to one or more different sample interference mechanisms such as 1, 5, 10, 20, 30, or more different interference mechanisms, those interfering substances, if present, will be captured by the nanoparticles and depleted from the sample after physical separation from the centrifugate or by the combination of centrifugation and magnetic separation described above.

[0056] These magnetic nanoparticles also do not require specificity to thrombi or cell debris that are separated via centrifugation, or a combination of centrifugation and magnetic separation in centrifugation. Their surfaces may be co-coated or immobilized with more than one type of antigen and / or antibody. In this case, one or more antibodies and / or antigens are specific to thrombi or cell debris, and other antigens and / or antibodies are specific to sample interferents. At this time, the nanoparticles will specifically bind to both sample interferents and thrombi and / or cell debris for subsequent physical separation or isolation via centrifugation or a combination of centrifugation and magnetic separation.

[0057] The use of nanoparticles specific to thrombi and / or cell debris increases the clotting rate by specifically binding with magnetic nanoparticles and pulling everything towards the magnetic body for magnetic separation and isolation. This bead-based pellet formed by the magnetic field and intensity also promotes thrombus formation based on the forced proximity of the thrombus or clotting factors specifically captured by the nanoparticles and subsequent magnets.

[0058] Method for chemical separation of particles

[0059] In some embodiments, the particles described herein are separated, isolated, or removed from a biological sample by a chemical separation method. In some embodiments, the chemical separation method is used within a primary blood collection tube, a custom sample collection device, a secondary transfer tube, or a custom sample device before a diagnostic test method is applied.

[0060] In one aspect, provided is a method for chemical separation of particles, including providing one or more salts, solvents, polymers, or surfactants.

[0061] In some embodiments, a chemical separation method, such as liquid-liquid phase separation, fractionates particles into phase A and a sample without nanoparticles into phase B to be tested. The agent for liquid-liquid phase separation (chemical phase separation) can be a salt, a soluble polymer, a surfactant, or the like.

[0062] For example, liquid-liquid phase separation can be caused by adding a nonpolar solvent such as hexane to a polar aqueous sample, leaving an aqueous phase without nanoparticles for testing by a diagnostic test as described herein, and the particles are fractionated into the nonpolar phase. 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.

[0063] A method for isolating particles in a biological sample, comprising supplying a nonpolar solvent and an aqueous polar solvent to the particles and the biological sample to provide a nonpolar solvent layer and a polar solvent layer, removing the nonpolar solvent layer containing the nonpolar solvent, and isolating the aqueous polar solvent containing the particles.

[0064] Before aspirating and discarding the nonpolar phase, the recovery of the sample can be adjusted or corrected by adding and using an internal standard substance such as a deuterated internal control substance or internal reference particles for LC-MS / MS.

[0065] In some embodiments, the separation is a physical separation used in combination with magnetic separation. For example, from one perspective, provided is an apparatus (e.g., a magnetized centrifuge, or a centrifuge equipped with a magnet that aids in separation by both gravity and the magnetic force of the magnet). From one perspective, provided herein is an apparatus comprising a magnet and a centrifuge for separating the particles described herein. In some embodiments, the apparatus significantly reduces the centrifugation time.

[0066] (Method for removing or concentrating biomarkers) Described herein is a method for concentrating or increasing the concentration of a biomarker in a biological sample. "Concentrating" is defined as, fully or partially, capturing particles and binding a target analyte or biomarker to the particles from a biological sample (e.g., human or animal serum, plasma, blood, whole blood, processed blood, urine, saliva, feces (liquid and solid), semen or seminal plasma, cells, tissues, biopsy materials, DNA, RNA, or any liquid or solid). In some embodiments, concentrating includes washing and concentrating the biological sample, for example, by washing biomarker-specific nanoparticles and isolating them to remove or minimize interfering substances prior to the characterization and measurement steps of the biomarker.

[0067] In some embodiments, the methods described herein are used to separate and purify a specific target (e.g., a biomarker) in a biological sample to concentrate or increase the concentration of the biomarker for subsequent elution and testing or prior to a diagnostic test.

[0068] After washing or isolating the biomarker-specific particles and prior to the characterization or measurement step, the particles can be dispersed, reconstituted, or resuspended in a buffer such as phosphate-buffered saline (i.e., PBS pH 7.2) or an LC-MS / MS compatible buffer. This means that the important characterization or measurement steps of the biomarker captured and concentrated by the particles are performed within a buffer system and do not introduce or cause matrix effects or biases between the biomarkers measured in the blood, plasma, serum, or urine of an animal compared to the same biomarker measured in the blood, plasma, serum, or urine of an animal or human within the matrix of the animal or human, where the same characterization, measurement, or test method or system was used. Washing can wash away the sample matrix, components, proteins, cell components, and associated interferences or matrix effects.

[0069] Concentration is defined as complete when a sufficient amount of analyte has been captured for subsequent diagnostic testing, such as quantitative, semi - quantitative, or qualitative analysis, and as partial when a sufficient amount of analyte or biomarker has been captured for subsequent semi - quantitative or qualitative analysis. Also, when a sufficient amount of the target analyte or biomarker and internal standard are captured by a measurement method that can adjust the recovery of the analyte or biomarker of interest using an internal standard, such as LCMS and LC - MS / MS (i.e., deuterated internal standard) and HPLC (internal standard of C14 or tritium internal radioisotope), it is also defined as partial.

[0070] A method for concentrating a biomarker in a sample prior to a diagnostic test is provided herein, which includes: a) adding particles (e.g., nanoparticles, microparticles) to the sample; b) mixing the particles (e.g., nanoparticles, microparticles) with the sample; 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) isolating 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) using a suitable diluent to remove non - specific materials; g) measuring the amount, mass, molar concentration, concentration, or yield of the biomarker captured by the particles (e.g., nanoparticles, microparticles) using a biomarker - specific quantitative, semi - quantitative, or qualitative diagnostic test. In some embodiments, the diluent includes water (e.g., deionized water, water for injection, physiological saline, buffered aqueous solution).

[0071] In some embodiments, the concentration methods described herein include a washing step. In the washing step, interfering substances as described herein are removed and / or the biomarker of interest that has been washed, purified, or isolated (e.g., a biomarker as described herein) is provided. In some embodiments, the concentration methods described herein are used prior to a diagnostic test that compares two biological samples of different origins. In some embodiments, the concentration methods described herein are used prior to a diagnostic test that compares an animal sample and a human sample. In some embodiments, the concentration methods described herein are used prior to a diagnostic test that compares a serum sample and a plasma sample. In some embodiments, the concentration methods described herein are used for high-viscosity samples.

[0072] The concentration methods described herein can be used to reduce, decrease, or manage known pre-analytical or analytical causes of test errors due to interfering substances such as hemolysis, lipemia, jaundice, bilirubin, microfibrin clots, cell debris, blood cells, fibrinogen, drug metabolites, supplements, herbal remedies, and multivitamins by washing or isolating biomarker-specific nanoparticles prior to the biomarker characterization and measurement steps to remove or minimize the aforementioned interfering substances.

[0073] In some embodiments, the concentration method includes combining a first biological sample in which the biomarker is concentrated with a second biological sample in which the biomarker is concentrated.

[0074] Provided herein is a method for measuring the amount, mass, molar concentration, concentration, or yield of a target biomarker captured and concentrated by particles (e.g., nanoparticles, microparticles) by eluting, dissociating, or liberating the target biomarker from the particles (e.g., nanoparticles, microparticles) by disrupting the binding interaction using elution strategies such as elution buffers at common pH values (e.g., pH increased with a base such as sodium bicarbonate, pH decreased with an acid such as acetic acid, trichloroacetic acid, sulfosalicylic acid, hydrochloric acid, formic acid, 100 mM glycine HCl at pH 2.5 - 3.0, 100 mM citric acid at pH 3.0, 50 - 100 mM triethylamine or triethanolamine at pH 11.5, 150 mM ammonium hydroxide at pH 10.5), a displacer or substitute, competitive elution (e.g., a counterligand or analog greater than 0.1 M), ionic strength and / or chaotropic effect (e.g., NaCl, KCl, 3.5 - 4.0 M magnesium chloride in 10 mM Tris at pH 7.0, 5 M lithium chloride in 10 mL of phosphate buffer at pH 7.2, 2.5 M sodium iodide at pH 7.5, 0.2 - 3.0 M sodium thiocyanate), surfactants, detergents, concentrated inorganic salts, denaturation (e.g., 2 - 6 M guanidine HCl, 2 - 8 M urea, 1% deoxycholate, 1% SDS), organic solvents (e.g., alcohol, chloroform, ethanol, methanol, acetonitrile, hexane, DMSO, 10% dioxane, 50% ethylene glycol at pH 8 - 11.5 (also chaotropic), radiation or heat (high temperature), structural changes, disulfide bond reducing agents (2 - mercaptoethanol, dithiothreitol, tris(2 - carboxyethyl)phosphine), enzyme inactivation, chaotropic agents (urea, guanidine chloride, lithium perchlorate), mechanical agitation, sonication, and proteolytic enzymes (pepsin, trypsin), and combinations thereof).

[0075] Unless otherwise stated or implied by the present disclosure, any of the embodiments described in connection with a particular method or composition described herein can be used in combination with any of the other embodiments described herein.

[0076] The methods and compositions of the present invention can be used in combination with any suitable assay known in the art, such as, 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 (ELISA), radioimmunoassays (RIA), and enzyme immunoassays (EIA), any suitable affinity assay or immunoassay known in the art, including direct or indirect assays, competitive assays, sandwich assays, CLIA or CLIA-waived tests, LC-MS / MS, analytical assays, etc.

[0077] Prior to a diagnostic test, methods for depleting sample interferents and concentrating biomarkers from the same sample both consist of: a) adding chemical and / or biological reagents, additives or compositions (e.g., nanoparticles, microparticles) to the sample to block or deplete sample-specific interference before adding biomarker-specific particles (e.g., nanoparticles, microparticles) to the sample; b) adding biomarker-specific particles (e.g., nanoparticles, microparticles) to the sample after pretreating or incubating the sample with chemical and / or biological reagents, additives or compositions; c) incubating biomarker-specific particles (e.g., nanoparticles, microparticles) with the sample to bind and capture the target biomarker to the particles (e.g., nanoparticles, microparticles); d) washing the particles (e.g., nanoparticles, microparticles) or isolating them from the sample, and chemical and / or biological reagent additives or compositions; e) using a diagnostic test to evaluate the characteristics of the biomarker captured and concentrated on the particles (e.g., nanoparticles, microparticles).

[0078] For example, in one embodiment, particles conjugated to streptavidin will bind to biotin in a sample at neutral pH. Biotin conjugated to streptavidin will release biotin when the pH is raised to 10.

[0079] Biomarker

[0080] Described herein is an isolation method, or a method for isolating or enriching one or more biomarkers present in a biological sample. As used herein, "biomarker(s)" is defined as a characteristic biological or biologically-derived indicator (e.g., metabolite) of a progression, event, or condition such as aging or disease. Biomarkers can be endogenous and / or exogenous analytes, antigens, small molecules, macromolecules, 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, molecular imprinted polymers, and aptamers. In some embodiments, the biomarker is an interfering substance described herein (e.g., a substance present in a patient's sample that can alter the correct value of the result of a diagnostic test by inhibiting antibody binding or can increase or decrease an assay signal by a mechanism of cross-linking, steric hindrance, or autoantibodies).As used herein, "interfering substances" include, but are not limited to, heterophilic or heterophilic-like interfering substances such as autoantibodies, rheumatoid factor (RF), human anti-mouse antibody (HAMA), human anti-animal antibodies (HAAA) such as polyclonal and / or monoclonal antibodies of goat, rabbit, sheep, bovine, mouse, horse, pig, and donkey, and chemiluminescent substrates (luminol, isoluminol, isoluminol derivatives, ABEI, ABEI derivatives, ruthenium, acridinium ester), fluorescein or other fluorescent dye molecules, and fluorescent labels such as staining agents, capture sites (streptavidin, neutravidin, avidin, captavidin, polyA, polyDT, aptamer, antibody, Fab, F(ab’)2, antibody fragments, recombinant proteins, enzymes, proteins, biomolecules, polymers, molecular imprint polymers) and their binding partners (i.e., biotin, fluorescein, polyDT, polyA, antigen, etc.), binding linkers (LC, LC-LC, PEO, PEOn), bovine serum albumin, human serum albumin, ovalbumin, gelatin, purified poly and monoclonal IgG of mouse, goat, sheep, and rabbit, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Tween-20, Tween-80, TritonX-100, triblock copolymers such as Pluronic® and Tetronic, and assay-specific interfering substances used in test design or assay formulations such as blocking agents, blocking proteins, and polymeric blocking reagents commercially available from companies such as Surmodics and Scantibodies, typically used in the design of sample pretreatment methods and devices for antibody-based diagnostic tests, non-antibody-based diagnostic tests, or subsequent analysis, as well as mass spectrometry (i.e., HPLC, MS, LCMS, LC-MS / MS), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), molecular diagnostics, lateral flow, point-of-care (PoC), CLIA and CLIA-waived tests and devices. In some embodiments, biomarkers are identified in the biological samples described herein.

[0081] (Fibrinogen) Fibrinogen is converted to fibrin by thrombin upon injury to tissue or blood vessels, resulting in the formation of fibrin-based thrombi. In some embodiments, the particles described herein (e.g., particles derivatized with anti-fibrinogen (e.g., mouse anti-fibrinogen)) used in the methods described herein bind to fibrinogen in whole blood and allow for separation (e.g., chemical separation). Particles bound to thrombi via fibrin can be isolated and removed from the serum after centrifugation for serum testing without particles. In some embodiments, the biomarker is fibrinogen. In some embodiments, the methods described herein use anti-fibrinogen-derived particles to eliminate the need for centrifugation of a sample (e.g., a blood sample).

[0082] For example, in some embodiments, these magnetic nanoparticles also do not require specificity for thrombi or cell debris separated via centrifugation or a combination of centrifugation and magnetic separation in centrifugation, and their surfaces may be co-coated or immobilized with more than one type of antigen and / or antibody, where the antibody(ies) is / are specific for thrombi or cell debris and the other antigen(s) and / or antibody(ies) is / are specific for sample interferents. In that case, the nanoparticles will specifically bind to both sample interferents and thrombi and / or cell debris for subsequent physical separation or isolation via centrifugation or a combination of centrifugation and magnetic separation.

[0083] The use of nanoparticles specific for thrombi and / or cell debris may increase the clotting rate by specifically binding with magnetic nanoparticles and pulling everything towards the magnet for magnetic separation and isolation. This bead-based pellet formed by the magnetic field and intensity may also promote thrombus formation based on the forced proximity of thrombi or clotting factors specifically captured by the nanoparticles and subsequent magnets.

[0084] (Traumatic Brain Injury) In one embodiment, the biomarker is for traumatic brain injury. There are nine (9) biomarkers related to the severity and magnitude of acute brain injury and the integrity of the blood-brain barrier (BBB), but they exist at very low blood circulation concentrations and are very difficult to detect and quantify using existing immunoassay techniques and test platforms. The Banyan BTI test (FDA approved on February 14, 2018) measures only two of those biomarkers, but the methods and devices described herein (e.g., concentration methods, devices for concentration) can simultaneously measure all nine biomarkers of a patient and assist in the diagnosis and prognosis of the patient. Particles derivatized at capture sites for each of the nine biomarkers may be added to a biological sample from a patient suspected of having TBI. In some embodiments, the biomarker for traumatic brain injury is selected from the group consisting of S100B, GFAP, NFL, NFH, γ-enolase (NSE), α-II spectrin, UCH-L1, total tau, and phosphorylated tau. In some embodiments, the biomarker for traumatic brain injury is selected from GFAP and UCH-L1.

[0085] In some embodiments, the methods described herein (e.g., concentration methods) are used to isolate or concentrate one, two, three, four, five, six, seven, eight, or nine biomarkers for traumatic brain injury selected from the group consisting of S100B, GFAP, NFL, NFH, γ-enolase (NSE), α-II spectrin, UCH-L1, total tau, and phosphorylated tau.

[0086] (Alzheimer's disease) In one embodiment, the biomarker is for Alzheimer's disease. There are two (2) biomarkers related to the severity and size of Alzheimer's disease. In some embodiments, the biomarker for Alzheimer's disease is selected from the group consisting of amyloid β, BACE1, and soluble Aβ precursor protein (sAPP). In some embodiments, the biomarker for Alzheimer's disease 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 one, two, or three biomarkers for Alzheimer's disease 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 biomarker for Alzheimer's disease is identified in a biological sample (e.g., CSF).

[0087] (Sexually Transmitted Infection) In certain embodiments, the biomarker is for sexually transmitted diseases (STDs). There are at least 10 biomarkers characteristic of STD infections. 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, HIV1 and 2, and HIV antibodies. In some embodiments, the methods described herein (e.g., concentration methods) are used to isolate or concentrate 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, HIV1 and 2, and HIV antibodies. In some embodiments, the biomarker is present in urine (e.g., Chlamydia, gonorrhea, Trichomonas). In some embodiments, the biomarker is present in blood, serum, or plasma (e.g., syphilis, HPV, herpes 1 and 2, HSV, hepatitis A, hepatitis B, hepatitis C, HIV1 and 2, and HIV antibodies).

[0088] (Bacterial infection) In certain embodiments, the biomarker is for bacterial infection. The current gold standard test for bacterial infection is blood culture, which can take 24 - 48 hours until a positive result is reflected in a confirmatory test such as molecular diagnosis. Described herein is a method for making a definitive diagnosis / rule - out diagnosis of bacterial infection in a shortened time that is important for successfully treating a patient to prevent or manage sepsis, for example, in less than 60 minutes (e.g., 50 minutes, 40 minutes, 30 minutes, 20 minutes or less), and at least 30 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 of bacterial species that cause sepsis (e.g., Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus). In some embodiments, the biomarker is a biomarker of Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. In some embodiments, the biomarker is a biomarker of gram - positive bacteria or gram - negative bacteria. In some embodiments, the biomarker is a biomarker of yeast pathogens (e.g., yeast pathogens associated with bloodstream pathogens).

[0089] In some embodiments, the Gram-positive bacteria are Enterococcus, Listeria monocytogenes, Staphylococcus, Staphylococcus aureus, Streptococcus, Streptococcus agalactiae, Streptococcus pneumoniae, or Streptococcus pyogenes.

[0090] In some embodiments, the Gram-negative bacteria are Acinetobacter baumannii, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Enterobacteriaceae, Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, or Serratia marcescens.

[0091] In some embodiments, the yeast pathogens are Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, or Candida tropicalis.

[0092] In some embodiments, mass spectrometry follows. A method is envisioned in which a cleaving agent (e.g., a reducing agent such as DTT or TCEP) is added to the bacterium-particle binding complex to cleave the linker (i.e., the linker that binds the particle to the surface capture moiety). The resulting bacteria are cultured or analyzed by MALDI-TOF mass spectrometry or molecular diagnostic methods.

[0093] A method is envisioned in which a cleaving agent (e.g., a reducing agent such as DTT or TCEP) is added to the bacterium-particle binding complex to cleave the linker (i.e., the linker that binds the particle to the surface capture moiety). The resulting bacteria are cultured and analyzed by MALDI-TOF mass spectrometry or molecular diagnostic methods.

[0094] (Thyroid function) TSH concentration is measured as part of a thyroid function test in patients suspected of having an excess (hyperthyroidism) or deficiency (hypothyroidism) of thyroid hormones. The methods described in some embodiments herein are used to evaluate thyroid function. In some embodiments, the biomarker is an antigen (e.g., TSH). In some embodiments, the capture site is an autoantibody (e.g., free autoantibody, complex autoantibody) having specificity for the antigen (e.g., TSH).

[0095] (Cardiac function) The methods described in some embodiments of this specification are used to evaluate cardiac function. An increase in the circulating blood concentration of troponin is a biomarker for heart diseases such as myocardial infarction. Cardiac I and T are specific indicators of myocardial injury. Subunits of troponin are also markers of the health status of the heart. In particular, cTnI and cTnT are biomarkers for acute myocardial infarction (AMI), such as type 1 and type 2 myocardial infarctions, unstable angina, postoperative myocardial trauma, and related diseases. In some embodiments, the biomarker is free cTnI, free cTnT, a two-component cTnI-TnC, or a three-component 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, the entireties of which are incorporated herein by reference). In some embodiments, the biomarker is an indicator of fibrosis (e.g., as described in http: / / www.onlinejacc.org / content / 65 / 22 / 2449, the entirety of which is incorporated herein by reference). In some embodiments, the biomarker is for the diagnosis of acute coronary syndrome (ACS). In some embodiments, the biomarker is for cardiac troponin (I, I-C, I-C-T, T) and other cardiac troponin fragments, natriuretic peptides (BNP, ANP, CNP), N-terminal fragments (e.g., NT-proBNP, NT-proCNP), glycosylated, non-glycosylated, CRP, myoglobin, creatine kinase (CK), CK-MB, sST2, GDF-15, galectin-3.

[0096] (Obstructive sleep apnea (OSA)) Urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, and kallikrein 1 peptide can be used to diagnose OSA. Methods and biomarkers for diagnosing OSA are described in U.S. Patent Application Publication No. 2006 / 0029980, U.S. Patent Application Publication No. 2016 / 0161489, U.S. Patent No. 8,999,658, and U.S. Patent No. 9,435,814, which are hereby incorporated by reference in their entireties. Biomarkers of OSA are also described in De Luca Canto et al., Sleep Med. Rev. October 2015; 23:28-45, which is hereby incorporated by reference in its entirety. The methods described in some embodiments herein relate to methods for diagnosing improved OSA. In some embodiments, biomarkers of OSA are urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein, or combinations thereof. In some embodiments, the biomarker (e.g., uromodulin peptide) is present at low concentration in the sample. In some embodiments, the capture site is an antibody specific for urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, or combinations thereof.

[0097] In one aspect, described herein is a method for detecting a urocortin III peptide, a uromodulin peptide, an orosomucoid 1 peptide, a kallikrein 1 peptide, IL-6, IL-10, a high-sensitivity C-reactive protein, or a combination thereof in a patient, the method comprising: a. obtaining a sample from a human patient; and b. detecting, by a plurality of particles, whether a urocortin III peptide, a uromodulin peptide, an orosomucoid 1 peptide, a kallikrein 1 peptide, IL-6, IL-10, a high-sensitivity C-reactive protein, or a combination thereof is present in the sample, and detecting a binding between the urocortin III peptide, the uromodulin peptide, the orosomucoid 1 peptide, the kallikrein 1 peptide, IL-6, IL-10, the high-sensitivity C-reactive protein, or a combination thereof and the plurality of particles. In one aspect, described herein is a method for diagnosing obstructive sleep apnea in a patient, the method comprising: a. obtaining a sample from a human patient; and b. detecting, by contacting the sample with a plurality of particles, whether a urocortin III peptide, a uromodulin peptide, an orosomucoid 1 peptide, a kallikrein 1 peptide, IL-6, IL-10, a high-sensitivity C-reactive protein, or a combination thereof is present in the sample, and detecting a binding between the urocortin III peptide, the uromodulin peptide, the orosomucoid 1 peptide, the kallikrein 1 peptide, IL-6, IL-10, the high-sensitivity C-reactive protein, or a combination thereof and the plurality of particles; and c. diagnosing the patient as having obstructive sleep apnea when a urocortin III peptide, a uromodulin peptide, an orosomucoid 1 peptide, a kallikrein 1 peptide, IL-6, IL-10, or a high-sensitivity C-reactive protein is detected in the sample. In some embodiments, the patient is human. In some embodiments, the human is from about 9 years old to less than about 2 years old.In some embodiments, the method includes simultaneously detecting the presence or absence of two or more of the group consisting of urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, and high-sensitivity C-reactive protein.

[0098] In some embodiments, the sample is a urine sample. In some embodiments, the sample is a blood sample. In some embodiments, a multimarker approach (uromodulin, urocortin-3, orosomucoid-1, kallikrein) in urine samples of pediatric patients is used. In some embodiments, measurement of urinary lipocalin-type prostaglandin D synthase (L-PGDS) concentration in the adult population is used as a marker for identifying patients with severe OSA. In some embodiments, an oxidative stress multimarker approach (L-PGDS, F2-isoprostane, etc.) in the adult population can be used to predict OSA. In some embodiments, biomarkers for OSA are evaluated using chromatography and / or MS methods. In some embodiments, proteins and metabolites including lipid profiles, adrenergic / dopaminergic biomarkers and derivatives, amino acids, oxidative stress biomarkers, and other small molecules are used for the diagnosis and / or effective treatment of OSA. In some embodiments, IL-6 and / or hsCRP are used as biomarkers for discriminating regardless of the presence or absence of the pathological condition of adult OSA patients. In some embodiments, myeloid-related protein (MRP) 8 / 14 is used for discrimination regardless of the presence or absence of the pathological condition in pediatric patients. In some embodiments, serum sLOX-1 concentration is used as an independent predictor of the presence of OSA. In some embodiments, an increase in serum YKL-40 concentration is used as an independent risk factor for the presence of OSA. In some embodiments, an increase in serum chymeryn concentration is used as an independent predictive marker for the presence and severity of OSA.

[0099] In some embodiments, the sample is urine and the biomarkers for OSA are uromodulin, urocortin-3, orosomucoid-1, kallikrein, lipocalin-type prostaglandin D synthase (L-PGDS), F2-isoprostane, or combinations thereof. In some embodiments, the sample is serum or blood and the biomarkers for OSA are IL-6, hsCRP, sLOX-1, YKL-40, myeloid-related protein (MRP) 8 / 14, chymeryn, or combinations thereof.

[0100] In some embodiments, accuracy and precision can be improved by testing samples with large sample volumes (i.e., 1 mL, 10 mL, 100 mL, 1000 mL, etc.) and very small sample volumes (i.e., neonates, pediatric, geriatric), typically samples that cannot currently be tested or require dilution of the sample prior to testing, which reduces the sensitivity, accuracy, and precision of the test, to detect very dilute or low-concentration biomarkers. In some embodiments, the biological sample is in a volume of 1 mL, 10 mL, 100 mL, 1000 mL, or even larger. In some embodiments, the biological sample is in a volume of 0.5 mL, 0.25 mL, 0.1 mL, 0.05 mL, or less.

[0101] Also provided herein is a method for assisting in the enrichment of a biomarker using pretreatment of a particle sample prior to a diagnostic test by selectively releasing or eluting a captured biomarker from particles, or selectively releasing or eluting a capture site-biomarker complex, after a washing step or particle separation and prior to a biomarker characterization step or test method.

[0102] As used herein, a "cleavage reagent" or "releasing agent" is an acidic or basic pH, high molarity salt, sugar, chemical substitute, detergent, surfactant, and / or chelating agent, or a combination thereof, that disrupts the binding of a capture site on the particle surface to a biomarker and selectively releases only the biomarker into solution, for example, without replacing or eluting the capture site. After washing or isolating the particles from the sample matrix by a magnet, the particles can be treated with an elution solution containing the releasing agent to selectively release the biomarker into the solution. The particles can be rapidly (less than 2 minutes, ideally less than 30 seconds) isolated to the side and / or bottom of a sample device (vial, test tube, etc.) to form a sample supernatant that is essentially free of particles. The particle-free sample supernatant can then be aspirated without disrupting the pellet containing the particles, dispensed into another transfer tube, or placed directly into an analytical system (i.e., LC-MS / MS or MALDI-TOF) to test for the biomarker.

[0103] For example, the cleavage reagents or release agents described herein can be elution buffers at common pHs (e.g., increased pH with a base such as sodium bicarbonate, decreased pH with an acid such as acetic acid, trichloroacetic acid, sulfosalicylic acid, hydrochloric acid, formic acid, 100 mM glycine HCl at pH 2.5 - 3.0, 100 mM citric acid at pH 3.0, 50 - 100 mM triethylamine or triethanolamine at pH 11.5, 150 mM ammonium hydroxide at pH 10.5), a displacer or substitute, competitive elution (e.g., a counterligand or analog greater than 0.1 M), ionic strength and / or chaotropic effect (e.g., NaCl, KCl, 3.5 - 4.0 M magnesium chloride in 10 mM Tris at pH 7.0, 5 M lithium chloride in 10 mL of phosphate buffer at pH 7.2, 2.5 M sodium iodide at pH 7.5, 0.2 - 3.0 M sodium thiocyanate), surfactant, detergent, concentrated inorganic salt, denaturation (e.g., 2 - 6 M guanidine HCl, 2 - 8 M urea, 1% deoxycholate, 1% SDS), organic solvent (e.g., alcohol, chloroform, ethanol, methanol, acetonitrile, hexane, DMSO, 10% dioxane, 50% ethylene glycol (also chaotropic) at pH 8 - 11.5), radiation or heat (high temperature), structural change, disulfide bond reducing agent (2 - mercaptoethanol, dithiothreitol, tris(2 - carboxyethyl)phosphine), enzyme inactivation, chaotropic agent (urea, guanidine chloride, lithium perchlorate), mechanical agitation, sonication, and proteolytic enzymes (pepsin, trypsin), and combinations thereof, to disrupt the binding interaction or cleavable bond described herein between the particles described herein and the capture sites described herein.

[0104] (Method for Characteristic Evaluation) Described herein are methods for depleting and / or concentrating biomarkers for subsequent characteristic evaluation or diagnostic testing. The characteristic evaluation of the biomarkers (e.g., interferents) described herein includes the identification and / or quantification of the biomarkers (e.g., interferents) described herein.

[0105] The characterization of the biomarkers described in this specification can include signal amplification techniques (e.g., chemiluminescence, fluorescence, metal-enhanced fluorescence) to improve detection sensitivity.

[0106] For example, the characterization of biomarkers can be used in single or multiplex characterization approaches where detection can measure specific signals (UV / vis absorbance, fluorescence, chemiluminescence, electrochemiluminescence, turbidimetry, etc.) using visual (color beads) or labeled particles.

[0107] 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 diagnostic methods. In some embodiments, the presence of the biomarker is determined by immunoassay.

[0108] (Particles of the present invention) Described herein are particles for the isolation, depletion, and / or concentration of a biological sample. In some embodiments, the particles include non-cleavable linkages and capture sites (e.g., a particle surface having a function of presenting one or more capture sites). In some embodiments, the particles described herein include capture sites (e.g., capture sites having high specificity for the biomarkers described herein). In some embodiments, the particles described herein (e.g., the surface of the particles described herein, the particle surface not bound to the capture sites described herein) are inert (e.g., do not show significant binding to the biomarkers described herein). In some embodiments, the particles described herein can be used in the diagnostic tests described herein without further modification of the particles or the diagnostic test. In some embodiments, the particles described herein can be added to or removed from a sample without changing the sample (e.g., without adding or removing additional biomarkers (e.g., interference)). The particles are also referred to herein as beads.

[0109] The particles described herein have an average diameter of from 0.05 micrometers to a maximum of 3.00 micrometers, or preferably a diameter of from 0.100 micrometers to 1.1, more preferably from 0.200 micrometers to 0.600 micrometers, and even more preferably a diameter of from 0.100 micrometers to 0.500 micrometers and are small enough. In some embodiments, the particles have a diameter of 5 nm to 100 nm. In some embodiments, the particles have a diameter of 50 nm to 100 nm. In some embodiments, the particles have a diameter of 100 nm to 500 nm.

[0110] In some embodiments, the particles described herein (e.g., microparticles, nanoparticles) include a core or support, which is a paramagnetic or superparamagnetic material selected from the group consisting of iron oxide, ferromagnetic iron oxide, Fe2O3, and Fe3O4, maghemite, or combinations thereof.

[0111] In some embodiments, the surface of the particles comprises a hydrophobic organic polymer or copolymer. In some embodiments, the surface of the particles (e.g., nanoparticles, microparticles) comprises, but is not limited to, colloidal metals such as ceramics, glass, polymers, copolymers, metals, latex, silica, gold, silver, or alloys, polystyrene, derivatized polystyrene, poly(di vinyl benzene), styrene acyl acid ester copolymer, styrene butadiene copolymer, styrene divinyl benzene copolymer, poly(styrene oxyethylene), polymethyl methacrylate, polymethacrylate ester, polyurethane, polyglutaraldehyde, polyethyleneimine, polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, N,N'-methylenebisacrylamide, polyolefin, polyethylene, polypropylene, polyvinyl chloride, polyacrylonitrile, polysulfone, poly(ether sulfone), pyrolyzates, block copolymers, and the aforementioned copolymers, silicone, or silica, methylol melamine, dextran or biodegradable polymers such as poly(ethylene glycol) dextran (PEG-DEX), or an organic polymer or copolymer such as a material selected from the group consisting of combinations thereof.

[0112] In some embodiments, the particle surface has a covalent bond (coupling, conjugation, binding) click chemistry functionality of its capture site such as carboxyl, tosyl, epoxy, amine, sulfhydryl, hydroxyl, ester, methyl chloride, maleimide, etc. [copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), strain-promoted azide-alkyne cycloaddition reaction (SPAAC), strain-promoted alkyne-nitrone cycloaddition reaction (SPANC), and [3+2] cycloaddition reaction of alkene and azide, inverse electron demand Diels-Alder reaction of alkene and tetrazine, and photo-click reaction of alkene and tetrazole, etc., strained alkene reactions] functional group or a plurality of functional groups, S-HyNic (succinimidyl-6-hydrazino-nicotinamide) and S-4FB (N-succinimidyl-4-formylbenzamide) heterobifunctional crosslinking agents, and photoreactive chemicals are included.

[0113] As used herein, "blocking agent" refers to a protein, polymer, surfactant, detergent, or a combination thereof. In some embodiments, the binding of the capture site to the particles described herein is blocked by a blocking agent such as a protein, polymer, surfactant, detergent, or a combination thereof. Blocking agents include proteins such as albumin, bovine serum albumin, human serum albumin, ovalbumin, gelatin, casein, acid-hydrolyzed casein, gamma globulin, purified IgG, animal sera, polyclonal antibodies, and monoclonal antibodies, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP ) Combinations of proteins and polymers, peptides, PEGylation reagents such as (PEO)n-NHS or (PEO)n-maleimide, polymers such as triblock copolymers like Pluronic® F108, F127, and F68, nonionic surfactants such as Triton X-100, polysorbate 20 (Tween-20), and Tween 80 (nonionic), zwitterionic surfactants such as CHAPS, ionic surfactants such as sodium dodecyl sulfate (SDS), deoxycholate, cholate, and sarcosyl, surfactants, sugars such as sucrose, and commercially available blockers such as heterophile antibody blocking reagents (Scantibodies), MAK33 (Roche Diagnostics), immunoglobulin inhibitory reagents (IIR) (Bioreclamation), heteroblock (Omega Biologicals), Block Master (JSR), TRU Block (Meridian Life Sciences), and StabilCoat® and StabilGuard® (Surmodics). In some embodiments, the blocker is bound to the particles described herein (e.g., a cleavable bond, a non-cleavable bond). In some embodiments, the blocker is not bound to the particles described herein (e.g., a cleavable bond, a non-cleavable bond).

[0114] (Capture site) Described herein are particles comprising one or more capture sites that bind to interferents as described herein or biomarkers as described herein. As described herein, a "capture site" is selected from the group consisting of antibodies, binding fragments of antibodies, IgG, IgM, IgA, IgE, IgD, receptors, ligands of receptors, hormones, hormone receptors, enzymes, substrates of enzymes, single-stranded oligonucleotides, single-stranded polynucleotides, double-stranded oligonucleotides, double-stranded polynucleotides, antigens, peptides, polymers, molecularly imprinted polymers, aptamers, and proteins.

[0115] In some embodiments, the capture moiety is a protein. The protein can be, for example, a monomer, dimer, multimer, or fusion protein. In certain embodiments, the protein includes, for example, an antibody, an antibody fragment, BSA, ovalbumin, a BSA fragment, an ovalbumin fragment, mouse IgG, polymeric mouse IgG, antibody fragments (Fc, Fab, F(ab’)2) against target HAMA and RF interference mechanisms, and mouse IgG of different subclasses (IgG1, IgG2a, IgG2b, IgG3, IgE, IgD), purified animal polyclonal antibodies against target HAAA interference (i.e., bovine, goat, mouse, rabbit, sheep), streptavidin, ALP, HRP, at least one albumin such as BSA (conjugated with isoluminol, ruthenium, acridinium) against target MASI interference, or mixtures thereof.

[0116] In at least one embodiment, the present invention provides two or more different capture moieties on a binding surface.

[0117] (Generation of Capture Moiety) In one aspect, provided is a method of manufacturing a capture moiety that includes producing or generating a complex-specific or structure-specific antibody against a free autoantibody or autoantibody complex. A free autoantibody is an autoantibody that has not yet complexed with an antigen target. A complex autoantibody is an autoantibody that has formed a complex with an antigen target.

[0118] From one perspective, provided is a method of manufacturing a capture site, including producing or generating a complex-specific or structure-specific antibody against an autoantibody complex such as MTSH. In some embodiments, the autoantibody is triiodothyronine (T3) or thyroxine (T4). In some embodiments, the autoantibody complex is MTSH. For example, a complex-specific or structure-specific antibody can be produced against an autoantibody complex such as MTSH, purified from human serum, and used as a capture site. In this way, the antibody will have specificity only for the complex of hIgG or hIgM and TSH. MTSH can be purified based on techniques and published methods, or by those skilled in the art in the field of protein biochemistry and purification. In some embodiments, patients with autoimmune diseases who are most likely to interfere with autoantibody assays are used to produce or generate autoantibodies. See, for example, the HyTest SES assay for BNP in International Publication No. WO 2014 / 114780, International Publication No. WO 2016 / 113719, and International Publication No. WO 2016 / 113720, which are hereby incorporated by reference in their entirety.

[0119] (Thyroid-specific antibody) For example, in one embodiment, the autoantibody is an anti-thyroid autoantibody (anti-thyroid peroxidase antibody, thyrotropin receptor antibody, thyroglobulin antibody). Anti-thyroid autoantibodies are autoantibody targets that target one or more components of the thyroid gland.

[0120] In some embodiments, the autoantibody is a free autoantibody (e.g., thyrotropin (TSH)).

[0121] In some embodiments, the autoantibody is a complex autoantibody (e.g., MTSH). In some embodiments, the capture site described herein is an antibody generated with specificity for a complex autoantibody or structural specificity for hIgG and / or hIgM already bound to an antigen target such as MTSH. In some embodiments, the autoantibody is T3 and T4.

[0122] The following is a non-limiting list of substances that can function as either a member of a binding pair consisting of an analyte binder (capture site) and an analyte, depending on the use for which the affinity assay is designed, or as the other member. Such substances can, for example, be used as a capture site (analyte binder), or can be used to generate a capture site that can be used in the present invention (e.g., by use as a hapten / antigen for generating a specific antibody). Affinity assays, including immunoassays, can be designed in accordance with the present invention to detect the presence and / or concentration of such substances that are analytes in a sample. In certain embodiments, the capture sites of the present invention that bind to an analyte can be used to detect these substances as analytes in a sample. Alternatively, the substances listed below can be associated with the surface of a solid support in accordance with the present invention and used to capture a capture molecule (e.g., an antibody or fragment thereof, a binding protein, or an enzyme, etc.) that interacts with them.

[0123] A non-limiting list of substances that can function as one member or the other member of a binding pair consisting of an analyte binder (capture site) and an analyte includes inducible nitric oxide synthase (iNOS), CA19-9, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-t, IL-5, IL-7, IL-10, IL-12, IL-13, sIL-2R, sIL-4R, sIL-6R, SIV core antigen, IL-1RA, TNF-α, IFN-gamma, GM-CSF; PSA, pPSA, BPSA, inPSA, non-α1-antichymotrypsin complexed PSA, α1-antichymotrypsin complexed PSA, etc. (prostate-specific antigen, PSA), hK2, hK4, and hK15, ek-rhK2, Ala-rhK2, TWT-rhK2, Xa-rhK2, HWT-rhK2, and other isoforms of prostate kallikrein such as other kallikreins; HIV-1p24; ferritin, L-ferritin, troponin I, BNP, leptin, digoxin, myoglobin, B-type natriuretic peptide or brain natriuretic peptide (BNP), N-terminal proBNP, CNP, N-terminal proCNP(1-50), N-terminal 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, Bacillus anthracis protective antigen, Bacillus anthracis lethal factor, Bacillus anthracis spore antigen, Francisella tularensis LPS, Staphylococcus aureus enterotoxin B, Yersinia pestis capsular F1 antigen, insulin, alpha-fetoprotein (e.g., AFP300), 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, haptoglobin, anti-cardiolipin, anti-dsDNA, anti-Ro, Ro, anti-La, anti-SM, SM, anti-nRNP, anti-histone, anti-Scl-70, Scl-70, antinuclear antibody, anti-centromere antibody, SS-A, SS-B, Sm, U1-RNP, Jo-1, CK, CK-MB, CRP, ischemic modified albumin, HDL, LDL, oxLDL, VLDL, troponin T, troponin I, troponin C, microalbumin, amylase, ALP, ALT, AST, GGT, IgA, IgG, prealbumin, anti-streptolysin, Chlamydia, CMVIgG, toxo IgG, toxo IgM, apolipoprotein A, apolipoprotein B, C3, C4, properdin factor B, albumin, α1-acid glycoprotein, α1-antitrypsin, α1-microglobulin, α2-microglobulin, anti-streptolysin-O, antithrombin III, apolipoprotein A1, apolipoprotein B, β2-microglobulin, ceruloplasmin, complement component C3, complement component C4, C-reactive protein, DNaseB, ferritin, free kappa L chain, free lambda L chain, haptoglobin, immunoglobulin A, immunoglobulin A (CSF), immunoglobulin E, immunoglobulin G, immunoglobulin G (CSF), immunoglobulin G (urine), immunoglobulin G subclass, immunoglobulin M, immunoglobulin M (CSF), kappa L chain, lambda L chain, lipoprotein(a), microalbumin, prealbumin, properdin factor B, rheumatoid factor, ferritin, transferrin, transferrin (urine), rubella IgG, thyroglobulin antibody, toxoplasma IgM, toxoplasma IgG, IGF-I, IGF binding protein (IGFBP)-3, hepsin, pim1 kinase, E-cadherin, EZH2, a-methylacyl CoA racemase, TGF beta, IL6SR, GAD, IA-2, CD-64, neutrophil CD-64, CD-20, CD-33, CD-52, cytochrome P450 isoforms, s-VCAM-1, sFas, sICAM, hepatitis B surface antigen, thromboplastin, HIV p24, HIV gp41 / 120, HCV C22, HCV C33, hemoglobin A1c, GAD65, IA2, vitamin D, 25-OH vitamin D, 1,25(OH)2 vitamin D, 24,25(OH)2 vitamin D, 25,26(OH)2 vitamin D, 3-epimer of vitamin D, FGF23, sclerostin, procalcitonin, calcitonin, Clostridioides difficile (C. difficile) toxin A and B, Helicobacter pylori (h. pylori), HSV1, HSV2 are included.

[0124] Depending on the use for which the affinity assay is designed, it can function as one member or the other member of a binding pair consisting of an analyte binder (capture site) and an analyte, and suitable substances that can be used in the present invention include, for example, those held, characterized, and / or distributed by the WHO International Institute for Biological Standards for biological reference purposes, such as antibodies or fragments thereof, specific sites for any of the WHO International Biological Reference Materials (updated on June 30, 2005, listing substances well known in the art and available at http: / www.who.int / bloodproducts / re_materials, which is hereby incorporated by reference into this specification).

[0125] A partial list of such appropriate international standards, identified by the WHO code in parentheses following the substance, includes: human recombinant thromboplastin (rTF / 95), rabbit thromboplastin (RBT / 90), thyroid stimulating antibody (90 / 672), human recombinant tissue plasminogen activator (98 / 714), high molecular weight urokinase (87 / 594), prostate specific antibody (96 / 668), prostate specific antibody 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 combined thromboplastin (OBT / 79), anti-D positive control intravenous immunoglobulin (02 / 228), islet cell antibody (97 / 550), lipoprotein a (IFCC SRM2B), human parvovirus B19 DNA (99 / 800), human plasmin (97 / 536), human plasminogen activator inhibitor 1 (92 / 654), platelet factor 4 (83 / 505), prekallikrein activator (82 / 530), human brain CJD control and human brain sporadic CJD preparation 1 and human brain sporadic CJD preparation 2 and human brain variant CJD (none; each cited in WHO TRS ECBS report No. 926, 53sup.rd report, brain homogenate), human serum complement components including C1q, C4, C5, factor B, and total functional complement CH50 (W1032), human serum immunoglobulin E (75 / 502), human serum immunoglobulins G, A, and M (67 / 86), human serum proteins albumin, alpha1-antitrypsin, alpha2-macroglobulin, ceruloplasmin, complement component C3, transferrin (W1031), anti-D negative control intravenous immunoglobulin (02 / 226), hepatitis A RNA (00 / 560), hepatitis B surface antigen subtype adw2 genotype A (03 / 262 and 00 / 588), hepatitis B virus DNA (97 / 746), hepatitis C virus RNA (96 / 798), HIV-1 p24 antigen (90 / 636), HIV-1 RNA (97 / 656), HIV-1 RNA genotype (set of 10 I01 / 466), human fibrinogen concentrate (98 / 614),Human plasma fibrinogen (98 / 612), elevated A2 hemoglobin (89 / 666), elevated F hemoglobin (85 / 616), hemoglobin cyanide (98 / 708), low molecular weight heparin (85 / 600 and 90 / 686), unfractionated heparin (97 / 578), blood coagulation factors VIII and von Willebrand factor (02 / 150), human blood coagulation factor VIII concentrate (99 / 678), human blood coagulation factor XIII plasma (02 / 206), human blood coagulation factors II, VII, IX, X (99 / 826), human blood coagulation factors II and X concentrate (98 / 590), human carcinoembryonic antigen (73 / 601), human C-reactive protein (85 / 506), recombinant human ferritin (94 / 572), apolipoprotein B (SP3-07), beta2 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 sample 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 anti-syphilis serum (HS), human anti-tetanus immunoglobulin (TE-3), human anti-thrombin concentrate (96 / 520), human plasma anti-thrombin (93 / 768), human anti-thyroglobulin serum (65 / 93), anti-toxoplasma serum (TOXM), human anti-toxoplasma serum (IgG) (01 / 600), human anti-varicella zoster immunoglobulin (W1044), apolipoprotein A-1 (SP1-01), human anti-interferon beta serum (G038-501-572), human anti-measles serum (66 / 202), anti-nuclear ribonucleoprotein serum (W1063), anti-nuclear endoplasmic reticulum (homogeneous) serum (66 / 233), anti-parvovirus B19 (IgG) serum (91 / 602), type 1, 2, 3 anti-parvovirus serum (66 / 202), human anti-rabies immunoglobulin (RAI), human anti-rubella immunoglobulin (RUBI-1-94), anti-smooth muscle serum (W1062), human anti-double-stranded DNA serum (Wo / 80), human anti-E complete blood type-specific 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 alpha serum (B037-501-572), human alpha-fetoprotein (AFP), ancrod (74 / 581), human anti-A blood group typing serum (W1001), human anti-B blood group typing serum (W1002), human anti-C complete blood group typing serum (W1004), anti-D (anti-Rh0) complete blood group typing serum (99 / 836), human anti-D (anti-Rh0) incomplete blood group typing serum (W1006), and human anti-D immunoglobulin (01 / 572) are included.

[0126] Depending on the use for which the affinity assay is designed, other examples of suitable substances that can function as one member of a binding pair consisting of an analyte binder (capture site) and an analyte, or as the other member, include compounds that can be used as haptens to generate antibodies that recognize the compound, including, but not limited to, the following: hormones including, but not limited to, progesterone, estrogen, testosterone, progestin, corticosteroids, and dehydroepiandrosterone, and any salt, ester, or ether of a non-protein / non-polypeptide antigen listed by the WHO as an international reference standard. A partial list of such suitable international reference standards identified by the WHO code in parentheses following the substance includes vitamin B12 (WHO81.563), folate (WHO95 / 528), homocysteine, transcobalamin, T4 / T3, and other substances described in the WHO catalog of international biological reference materials incorporated herein by reference (available on the WHO web, e.g., the page at http: / / www.who.int / bloodproducts / ref_materials / updated on June 30, 2005). The methods and compositions described herein can include one or more of the aforementioned WHO reference standards or mixtures containing reference standards.

[0127] Other examples of substances that can function as either one member of a binding pair consisting of an analyte binder (capture site) and an analyte, or as the other member, depending on the use for which the affinity assay is designed, include drugs of abuse.Drug abuse includes, for example, the following list of drugs: heroin, morphine, hydromorphone, codeine, oxycodone, hydrocodone, fentanyl, demerol, methadone, darvon, stadol, talwin, paregoric, buprenex; stimulants such as, for example, amphetamine, methamphetamine, etc.; methylamphetamine, ethylamphetamine, methylphenidate, ephedrine, pseudoephedrine, ephedra, ma huang, methylenedioxyamphetamine (MDA), phentermine, phenylpropanolamine; amiphenazole, bemegride, benzfetamine, bromantan, chlorphentermine, clopropamide, clotetamide, diethylpropion, dimethylamphetamine, doxapram, ethamivan, fencamfamine, meclofenoxate, methylphenidate, nikethamide, pemoline, pentetrazol, phentermine, phenylpropanolamine, picrotoxin, pipradol, prolintane, strychnine, synephrine, fencyclidine, and analogs such as angel dust, PCP, ketamine; antidepressants such as, for example, barbiturates, glutethimide, methaqualone, and meprobamate, methohexital, thiamyl, thiopental, amobarbital, pentobarbital, secobarbital, butalbital, butabarbital, talbutal, and aprobarbital, phenobarbital, mephobarbital; benzodiazepines such as, for example, estazolam, flurazepam, temazepam, triazolam, midazolam, alprazolam, chlordiazepoxide, chlorazepate, diazepam, halazepam, lorazepam, oxazepam, prazepam, quazepam, clonazepam, flunitrazepam; GHB drugs such as gamma-hydroxybutyric acid and gamma-butyrolactone; glutethimide, methaqualone, meprobamate, carisoprodol, zolpidem, zaleplon; cannabinoid drugs such as tetrahydrocannabinol and analogs; cocaine, 3,4-methylenedioxymethamphetamine (MDMA); hallucinogens such as, for example, mescaline and LSD, and their metabolites (for example, metabolites present in blood, urine, and other biological substances), and any salts, esters, or ethers thereof are included.

[0128] (Example) (Example 1: Concentration of a small amount of biomarker) A very low concentration of biomarker (0.0195 μIU TSH / mL or 0.497 pg PTH / mL) in 40 mL of PBS was concentrated using streptavidin-coated 550 nm superparamagnetic nanoparticles and then coated with biotinylated anti-TSH antibody (VERAPREP Concentrate TSH reagent) or biotinylated anti-PTH monoclonal antibody (VERAPREP Concentrate PTH reagent).

[0129] In the first study, the VERAPREP Concentrate TSH reagent was prepared by coating 550 nm VERAPREP Biotin with biotinylated anti-TSH capture antibody. 0.08 mL of TSH antigen (10 μIU / mL, for ELISA calibration) was diluted to 0.0195 μIU / mL with 41 mL of PBS buffer, which is less than the functional sensitivity (less than 0.054 μIU / mL) of DRG TSH Ultrasensitive ELISA (product number EIA-1790, lot number RN58849), and 1 mL was stored as a baseline sample (before concentration). 40 mL of the sample was processed using the protocol of VERAPREP Concentrate TSH, and then 1.0 mL of the concentrated sample was prepared for the subsequent TSH ELISA test. 1. Dilute 80 μL of 10 μIU / mL TSH standard solution to 0.0195 μIU / mL with 41.0 mL of PBS and store 1.0 mL as a baseline sample (before concentration). 2. Dilute 80 μL of 10 μIU / mL TSH standard solution to 0.80 μIU / mL with 1.0 mL of VERAPREP Cleave as Control. 3. Add 40 μL of 0.0195 μIU / mL TSH in PBS to a 50 mL Falcon tube. 4. Add the VERAPREP Concentrate TSH mixture. 5. Incubate with mixing for 60 minutes at room temperature. 6. Use Dexter LifeSep® 50SX to magnetically separate VERAPREP Concentrate TSH for 60 minutes. 7. Decant and discard 40 mL of PBS. 8. Add 4.0 mL of PBS wash buffer and mix. 9. Use Dexter LifeSep® 50SX to magnetically separate VERAPREP Concentrate TSH in 4 mL of PBS wash buffer for 30 minutes. 10. Decant and discard 4 mL of PBS. 11. Add 1 mL of PBS wash buffer and mix. 12. Transfer 1 mL of VERAPREP Concentrate TSH to a 1.75 mL conical bottom snap cap vial. 13. Use Dexter LifeSep® 1.5S to magnetically separate VERAPREP Concentrate TSH in 1 mL of PBS wash buffer for 10 minutes. 14. Aspirate and discard 1 mL of PBS. 15. Add 1 mL of VERAPREP Cleave and mix. 16. Use Dexter LifeSep® 1.5S to magnetically separate VERAPREP Concentrate TSH in 1 mL of VERAPREP Cleave for 10 minutes. 17. Aspirate and save 1 mL of the supernatant (concentrated sample) and test the control, baseline sample, and concentrated sample.

[0130] 0.08 mL of TSH antigen (10 μIU / mL, for ELISA calibration) was also diluted to 0.800 μIU / mL with 1 mL of VERAPREP Cleave buffer as a control. The baseline sample, concentrated sample, and control were measured by DRG TSH Ultrasensitive ELISA, and the TSH recovery rate of the concentrated sample was calculated as [result of concentrated sample] / [result of control] × 100%. As expected, the diluted TSH baseline sample was not detected by Ultrasensitive ELISA and was shown as 0.00 μIU / mL. Using only 0.80 mg of reagent, VERAPREP Concentrate TSH successfully concentrated the diluted TSH from an undetectable state to 0.73 μIU / mL (Table 1). The recovery rate was 98.6% compared to the control, but the assay signal may have been suppressed by the matrix effect of VERAPREP Cleave buffer in TSH ELISA (Table 2).

[0131]

Table 1

[0132]

Table 2

[0133] In the second study, VERAPREP Concentrate PTH reagent was prepared by coating 550 nm VERAPREP Biotin with the biotinylated anti-PTH capture site. 0.021 mL of PTH antigen (971 pg / mL, for ELISA calibration) was also diluted to 0.497 pg / mL with 41 mL of PBS buffer less than the functional sensitivity of DRG PTH (Parathyroid) Intact ELISA (product number EIA-3645, lot number 2896) (less than 1.56 pg / mL), and 1 mL was stored as the baseline sample (before concentration). 40 mL of the sample was processed using the protocol of VERAPREP Concentrate PTH, and then 1.0 mL of the concentrated sample was prepared for subsequent PTH ELISA testing. 1. Dilute 21 μL of 971 pg / mL PTH standard solution with 41.0 mL of PBS to 0.497 pg / mL, and store 1.0 mL as the baseline sample (before concentration). 2. Dilute 21 μL of 971 pg / mL PTH standard solution to 20.4 pg / mL with 1.0 mL of VERAPREP Cleave as a control. 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 for 30 minutes at room temperature with mixing. 6. Magnetically separate VERAPREP Concentrate PTH for 15 minutes using Dexter LifeSep® 50SX. 7. Decant and discard 40 mL of PBS. 8. Add 4.0 mL of PBS wash buffer and mix. 9. Magnetically separate VERAPREP Concentrate PTH in 4 mL of PBS wash buffer for 10 minutes using Dexter LifeSep® 50SX. 10. Decant and discard 4 mL of PBS. 11. Add 1.0 mL of PBS wash buffer and mix. 12. Transfer 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 for 10 minutes using Dexter LifeSep® 1.5S. 14. Aspirate and discard 1 mL of PBS. 15. Add 1 mL of VERAPREP Cleave and mix. 16. Use Dexter LifeSep® 1.5S to magnetically separate VERAPREP Concentrate PTH in 1 mL of VERAPREP Cleave for 10 minutes. 17. Aspirate and store 1 mL of the supernatant (concentrated sample), and test the control, baseline sample, and concentrated sample.

[0134] 0.021 mL of PTH antigen (971 pg / mL, for ELISA calibration) was also diluted to 20.4 pg / mL with 1 mL of VERAPREP Cleave buffer as a control. The baseline sample, concentrated sample, and control were examined by DRG PTH (Parathyroid) Intact ELISA, and the PTH recovery rate of the concentrated sample was calculated as [concentrated sample result] / [control result] × 100%. The diluted PTH baseline sample became 13.5 pg / mL due to the matrix effect of VERAPREP Cleave buffer in the ELISA assay. This matrix effect improved the assay signal. VERAPREP Concentrate PTH, using only 0.80 mg of reagent, successfully concentrated the diluted PTH to 42.3 pg / mL (Table 3). This was a 109% recovery compared to the control (Table 4).

[0135]

Table 3

[0136]

Table 4

[0137] (Example 4: Concentration of Low-Concentration Biomarkers from Urine for Subsequent Mass Spectrometry (LC-MS / MS or MALDI-MS) Analysis) The following describes a mass spectrometry sample pretreatment protocol for concentrating low-concentration biomarkers and spiked internal standards (ISTDs) from a large volume of urine samples using superparamagnetic nanoparticles coated with capture sites specific to the biomarkers. The exact same protocol can also use multiple different populations of superparamagnetic nanoparticles, each population coated with a different capture site, to multiplex and concentrate two or more biomarkers and their corresponding spiked ITDs from the same sample. The concentration and characterization of two or more biomarkers enables the easy use of algorithms for clinical diagnosis and / or prognosis of diseases that are not possible with the characterization of a single biomarker. For example, to diagnose obstructive sleep apnea (OSA) from urine, the VERAPREP Concentrate reagent can consist of four antibodies for capturing and concentrating kallikrein-1, uromodulin, urocortin-3, and orosomucoid-1, or seven antibodies for capturing and concentrating 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 a urine collection cup). 2. Mix the urine sample. 3. Add 40 mL of urine to a 50 mL Falcon tube. 4. Add the deuterated internal standard for the biomarker for concentration and mix. 5. Add VERAPREP Condition and mix. 6. Add VERAPREP Concentrate and mix. 7. Incubate: The biomarker and deuterated internal standard are captured by VERAPREP Concentrate. 8. Magnetically separate VERAPREP Concentrate in 40 mL of urine using Dexter LifeSep® 50SX. 9. Aspirate and discard the urine. 10. Add 4 mL of PBS wash buffer and mix. 11. Using Dexter LifeSep® 50SX, magnetically separate VERAPREP Concentrate in 4 mL of PBS wash buffer. 12. Aspirate and discard the urine. 13. Repeat step 12 two more times (2×). 14. Add 1 mL of VERAPREP Cleave and mix (mass spectrometry compatible buffer). 15. Using Dexter LifeSep® 1.5S, magnetically separate VERAPREP Concentrate in 1 mL of VERAPREP Cleave. 16. Aspirate 1 mL of the supernatant sample and test by LC-MS. 17. The final biomarker concentration was determined based on 1) a 40 mL urine sample size, 2) LC-MS quantification of the biomarker, and 3) adjustment of the reported biomarker value based on the recovery rate of the deuterated internal standard.

[0138] Selective release or cleavage of the captured and concentrated biomarker(s) can be achieved by a change in pH (acidic pH such as neutralization after elution with glycine pH 2.5 or alkaline pH of pH 10.0 or higher), use of a cleavable linker such as a disulfide bond cleaved with a reducing agent like TCEP or DTT, or use of competitive elution with a molar excess of D-biotin with monomeric avidin to compete for the binding site on concanavalin A or a molar excess of sugar with concanavalin A.

[0139] (Example 5: Sample Pretreatment for Depletion of Sample Interferents Before Concentration of Low-Concentration Biomarkers) The following describes a sample pretreatment protocol for depleting sample interferents before concentrating low-concentration biomarkers. In this protocol, the sample is pretreated with Reagent A, which contains exactly the same microparticles as Reagent B, except that the capture sites on the microparticles of Reagent A do not have specificity for the biomarker to be concentrated. After performing sample pretreatment to deplete the sample interferents, the beads of Reagent A are removed from the sample magnetically, physically, or chemically. Next, the sample depleted of interferents or free of interferents is added to or mixed with Reagent B to capture and concentrate the biomarker in the absence of interferents.

[0140] This method is automated on liquid handling systems such as Hamilton and Tecan using magnets on the deck to capture magnetic, paramagnetic, or superparamagnetic microparticles such as 96-well or 384-well plate magnets. 1. Place the sample rack on the system. 2. Aspirate the sample and dispense it into reaction vessel A such as a tube, 96-well plate, or 384-well plate. 3. Aspirate and dispense the mixed Reagent A (magnetic microparticles) into reaction vessel A, mix, and incubate to capture and deplete sample-specific interferents. 4. Move reaction vessel A to the magnet position (e.g., a single magnet for a tube or a 96-well or 384-well magnetic separator) to separate Reagent A. 5. Aspirate the pretreated sample and dispense it into reaction vessel B such as a tube, 96-well plate, or 384-well plate. 6. Add the mixed Reagent B to the reaction vessel, mix, and incubate to capture the biomarker or concentrate the biomarker in the absence of interferents (depleted and removed by sample pretreatment with Reagent A). 7. Move reaction vessel B to the magnet position (e.g., a single magnet for a tube or a 96-well or 384-well plate magnetic separator) to separate Reagent A. 8. Aspirate / remove the sample supernatant and matrix, discard, and wash Reagent B. 9. To measure the captured biomarker, reagent B is tested directly in the measurement system, or for measurement in the test system, the biomarker is cleaved, eluted, or selectively released from reagent B, or the capture site-biomarker complex is cleaved, eluted, or selectively released from reagent B, or the (pre-labeled capture site)-biomarker complex is cleaved, eluted, or selectively released from reagent B.

[0141] (Abbreviations) ABEI N-(4-aminobutyl)-N-ethylisouramil ALP Alkaline phosphatase BSA Bovine serum albumin Fab fragment antibody binding Fc fragment, crystallizable HAAA Human anti-animal antibody HAMA Human anti-mouse antibody HASA Human anti-sheep antibody IFU Instructions for use IgG Antibody or immunoglobulin IgM Immunoglobulin M HRP Horseradish peroxidase LC-MS / MS Liquid chromatography-tandem mass spectrometry LDT Laboratory-developed test Mab Monoclonal antibody MASI Manufacturing assay-specific interference MFG IVD manufacturer PMP Superparamagnetic microparticles PBCT Primary blood collection tube RF Rheumatoid factor RLU Relative light unit or assay response signal RUO For research use only SAv Streptavidin STT Secondary transfer tube TAT Turnaround time WF Workflow

[0142] (Definitions) As used herein, "sample" or "biological sample" refers to human or animal serum, plasma (i.e., EDTA, lithium heparin, sodium citrate), blood, whole blood, processed blood, urine, saliva, feces (liquid and solid), semen or seminal plasma, amniotic fluid, cerebrospinal fluid, cells, tissues, biopsy materials, DNA, RNA, or any liquid or dissolved solid, or processed solid material that is tested for monitoring such as diagnosis, prognosis, screening, risk assessment, severity classification, and therapeutic drug monitoring. In some embodiments, the sample is a large volume sample. In some embodiments, the sample includes a plurality of samples (e.g., two or more samples from the same or different subjects). In some embodiments, the sample includes a biomarker present at a low concentration in the sample.

[0143] In some embodiments, the sample is a primary blood collection tube (PBCT), a secondary transfer tube (SST), a 24-hour (24hr) urine collection device, a BD Vacutainer Barricor tube, a nanotainer, a saliva collection tube, a blood spot filter paper, or any collection tube or device for feces and semen, a light green or green top plasma separation tube (PST) containing sodium heparin, lithium heparin, or ammonium heparin, a light blue top tube containing sodium citrate (i.e., 3.2% or 3.8%) or citrate, theophylline, adenosine, dipyridamole (CTAD), a red top tube for serology or immunohematology for collecting serum in a glass tube (without additive) or a plastic tube (containing a thrombin activator), a red top tube for chemistry for collecting serum in a glass tube (without additive) or a plastic tube (containing a thrombin activator), a purple lavender top tube or an EDTA K2 / gel tube containing EDTA K2, EDTA K3, a liquid EDTA solution (i.e., 8%) for testing plasma in molecular diagnostics and detection of viral load, a pink top tube for blood bank EDTA, a gray top tube containing potassium oxalate and sodium fluoride, sodium fluoride / EDTA, or sodium fluoride (for non-anticoagulated, serum samples), a yellow top tube containing ACD solution A or ACD solution B, a royal blue top tube (serum, without additive or containing sodium heparin), a white top tube, or any color or any type of tube for blood collection containing no additive or any additive, or combinations thereof, for any use or type of diagnostic test.

[0144] In some embodiments, the sample is urine, 24-hour urine, saliva, and feces, or a difficult type of sample such as one in which the biomarker of interest is diluted or difficult to measure. For example, the biological sample may be difficult for a population of patients (e.g., neonates, children, the elderly, pregnant women, tumors, autoimmune diseases). For example, some biomarkers are too diluted or too low in concentration, either in circulation or in urine, to be reliably detected by current POCT and major laboratory analyzers and cannot be measured accurately and precisely. In some embodiments, the difficult sample is cerebrospinal fluid (CSF).

[0145] As used herein, the "collection device" can be a primary blood collection tube (PBCT), 24-hour urine collection device, urine collection device, saliva collection tube, feces collection device, semen collection device, blood collection bag, or any sample collection tube or device prior to adding the sample.

[0146] PCBT and secondary transfer tubes (SSTs) can be standard or custom collection tubes (with or without gel separator), glass tubes, plastic tubes, green or light green top plasma separation tubes (PSTs) containing sodium heparin, lithium heparin, or ammonium heparin, tubes from companies such as Becton Dickinson (BD), Greiner, VWR, and Sigma Aldrich, light blue top tubes containing sodium citrate (i.e., 3.2% or 3.8%) or citrate, theophylline, adenosine, dipyridamole (CTAD), red top tubes for serology or immunohematology for collecting serum in glass tubes (without additives) or plastic tubes (containing thrombin activators), red top tubes for chemistry for collecting serum in glass tubes (without additives) or plastic tubes (containing thrombin activators), purple lavender top tubes containing EDTA K2, EDTA K3, liquid EDTA solution (i.e., 8%) or EDTA K2 / gel tubes for testing plasma in molecular diagnostics and detection of viral load, pink top tubes for blood bank EDTA, gray top tubes containing potassium oxalate and sodium fluoride, sodium fluoride / EDTA, or sodium fluoride (for non-anticoagulated, serum samples), yellow top tubes containing ACD solution A or ACD solution B, royal blue top tubes (serum, without additives or with sodium heparin), white top tubes, or any color or any type of tube for blood collection containing no additives or any additives, or combinations thereof, for any application or type of diagnostic test.

[0147] As used herein, "storage device" or "transfer device" refers to a device capable of receiving samples 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, can, microtiter plate, ELISA plate, 96-well plate, 384-well plate, 1536-well plate, cuvette, reaction module, reservoir, or any container suitable for holding, storing, or processing liquid samples.

[0148] As used herein, "diagnostic test" includes, but is not limited to, diagnostic tests of antibody systems, non-antibody systems, sample pretreatment methods or devices for subsequent analysis by chromatography, spectrophotometry, and mass spectrometry (i.e., HPLC, MS, LCMS, LC-MS / MS) such as immunoprecipitation (IE) and solid-phase extraction (SPE), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), molecular diagnostics, lateral flow (LF), point-of-care (PoC), direct-to-consumer (DTC), CLIA and CLIA-waived tests and devices, research use only (RUO) tests, in vitro diagnostic (IVD) tests, laboratory-developed tests (LDT), companion diagnostics, and any tests for monitoring such as diagnosis, prognosis, screening, risk assessment, severity classification, and therapeutic drug monitoring. In some embodiments, diagnostic tests include diagnostic tests with short turnaround times (STAT), outpatient tests, lateral flow tests, point-of-care (PoC) tests, molecular diagnostic tests, HPLC, MS, LCMS, LC-MS / MS, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), CLIA and CLIA-waived tests, and any diagnostic tests for diagnosis, prognosis, screening, risk assessment, severity classification, treatment monitoring, and therapeutic drug monitoring.

[0149] (Appendix) (Appendix 1) A method for removing biomarkers from a biological sample, comprising: a) combining the sample with a plurality of types of particles, each particle independently comprising a capture site (i.e., a type or kind of capture site), to provide a mixture; b) mixing the mixture to provide one or more particle complexes with the biomarker; c) removing or isolating the particle complex to provide a depleted solution; thereby removing the biomarker from the biological sample. Method.

[0150] (Appendix 2) A method for isolating biomarkers from a biological sample, comprising: a) combining the sample with a plurality of types of particles, each particle independently comprising a capture site (i.e., a type or kind of capture site), to provide a mixture; b) mixing the mixture to provide one or more particle complexes with the biomarker; c) removing or isolating the particle complex to provide a depleted solution and a concentrated isolate; thereby isolating the biomarker from the biological sample. Method.

[0151] (Appendix 3) The method according to Appendix 1 or 2, wherein a conditioning agent is added to the biological sample before combining the sample with the plurality of types of particles.

[0152] (Appendix 4) The method according to Appendix 3, wherein the conditioning agent is a pH adjuster, a molarity adjuster, an interference blocker, or a releasing or liberating agent.

[0153] (Appendix 5) The method according to Appendix 1 or 2, which is performed on the biological sample before a diagnostic test is performed.

[0154] ​​ (Appendix 6) The method according to Appendix 1 or 2, wherein the plurality of types of particles include a plurality of types of capture sites (for example, a plurality of types of particles each independently covalently or non-covalently bonded to a plurality of types of capture sites).

[0155] (Appendix 7) The method according to Appendix 1 or 2, wherein the plurality of types of particles include a first particle having a first capture site.

[0156] (Appendix 8) The method according to Appendix 7, wherein the plurality of types of particles include a second particle having a second capture site.

[0157] (Appendix 9) The method according to Appendix 8, wherein the plurality of types of particles include a third particle having a third capture site.

[0158] (Appendix 10) The method according to Appendix 9, wherein the plurality of types of particles include a fourth particle having a fourth capture site.

[0159] (Appendix 11) The method according to Appendix 10, wherein the plurality of types of particles include a fifth particle having a fifth capture site.

[0160] (Appendix 12) The method according to Appendix 11, wherein the plurality of types of particles include a sixth particle having a sixth capture site.

[0161] (Appendix 13) The method according to Appendix 12, wherein the plurality of types of particles include a seventh particle having a seventh capture site.

[0162] (Appendix 14) The method according to Appendix 13, wherein the plurality of types of particles include an eighth particle having an eighth capture site.

[0163] (Appendix 15) The method according to appendix 14, wherein the plurality of types of particles includes a ninth particle having a ninth capture site.

[0164] (Appendix 16) The method according to appendix 15, wherein the plurality of types of particles includes a tenth particle having a tenth capture site.

[0165] (Appendix 17) The method according to appendix 16, further comprising removing or separating the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth biomarker from the biological sample.

[0166] (Appendix 18) The method according to appendix 1 or 2, further comprising adding a cleavage reagent or releasing agent to the mixture to provide an enriched isolate.

[0167] (Appendix 19) The method according to appendix 1 or 2, further comprising performing a diagnostic test on the biomarker (for example, after the removal method or isolation method described herein).

[0168] (Appendix 20) The method according to appendix 19, wherein the diagnostic test simultaneously detects the presence or absence of two or more biomarkers.

[0169] (Appendix 21) The method according to appendix 8, wherein the first particle differs from the second particle in size, shape, chemical properties, color, or other properties.

[0170] (Appendix 22) The method according to appendix 9, wherein the first particle differs from the third particle in size, shape, chemical properties, color, or other properties.

[0171] (Appendix 23) The method according to appendix 10, wherein the first particle differs from the fourth particle in size, shape, chemical properties, color, or other properties.

[0172] (Supplementary Note 24) The method according to Supplementary Note 11, wherein the first particle differs from the fifth particle in size, shape, chemical properties, color, or other properties.

[0173] (Supplementary Note 25) The method according to Supplementary Note 12, wherein the first particle differs from the sixth particle in size, shape, chemical properties, color, or other properties.

[0174] (Supplementary Note 26) The method according to Supplementary Note 13, wherein the first particle differs from the seventh particle in size, shape, chemical properties, color, or other properties.

[0175] (Supplementary Note 27) The method according to Supplementary Note 14, wherein the first particle differs from the eighth particle in size, shape, chemical properties, color, or other properties.

[0176] (Supplementary Note 28) The method according to Supplementary Note 15, wherein the first particle differs from the ninth particle in size, shape, chemical properties, color, or other properties.

[0177] (Supplementary Note 29) The method according to Supplementary Note 16, wherein the first particle differs from the tenth particle in size, shape, chemical properties, color, or other properties.

[0178] (Supplementary Note 30) The method according to any one of Supplementary Notes 21 to 29, wherein the property is selectivity, affinity, or binding property for a biomarker described in this specification.

[0179] (Supplementary Note 31) The method according to any one of Supplementary Notes 21 to 29, wherein the size is 50 to 1000 nm in diameter (for example, 100 to 500 nm, 200 to 600 nm).

[0180] (Supplementary Note 32) The method according to any one of Supplementary Notes 21 to 29, wherein the size is 1 to 3 microns in diameter.

[0181] (Supplementary Note 33) The method according to Supplementary Note 1 or 2, wherein the first particle population exists at a higher concentration than the second particle population.

[0182] (Supplementary Note 34) The method according to Supplementary Note 33, wherein the ratio of the first particle to the second particle is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0183] (Supplementary Note 35) The method according to Supplementary Note 7, wherein the first particle exists at a first concentration.

[0184] (Supplementary Note 36) The method according to Supplementary Note 8, wherein the second particle exists at a second concentration.

[0185] (Supplementary Note 37) The method according to Supplementary Note 9, wherein the third particle exists at a third concentration.

[0186] (Supplementary Note 38) The method according to Supplementary Note 10, wherein the fourth particle exists at a fourth concentration.

[0187] (Supplementary Note 39) The method according to Supplementary Note 11, wherein the fifth particle exists at a fifth concentration.

[0188] (Supplementary Note 40) The method according to Supplementary Note 12, wherein the sixth particle exists at a sixth concentration.

[0189] (Supplementary Note 41) The method according to Supplementary Note 13, wherein the seventh particle exists at a seventh concentration.

[0190] (Supplementary Note 42) The method according to Supplementary Note 14, wherein the eighth particle exists at an eighth concentration.

[0191] (Supplementary Note 43) The method according to appended note 15, wherein the ninth particles are present at a ninth concentration.

[0192] (Appended note 44) The method according to appended note 16, wherein the tenth particles are present at a tenth concentration.

[0193] (Appended note 45) The method according to appended note 7, wherein the first particles are control particles (for example, particles containing a label or an indicator (for example, a label or an indicator of a known amount or concentration)).

[0194] (Appended note 46) The method according to appended note 45, wherein the label or the indicator provides a measurement of the concentration or volume of the sample.

[0195] (Appended note 47) The method according to appended note 45, wherein the label or the indicator provides a measurement of the yield or the particle recovery rate.

[0196] (Appended note 48) The method according to appended note 45, wherein the label or the indicator provides an indicator of the lot number or batch number of the sample.

[0197] (Appended note 49) The method according to appended note 1 or 2, wherein the biomarker is biotin, HAMA, RF, heterophile, or anti-SAv.

[0198] (Appended note 50) The method according to appended note 1 or 2, wherein the biomarker is an indicator of a bacterial infection.

[0199] (Appended note 51) The method according to appended note 1 or 2, wherein the biomarker is a bacterial capture site.

[0200] (Appended note 52) The method according to appended note 1 or 2, wherein the removal or isolation of the particle complex includes cleavage, elution, or selective release of the capture site-biomarker complex.

[0201] (Appendix 53) The method according to Appendix 52, wherein the capture site contains a signal detection molecule for measurement in a diagnostic test system.

[0202] (Appendix 54) Before the combination step a), the sample is pretreated to remove or deplete interfering substances by: (i) combining the sample with particles containing a capture site that is not specific for the biomarker to provide a mixture; (ii) mixing the mixture to provide a particle complex with the interfering substance; and (iii) removing or completely removing the particle complex to provide a depleted solution. The method according to Appendix 1 or 2.

[0203] (Appendix 55) A kit comprising a plurality of types of particles, a magnet, a tube, and an instruction manual.

[0204] (Appendix 56) A method for detecting a patient's urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein, or a combination thereof, comprising: a. obtaining a sample from a human patient; and b. detecting whether urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein, or a combination thereof is present in the sample by a plurality of types of particles, and detecting the binding between urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein, or a combination thereof and the plurality of types of particles. A method comprising the above steps.

[0205] (Appendix 57) A method for diagnosing a patient's obstructive sleep apnea, comprising: a. Obtaining a sample from a human patient, and, b. Detecting whether urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein, or a combination thereof is present in the sample by contacting the sample with a plurality of types of particles, and detecting the binding between urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein, or a combination thereof and the plurality of types of particles, and, c. Diagnosing as a patient with obstructive sleep apnea when the presence of urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein is detected in the sample. A method comprising the above steps.

[0206] (Appendix 58) The method according to Appendix 56 or 57, wherein the patient is human.

[0207] (Appendix 59) The method according to Appendix 58, wherein the human is from about 9 years old to less than about 2 years old.

[0208] (Appendix 60) The method according to Appendix 56 or 57, comprising simultaneously detecting the presence or absence of two or more of the group consisting of urocortin III peptide, uromodulin peptide, orosomucoid 1 peptide, kallikrein 1 peptide, IL-6, IL-10, high-sensitivity C-reactive protein.

Claims

1. 1. A method for removing a biomarker from a biological sample, comprising: a) combining said sample with a plurality of particles, each particle independently comprising a capture site (i.e., a type or variety of capture site) to provide a mixture; b) mixing said mixture to provide one or more particulate complexes with said biomarkers; c) removing or isolating said particle complexes to provide a depleted solution; Including, thereby removing the biomarker from the biological sample. method.

2. 1. A method for isolating a biomarker from a biological sample, comprising: a) combining said sample with a plurality of particles, each particle independently comprising a capture site (i.e., a type or variety of capture site) to provide a mixture; b) mixing said mixture to provide one or more particulate complexes with said biomarkers; c) removing or isolating said particle complexes to provide a depleted solution and a concentrated isolate; Including, thereby isolating biomarkers from the biological sample; method.

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