Compositions and methods for detecting and depleting interferents in samples

Abstract

The present invention relates to compositions and methods for detecting and depleting interferents in a sample. The present invention provides a streptavidin (QSAv) saturated with biotin prepared by a method comprising the step of exposing the streptavidin to a 5: 1 to 11: 1 molar excess of biotin. Also provided are microparticles (QSAv beads) saturated with biotin and coupled to streptavidin prepared by a method comprising the step of exposing streptavidin to a 4: 1 to 6: 1 molar excess of biotin. Through biotin-saturated streptavidin (QSAv) and a magnetic nanoparticle technology, anti-biotin and anti-streptavidin interferents in a sample can be specifically recognized and depleted, and the accuracy of diagnostic testing is remarkably improved.

Description

[0001] This application is a divisional application of international application PCT / US2020 / 039503, which entered the Chinese national phase on February 24, 2022, with application number 202080060085.6 and the invention title "Composition and Method for Detecting and Depleting Interfering Substances in a Sample".

[0002] Cross-reference with related applications

[0003] This application claims priority to U.S. Provisional Application No. 63 / 006,630, filed April 7, 2020, and U.S. Provisional Application No. 62 / 866,318, filed June 25, 2019, the entire contents of each of which are incorporated herein by reference. Background Technology

[0004] One person dies every nine minutes due to a misdiagnosis or delay[1]. Doctors rely on diagnostic tests to guide treatment, but 2% or more of the tests may be inaccurate due to various interfering substances in blood or urine (such as biotin in blood tests)[2].

[0005] Biotin, also known as vitamin B7, vitamin H, and coenzyme R, is a water-soluble vitamin often found in high doses in over-the-counter (OTC) dietary supplements, multivitamins, and prenatal vitamins. Biotin is primarily used for health and beauty purposes, including hair, skin, and nail growth, as well as weight loss. It is also provided to patients in high therapeutic doses to treat certain medical conditions, such as multiple sclerosis. However, biotin can significantly interfere with certain laboratory tests and lead to incorrect test results, potentially becoming undetectable and causing misdiagnosis or delayed treatment [3-13].

[0006] In 2017, the FDA issued a safety warning due to the number of adverse events related to inaccurate test results and biotin supplementation.

[14] On June 13, 2019, the FDA issued a notice of a draft guidance document on “Testing for Biotin Interference in In Vitro Diagnostic Devices”.

[15]

[0007] In vitro diagnostic (IVD) companies are actively working to redesign or reformulate their tests to mitigate or increase the biotin interference threshold, requiring much higher biotin concentrations to interfere with the test. However, these biotin-based tests remain susceptible to secondary interference mechanisms associated with biotin or patient use of biotin or anti-biotin interfering agents [16-17], as well as interference mechanisms associated with the use of streptavidin in test design to capture biotin or anti-streptavidin interfering agents that have been conjugated to antibodies, proteins, or antigens [18-26].

[0008] Anti-biotin and anti-streptavidin antibodies and proteins can significantly interfere with certain laboratory tests and cause incorrect test results. Similar to biotin-interfering substances that cause a decrease in test signal or false low or high patient results depending on the assay design and format, anti-biotin and anti-streptavidin interfering substances also cause a decrease in test signal but by different mechanisms, and therefore they may be mistaken for biotin-interfering substances [16-26].

[0009] Although the FDA recently issued guidance requiring IVD companies to test biotin interferons at concentrations up to 1200 ng / mL in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI) standards, and to reflect current trends in biotin consumption, the FDA has not issued any safety warnings for adverse events associated with inaccurate test results caused by human antibiotin or human antistreptavidin interferons

[15] . Although human antistreptavidin and human antibiotin interferons have been reported in the literature, it is difficult to detect and confirm the mechanisms of these specific interferons or to distinguish them from biotin interferons.

[0010] Sample pretreatment using immobilized or covalently coupled binding surfaces to capture portions or interfering specific targets (i.e., magnetic beads, non-magnetic beads, nanoparticles, microtiter plates / wells, cuvettes, slides, sensors, chips, rods, filters, membranes, test tubes, or any other solid phase used to process the sample) can be used to deplete, enrich, and / or characterize interfering substances or biomarkers in samples prior to diagnostic testing to improve the quality and accuracy of test results

[27] . Summary of the Invention

[0011] A significant proportion of IVD assays utilize streptavidin-biotin binding. These assays are susceptible to heterophilic interferences from free biotin and reagents that compete for or otherwise interfere with the binding of the assay reagent to streptavidin or biotin, including anti-streptavidin antibodies and anti-biotin antibodies. Substances that interfere with the binding of the assay reagent to streptavidin or biotin are referred to herein as anti-biotin substances or anti-streptavidin substances, regardless of whether the substance is an antibody. The reagents (beads) disclosed herein can be used to: 1) detect or quantify these different types of interfering substances, and 2) remove or deplete interfering substances that may be present in the assay reagent, sample, or reaction mixture to obtain more accurate assay results. Methods for preparing and using these reagents are also provided.

[0012] Some of the reagents disclosed herein comprise nanoparticles coated with streptavidin to form streptavidin beads. In some embodiments, some or all of the streptavidin are covalently coupled to the nanoparticles. In some embodiments, the nanoparticles are magnetic to facilitate separation of the beads from storage solutions and treated samples, assay reagents, etc. In some embodiments, the nanoparticles may or may not be magnetic, and separation of the beads from storage solutions and treated samples is achieved by sedimentation (e.g., centrifugation) or filtration. Other embodiments comprise free (or soluble) streptavidin. In either type of free or coated bead embodiment, the streptavidin is saturated (or quenched) with a minimum excess of biotin, preferably saturated, such that the streptavidin cannot bridge between biotinylated assay reagent molecules and become a source of heterophilic interference. Saturation means that all accessible biotin-binding sites on the streptavidin are occupied by biotin. The free biotin-saturated streptavidin is suitable as a blocking agent against streptavidin heterophile interference, and can be added, for example, to the assay reaction mixture (present therein). The biotin-saturated streptavidin-coated beads are suitable as a cleaning agent against streptavidin heterophile interference, and can be added, for example, to the biological fluid or extract (sample) to be tested, and then removed before adding the sample to the assay reaction mixture. In some uses, the cleaning agent can be added to the assay reagent or a portion of the assay reaction mixture and removed before completing the assay reaction mixture and starting the assay reaction.

[0013] Embodiments utilizing streptavidin are described throughout this disclosure. However, other embodiments incorporating alternatives such as streptavidin, deglycosylated streptavidin (neutral streptavidin), CaptAvidin, and monomeric streptavidin are also contemplated. Natural and recombinant versions of streptavidin and their alternatives are also contemplated. These agents may be referred to as biotin-binding means or means of binding anti-streptavidin interfering agents.

[0014] Embodiments utilizing biotin to quench or saturate the biotin-binding sites of streptavidin activity are described throughout this disclosure. However, the use of biotinylation agents such as biotin-PEG is also contemplated. n -COOH or biotin-PEG n -CH3 or biotin-PEG n Other embodiments of -OH or other biotin-R- (non-reactive terminal chemical groups) (where R is a carbon chain or ring structure). Biotin and these modified forms of biotin may be referred to as means of binding streptavidin or means of binding anti-biotin interferon.

[0015] In other embodiments, the streptavidin-coated beads, biotin-saturated streptavidin-coated beads, streptavidin, or quenched streptavidin are modified by coupling with one or more additional capture portions to remove other heterophilic or cross-reactive interfering agents (other than anti-streptavidin interfering agents). In one instance of these embodiments, the additional capture portion is biotin coupled to the biotin-saturated streptavidin-coated beads and is also suitable as a cleaning agent for anti-biotin heterophilic interfering agents. In other instances of these embodiments, the additional capture portion is ruthenium (an element); luminol, acridinium ester, ABEI, or cyclic ABEI (similar to biotin, a small organic molecule); or a protein, such as a signaling enzyme, such as alkaline phosphatase or horseradish peroxidase; streptavidin; an antibody, such as an antibody derived from a non-human species; or an antigen. In other instances, the capture portion can be any non-antibody peptide or protein. In all these cases, the additional capturing portion makes the streptavidin-coated beads or biotin-saturated streptavidin-coated beads suitable as a cleaning agent for removing or depleting heterophilic or cross-reactive interfering substances associated with the coupled molecule. Some embodiments specifically include one or more capturing portions. Some embodiments specifically exclude one or more capturing portions. For example, in some embodiments, the additional capturing portion is not biotin.

[0016] Several chemical methods exist for achieving coupling with streptavidin (soluble or bead-bound, saturated with or without biotin). Coupling can be performed using an amine-reactive agent bonded to the primary amine of streptavidin, typically an ester, such as an NHS-modified compound or protein. Alternatively, the primary amine of streptavidin can be thiolated. Standard thiolation agents are known in the art, but include trans-4-(maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) and 3-(2-pyridyldithio)propionic acid succinimide ester (SPDP). Coupling can then be performed using thiol or thiol-reactive agents, such as maleimide-modified compounds or proteins. In another alternative, the primary amine of streptavidin is reacted with maleimide using a standard ester-maleimide heterobifunctional crosslinking agent. Coupling can then be performed using thiol or thiol-modified (or containing) compounds or proteins. These chemical methods and related reagents can be referred to as coupling means, and the reaction itself is referred to as a coupling step.

[0017] Typically, the coupling of the additional capture portion with the streptavidin-coated beads, biotin-saturated streptavidin-coated beads, streptavidin, or quenched streptavidin involves the use of a heterobifunctional linker. A functional group at one end of the linker is covalently attached to the streptavidin, and a functional group at the other end is covalently attached to the additional capture portion. The chemistry of the specific functional groups is discussed later. In some embodiments, the capture portion attached to the linker may be commercially available. Some embodiments specifically include a specific functional group or a set of functional groups. Some embodiments specifically exclude a specific functional group or a set of functional groups. In some embodiments, the central portion of the heterobifunctional linker comprises polyethylene glycol (PEG) or polyethylene oxide (PEO). In some cases of this embodiment, the linker may comprise multiple PEG or PEO units, such as PEG. n or PEO n , where n is any integer from 1 to 36. In other cases, the PEG linker may be branched or dendritic, such as monodisperse PEG, trifunctional PEG, 4-arm PEG, 8-arm PEG, heterobifunctional PEG, homobifunctional PEG, rather than linear monofunctional PEG. Other linkers are disclosed below.

[0018] Various methods of conjugating biotin to biotin-saturated streptavidin are disclosed below. However, any other capture agent can be similarly conjugated to streptavidin (with or without biotin saturation, bead-bound or unbound).

[0019] In an alternative embodiment, the additional trapping portion is not covalently attached, but rather attached using a biotin linker. In some embodiments, the additional trapping portion is biotin, and the linker has a biotin molecule at each end, such as biotin-PEG. n - Biotin. In such embodiments, the dual biotin linker is added as a small fraction of the biotin used in the streptavidin saturation process (to avoid bridging between beads). Thus, biotin at one end binds to streptavidin, while biotin at the other end remains free to act as a capture portion. In other embodiments, a linker with biotin at one end and any other capture portion at the other end, such as biotin-(PEO)n-ruthenium, is used. In other embodiments, two or more distinct capture portions can be introduced in this way, for example, by co-coating streptavidin with biotin-(PEO)n-ruthenium and biotin-(PEO)n-alkaline phosphatase. In these embodiments, a potentially larger proportion of the biotin used in the streptavidin saturation process may be the biotin linked to the capture portions, as bridging between beads should not be a problem; however, spatial considerations based on the size of the capture portions and the length of the linker may limit this proportion.

[0020] Some embodiments relate to methods for mitigating interference in liquid biological samples. Other embodiments relate to methods for reducing interference in diagnostic assays. In some embodiments, biotin-saturated streptavidin (biotin-quenched streptavidin; QSAv) is combined with a liquid biological sample to form a mixture, which is then mixed to promote the binding of the interfering substance to the streptavidin, thereby blocking or reducing interference. Some embodiments also include performing a diagnostic assay. In some embodiments, the combination and mixing occur prior to the analytical phase of the assay. When used herein, the term "analytical phase of the assay" begins when the sample is mixed with the reagent to capture or detect the analyte and / or generate a signal indicating or quantifying the presence of the analyte, and continues until a measurement of the signal is obtained.

[0021] In other embodiments (for mitigating or reducing interference), streptavidin-containing particles, quenched with or without biotin, are combined with a liquid biological sample to form a mixture. This mixture is then mixed to promote binding of the interfering substance to the streptavidin, and the particles are separated from the sample to remove or reduce the interference. Some embodiments also include performing a diagnostic assay. In some embodiments, the merging, mixing, and separation occur prior to the analytical phase of the assay. In one case of these methods, the particles are magnetic, and separating the particles from the sample involves exposing the mixture to a magnet and collecting the liquid sample. In other cases, the sample is undiluted, and there is little or no sample loss.

[0022] In some embodiments of these methods for mitigating or reducing interference, the sample is used for a sandwich immunoassay. In other embodiments, the sample is used for a competitive immunoassay.

[0023] Some embodiments relate to methods for preparing quenched streptavidin. Some of these embodiments include exposing the streptavidin to a minimum molar excess of free biotin. In one case, this may include metered addition to combine a biotin solution with a streptavidin solution. Some of these embodiments include washing the quenched streptavidin with a hot buffer. In one case, this may include percolation. Some of these embodiments include blocking the streptavidin to prevent aggregate formation. Some embodiments include coupling an additional capture fraction to the quenched streptavidin. Some embodiments include quenched streptavidin prepared by any of these methods.

[0024] Some embodiments relate to methods for preparing particle-conjugated streptavidin. Some of these embodiments include exposing the particle-conjugated streptavidin to a minimum molar excess of free biotin. In one case, this may include metered addition to combine a biotin solution with a suspension of particle-conjugated streptavidin. Some of these embodiments include washing the quenched streptavidin with hot water. In one case, this may include magnetic separation of the particles. In other cases, this may include separating the particles by filtration or sedimentation. Some embodiments include coupling an additional capture fraction to the biotin-quenched or un-biotin-quenched particle-conjugated streptavidin. Some embodiments include biotin-quenched or un-biotin-quenched particle-conjugated streptavidin prepared by any of these methods. Attached Figure Description

[0025] Figure 1 The size distribution of biotinylated 100BS streptavidin beads was depicted. The data showed a uniform single peak at 883.9 nm and a polydispersity index of 12.2%.

[0026] Figure 2A -B depicts: (2A) the size distribution of biotinylated 100BS streptavidin beads after incubation with streptavidin for 30 minutes. Bead aggregation occurred, with peaks at 1,441.3 nm and 6,641 nm, and a polydispersity index of 173.3%; and (2B) the size distribution of biotinylated 100BS streptavidin beads after incubation with streptavidin for 4 hours. Bead aggregation occurred, with peaks at 1,512.6 nm and 14,536 nm, and a polydispersity index of 242.8%.

[0027] Figure 3 The size distribution of biotinylated 100BS streptavidin beads after overnight incubation with monoclonal anti-biotin conjugate antibody was depicted. Bead aggregation occurred, with a peak at 2,148 nm and a polydispersity index of 316.2%.

[0028] Figure 4A -C depicts the SEC-HPLC standard curve for (4A) anti-biotin antibody. Data points indicated by arrows correspond to the peak area and residual anti-biotin antibody (µg / mL) in the sample after pretreatment with biotinylated 100BS streptavidin beads to deplete the anti-biotin antibody. 4B-C depicts the SEC-HPLC analysis of anti-biotin antibody before and after depletion with biotinylated 100BS streptavidin beads (4B) and (4C). After depletion of the anti-biotin antibody, the peak area decreased from 1,384 to 318, and the concentration of the anti-biotin antibody decreased from 205 µg / mL to 44.62 µg / mL.

[0029] Figure 5A -D are chromatograms from HPLC-SEC depletion assays using biotinylated 100BS streptavidin beads, before and after treatment. 5A shows the depletion of affinity-purified goat IgG by the beads without affinity. 5B shows the depletion of affinity-purified goat IgG by the beads without biotinylation. 5C shows the depletion of goat anti-biotin Ab by the beads. 5D shows the depletion of goat anti-streptavidin antibody by the beads. Untreated and treated curves are indicated by marked arrows in all cases.

[0030] Figure 6Serum parathyroid hormone levels as detected by ELISA were depicted under various interfering agents and with and without the use of biotinylated 100BS streptavidin beads. No interfering agents; Biotin -> 250 ng / mL; Anti-Biotin IgG - 16.5 μg / mL Ab; Anti-SAv IgG - 16.5 μg / mL Ab; Anti-Biotin IgG / SAv IgG - 8.25 μg / mL for each Ab.

[0031] Figure 7 An apparatus for metered addition of biotin to streptavidin during a saturation process is described.

[0032] Figure 8 An apparatus for percolation, washing, and concentration of biotin-saturated streptavidin is described.

[0033] Detailed description

[0034] While methods exist for identifying or depleting compounds that cause assay interference, there remains a clinical need for rapid and easy-to-use product solutions for detecting and mitigating interference from avidin and streptavidin in patient samples. Such solutions would also facilitate epidemiological studies to help clinicians and laboratory medicine professionals better understand which patients and patient populations are at greatest risk for these interferences.

[0035] There is also a clinical need to mitigate anti-streptavidin interference in patient samples by using blocking reagents that specifically target the interference from anti-streptavidin in the assay formulation. This product solution will also have minimal impact on laboratory workflows, as anti-streptavidin interference will be mitigated by the diagnostic test design.

[0036] Interference in immunoassays can lead to falsely reported high or low levels of the analyte being measured. One type of interference involves signal generation and observation. These include factors such as turbidity, hemolysis, quenching, and inhibition of signal-generating enzymes. Generally, these interferences are directly observable or can be tested without specialized reagents. The embodiments disclosed herein do not address such signal generation / observation interferences, and such interferences are not included in the general designation of interference herein.

[0037] Another type of immunoassay interference involves the capture and physical detection of analytes. These include interferences that inhibit the interaction between the analyte and the capture or detection reagent, or interferences that cause binding of the capture and detection reagent regardless of the presence (or absence) of the analyte. This type of interference is called heterophilic interference; when used herein, “interference” should be understood to mean heterophilic interference unless the context otherwise specifies. The embodiments disclosed herein address various different specific heterophilic interferences. Generally, heterophilic interferences are not directly observable, and their presence cannot be readily confirmed using standard assay reagents. Some of the embodiments disclosed herein can be used to confirm the presence of or quantify specific heterophilic interferences. Heterophilic interfering agents include biotin, anti-biotinylate substances, anti-streptavidin substances, and anti-heteroantibody interfering agents, as well as interfering agents that bind to components (enzymes, fluorescent agents, etc.) of the assay signal generation system. Anti-heteroantibody interfering agents include human antibodies that recognize mouse, rat, rabbit, sheep, bovine, and / or goat immunoglobulins.

[0038] Another type of immunoassay interference involves cross-reactive antibodies. Cross-reactivity occurs between an antibody that successfully binds to a different antigen when an antibody against a specific antigen successfully binds to that of a different antigen. In other words, cross-reactivity involves the binding of an antibody to an antigen other than its immunogen. This can be particularly problematic in immunoassays, for example, designed to detect antibodies that recognize antigens from a specific strain of bacteria or virus. Such assays are often used to determine whether a subject has been exposed to (infected by) the pathogen or agent in question. If a subject has previously been exposed to the relevant strain, they may have antibodies that will cross-react with antigens from the strain that the assay is intended to detect, thus producing false positive results.

[0039] When used herein, "immunoassay" generally refers to an assay in which the detection or quantification of an analyte utilizes an antibody (or its antigen-binding fragment or derivative) that specifically binds to the analyte. However, assays can also be designed in which non-antibody reagents that can specifically bind the analyte are used, similar to anti-analyte antibodies. In some embodiments, the non-antibody reagent that can specifically bind the analyte is an aptamer or a molecularly imprinted polymer. Thus, in some embodiments, "immunoassay" can encompass assays in which non-antibody reagents provide analyte-specific binding activity typically provided by antibodies. Various different embodiments specifically include or exclude antibodies or non-antibody reagents as analyte-specific binding activity. Some embodiments specifically include or exclude aptamers or molecularly imprinted polymers. Immunoassays can be classified based on the technique and physical arrangement of components, and the assay format. One type involves combining a sample (potentially containing an analyte) with detection and / or signal generation reagents in a container, such as a microtube or microtiter plate, in which the assay reaction is performed. Reagents can be added to or removed from the container during the assay. (In some variations, a portion of the assay component is removed from the initial container and added to a second container, where the assay continues.) This type of assay will be referred to herein as a “pot” assay. In another type, certain detection and / or signal-generating reagents are immobilized to a specific area of ​​a solid substrate or matrix, such as a membrane. The sample (potentially containing the analyte) is applied to a specific location in the device containing the solid substrate or matrix and encounters the immobilized reagent by movement, for example, by lateral flow into and generally through the area where the reagent is immobilized. Other assay reagents will move with the mobile phase. This type of assay will be referred to herein as a “zonal” assay. The time point from the addition of the sample to the container where the assay reaction takes place in a pot assay, or the time point of adding the sample to a specific location in the device containing the solid substrate or matrix in a zonal assay, up to the measurement of the generated signal, is referred to as the analytical phase of the assay. In many embodiments, prior to the analytical phase, interference-removing or blocking reagents disclosed herein are added to the sample and the cleaning reagents are removed from the sample; that is, in these embodiments, the interference-removing reagents are used as a “pretreatment.”

[0040] Patients who ingest high doses of biotin for health and beauty purposes (5,000 to 20,000 mcg daily) or for treatment purposes (100,000 to 300,000 mcg daily) may have high circulating biotin concentrations in their blood, which can be as high as 1,000 ng / mL or higher, depending on the length of time since biotin ingestion, patient-specific biotin clearance time, and whether the patient has kidney disease or impaired renal function that may impair biotin clearance and increase circulating biotin levels. If a patient's free biotin has not been cleared to below the test-specific biotin interference threshold before drawing blood, serum, or plasma samples, or before collecting urine samples, any biotin in the sample above the test-specific biotin interference threshold will compete for and bind to the anti-biotinylate capture fraction (i.e., streptavidin, avidin, neutral avidin, monomeric avidin, CaptAvidin, or biotin-specific antibodies, antibody fragments / Fab / F(ab)'2, aptamers, and molecularly imprinted polymers), and subsequently interfere with the binding of the biotinylated antibody, protein, or antigen used in the assay formulation. This will produce a false low assay signal and, depending on the assay format, a false low dose (sandwich assay) or a false high dose (competitive inhibition assay).

[0041] Streptavidin is a protein of ~52,000–55,000 kDa, composed of four identical polypeptide chains. When used herein, monomeric streptavidin refers to the non-aggregated streptavidin protein, but not to the dissociated streptavidin polypeptide chain. Biotin binds to streptavidin (reported in the literature as 10...). −14 Or 10 −15 (The concentrations may vary by mol / L) represent one of the strongest known non-covalent interactions in nature. Recombinant streptavidin is a suitable tool for obtaining universal assay systems in immunology and molecular diagnostics, and is commonly used in diagnostic assays such as immunoassays to capture biotinylated antibodies, proteins, and antigens, or to attach various biomolecules to each other or to solid supports such as microplates, beads, and microarrays. The use of streptavidin also allows assay developers to utilize proven avidin-delayed capture assay formats to obtain improved assay kinetics, precision, and sensitivity, while also contributing to shorter assay incubation times and faster turnaround times (TAT) in STAT assays.

[0042] If the sample contains an interfering agent specific to streptavidin, the anti-streptavidin interfering agent can bind to streptavidin or its polypeptide chain, spatially blocking or impairing the binding of the conjugated biotin to the biotin-binding site of streptavidin. If streptavidin can no longer freely bind to the biotinylated antibody, protein, or antigen used in the test design or assay format, then, like the biotin interfering agent, the anti-streptavidin interfering agent will produce a false low assay signal, and may produce a false low dose (sandwich assay) or a false high dose (competitive inhibition assay). Similarly, if the sample contains an interfering agent specific to biotin, the anti-biotin interfering agent can bind to biotin, spatially blocking or impairing the binding of the conjugated biotin to the biotin-binding site of streptavidin. If biotin originates from biotinylated antibodies, proteins, or antigens used in the test design or assay format, anti-biotin interfering agents will produce false low assay signals, and can also produce false low doses (sandwich assays) or false high doses (competitive inhibition assays). Some implementations target anti-streptavidin interfering agents. Some implementations target both anti-streptavidin and anti-biotin interfering agents.

[0043] Although biotin-streptavidin interactions are commonly used in the capture portion of immunoassay mechanisms, heterophilic interference can also occur through interactions with commonly used detection components. These components can include fluorophores such as fluorescein or elemental ruthenium; chemiluminescent agents such as luminol, acridinium ester, ABEI, and cyclic ABEI; bioluminescent agents such as luciferin; and enzymes such as alkaline phosphatase or horseradish peroxidase. Interferences binding to these signal-generating molecules can cause cross-linking between the analyte-bound and non-analyte-bound detection antibodies (or other detection reagents), producing false high signals. Some implementations target both streptavidin-resistant and signal-generating molecule interferences.

[0044] Immunoassays typically utilize antiserum, polyclonal antibodies, or monoclonal antibodies derived from non-human species. Serum or other assay samples may contain interfering agents that recognize these heterologous antibodies, sometimes referred to as human anti-animal antibodies (HAAAs). Specifically, humans produce antibodies against a wide range of animal species. Typically, these are those with which they interact most frequently, such as mice, cows, horses, dogs, cats, goats, and sheep. Among these, antibodies from mice, goats, rabbits, and sheep, particularly IgG, are very commonly used in clinical immunochemical assay systems. However, similar heterophilic interferences can occur in samples from non-human subjects. Such anti-antibody interfering agents can cause cross-linking between capture and detection antibodies in the absence of bound analytes, or between detection antibodies bound to and unbound to analytes, producing false high or low signals. Some implementations target both anti-streptavidin and anti-heterologous antibody interfering agents.

[0045] There are two methods for addressing interference in immunoassays: blocking and cleaning. As used herein, a blocking agent is present in the assay reaction and prevents or reduces interference through its interaction with the interfering substance. Some embodiments include or utilize soluble, biotin-saturated streptavidin and are suitable for blocking anti-streptavidin interfering agents. In some embodiments, the soluble, biotin-saturated streptavidin is conjugated to a second molecule (e.g., biotin, a signaling molecule, or a heterologous antibody) that is readily bound to the interfering substance. Embodiments containing or utilizing soluble, biotin-saturated streptavidin conjugated to a second interfering target molecule are suitable for blocking both anti-streptavidin and anti-second molecule interfering agents. Some embodiments specifically include one or more genera or species of second interfering target molecules. Some embodiments specifically exclude one or more genera or species of second interfering target molecules. The blocking agent may be added during the analytical phase of the assay or added pre-analytical and retained during the analytical phase. In some embodiments, the blocking agent may also be encountered during the analytical phase of a regional assay, such as a lateral flow assay, or may be retained in a specific region of such an assay. When used herein, the analytical phase of the assay refers to the temporal and / or physical portion of the assay or assay system in which analyte capture, detection, and quantification occur.

[0046] It should be noted that the term "blocking" is used herein in more than one meaning, although these meanings are conceptually related. In the preparation of the reagents disclosed herein, "blocking" and the like are used to describe hindering or otherwise reducing the reactivity of chemically reactive sites and the effective affinity of specific and / or nonspecific binding sites. This can be referred to as preparative blocking. Therefore, detergent and polymer blocking reagents used herein to prevent the reaction and / or the nonspecific binding or aggregation of proteins with the core nanoparticles of streptavidin-coated beads disclosed herein involve this meaning of "blocking." Saturating streptavidin with biotin can also be considered a form of preparative blocking, where biotin is the blocking agent. Preparative blocking should not be confused with blocking as a different function of preventing or reducing assay interference.

[0047] When used herein, a cleaning agent is added to serum or other biological samples or other components of the immunoassay reaction mixture, and then removed from the sample or other components before the components of the immunoassay reaction mixture are mixed together. That is, the cleaning agent is used and removed in the pre-analytical phase of the assay and is not present in the analytical phase. Interference is prevented or reduced by depleting or removing interfering substances from the sample and / or other assay reagents. Some embodiments include or utilize biotin-saturated streptavidin coated on magnetic nanoparticles, i.e., biotin-saturated streptavidin beads. Such biotin-saturated streptavidin beads are suitable for cleaning streptavidin interference. In some embodiments, the biotin-saturated streptavidin of the beads is coupled to a second molecule (e.g., biotin, a signaling molecule, or a heterologous antibody or antigen) that is readily bound to interfering substances. Embodiments that include or utilize biotin-saturated streptavidin beads in which streptavidin is coupled to a second interfering target molecule are suitable for cleaning both anti-streptavidin and anti-second-molecule interfering substances. Some implementations specifically include one or more genera or species of second interfering target molecules. Some implementations specifically exclude one or more genera or species of second interfering target molecules.

[0048] The biotin-saturated streptavidin beads can be magnetically separated from their storage buffer, the storage buffer removed, and then the sample or reagent to be cleaned added to the beads, such that, unlike when using soluble blocking agents, the sample or reagent is not diluted during the cleaning process. In other embodiments, the beads are separated from the fluid phase by filtration or sedimentation.

[0049] Assays that use streptavidin in their test design, format, or formulation cannot simply use natural streptavidin as a specific blocker, additive, or component in the assay buffer to mitigate interference from anti-streptavidin substances in the sample. If used as a blocker in the assay, streptavidin may also compete with and bind to biotinylated antibodies, proteins, oligomers, or antigens, producing false low assay signals and false low doses (sandwich assays) or false high doses (competitive inhibition assays). This is particularly concerning for streptavidin, given its very strong binding constant and affinity for biotin. While some assays can mitigate interference from lower titers or concentrations of anti-streptavidin substances by increasing the total amount or overall concentration of streptavidin used in the assay, this is test-specific and assay format-specific, and increases test costs. This may also be ineffective if the sample contains high titers or levels of anti-streptavidin interfering substances exceeding the test-specific streptavidin interference threshold.

[0050] The interference blocking agent disclosed herein is based on biotin-saturated streptavidin, also known as quenched streptavidin (QSAv). The QSAv should be predominantly non-aggregated, i.e., based on monomeric streptavidin protein. In some cases, the predominantly non-aggregated QSAv has <5% aggregation, <1% dimers or aggregates as measured by size exclusion chromatography HPLC, wherein the average observed molecular weight of the monomer peaks is 52 to 55 KD. In other cases, the QSAv is at least 80, 90, 95, 97, 98, 99% monomeric, or any range defined by these values. In some embodiments, the streptavidin is blocked with, for example, a detergent or polymer blocking agent to maintain it in a monomeric, non-aggregated state. QSAv can be used to block streptavidin interferences. In some embodiments, the streptavidin can be modified with one or more additional capture moieties before, during, or after biotin saturation. The capture moieties can be covalently coupled to the streptavidin before or after the biotin saturation process. Alternatively, the capturing portion may be biotinylated before or during the biotin saturation process and bound to streptavidin via biotin-avidin binding. However, if the additional capturing portion is biotin, for example, achieved by using a dibiotin linker, it must be bound to the streptavidin in the presence of excess free biotin (i.e., during the saturation process). Embodiments comprising streptavidin modified with one or more additional capturing portions can be used to block interference from anti-streptavidin substances and interference caused by reagents binding the one or more capturing portions.

[0051] The interference cleaning agents disclosed herein are based on streptavidin coupled to microparticles (or nanoparticles) to form streptavidinized beads. The use of beads (particularly magnetic beads) facilitates cleaning of samples or assay reagents without loss or dilution. In some embodiments, the streptavidin is saturated with biotin, while in others it is not. Embodiments containing biotin-saturated streptavidin can be used as cleaning agents to remove or reduce anti-streptavidin interference. Embodiments containing unsaturated streptavidin can be used as cleaning agents to remove or reduce both biotin-interfering and anti-streptavidin interference. In some embodiments, the streptavidin may be modified with one or more additional capture portions before, during, or after biotin saturation. The capture portions may be covalently coupled to the streptavidin before or after the biotin saturation process. Alternatively, the capture portions may be biotinylated before or during the biotin saturation process and bound to the streptavidin via biotin-avidin binding. Embodiments comprising streptavidin modified with one or more additional capture portions can be used to block interference from anti-streptavidin substances and interference caused by reagents binding to the one or more capture portions. In embodiments utilizing biotin-saturated streptavidin, the one or more capture portions may include biotin.

[0052] The additional capture fraction can be any substance that causes heterophilic or cross-reactive interference, except that it cannot be biotin if the streptavidin is not saturated with biotin. In some embodiments, the additional capture fraction is ruthenium (an element); luminol, acridinium ester, ABEI, or cyclic ABEI (similar to biotin, a small organic molecule); or a protein such as a signal-generating enzyme, such as alkaline phosphatase or horseradish peroxidase; streptavidin; an antibody, such as an antibody derived from a non-human species; or an antigen. In some embodiments, the antigen is an antigen that can be recognized by an antibody capable of cross-reacting with the antigen to be used as the capture fraction in the immunoassay. In various embodiments, the antigen used as the capture fraction in the cleaning or blocking reagent or in the assay is an allergen, an antigen derived from a pathogen, or an antigen associated with a disease or disorder, such as peanut allergen, herpes simplex virus antigen, and an autoimmune antigen, such as cardiac troponin I or TSH, which have known autoantibody interference problems. In some embodiments, the antigen derived from a pathogen is a viral antigen, a bacterial antigen, or a protozoan antigen. In some embodiments, the capture portion removes cross-reactive antibodies against coronaviruses other than MERS, SARS, or SARS-CoV-2. Some embodiments specifically include capture portions of one or more of these genera or species. Some embodiments specifically exclude capture portions of one or more of these genera or species.

[0053] Biotin linkers or conjugated biotin with different linker types and lengths can be constructed or purchased, and biotin can be covalently attached to antibodies, antibody fragments, peptides, oligomers, antigens, and small molecules (conjugated biotin) using different functional groups. Common linkers and functional groups used with biotin (e.g., NHS esters, TFP esters, hydrazides, maleimides, thiols, etc.) are NHS-Biotin, NHS-LC-Biotin, TFP-LC-Biotin, NHS-LC-LC-Biotin, NHS-chromalink-Biotin, NHS-PEO4-Biotin, and NHS-(PEO) n -Biotin, TFP-(PEO) n -Biotin, Acylhydrazide-Biotin, Acylhydrazide-LC-Biotin, Acylhydrazide-PEO4-Biotin, Maleimide-(PEO) n -Biotin and SH-(PEO) n -Biotin. Biotin-labeled reagents can be amine-reactive, carboxyl-reactive, carbonyl-reactive, water-soluble, and cleavable. Examples include amine-reactive, carbonyl-reactive, carboxyl-reactive, cleavable biotin, click chemistry, desulfurized biotin, thiol-reactive, tetrazine-linked, biotinol, di-biotin-PEG, and D-biotin-PEG-thalidomide.

[0054] If the sample contains an anti-biotin interfering agent, this anti-biotin interfering agent can bind to the coupled biotin used in the test design or assay format, spatially blocking or impairing the accessibility of the coupled biotin-binding streptavidin solid phase or other anti-biotin capture moieties. If the coupled biotin can no longer freely bind to the anti-biotin capture moieties, as with both biotin and anti-streptavidin interfering agents, the anti-biotin interfering agent will cause false low assay signals and may result in false low doses (sandwich assays) or false high doses (competitive inhibition assays).

[0055] Tests using biotin conjugates in their test design, format, or formulation cannot simply use biotin or biotin-conjugated biotin as a specific blocker, additive, or component in the assay buffer to mitigate anti-biotin interference in the sample. If used as a blocker in the test, biotin may also compete with and bind to streptavidin used in the test, producing false low assay signals and false low doses (sandwich assays) or false high doses (competitive inhibition assays). While low concentrations of biotin below the test-specific biotin interference threshold can be used to block anti-biotin interference, this can be problematic if the patient sample also contains biotin interference close to the threshold, where the combination or total amount of sample biotin (endogenous biotin) and test biotin (biotin as a blocker) may exceed the test-specific biotin interference threshold, producing false low assay signals and false low doses (sandwich assays) or false high doses (competitive inhibition assays).

[0056] Streptavidin binds to biotin very rapidly and very strongly (the binding constant reported in the literature is 10). −14 Or 10 −15 (The concentration varies by mol / L). Although some studies suggest the presence of protein structural changes or cooperative binding of biotin to the four binding sites [28-29], other studies have concluded that there is no cooperative binding of biotin to the four subunits of the tetramer [30-31]. If streptavidin is exposed to a molar excess of free biotin, very rapid and strong binding interactions occur between biotin and all four binding sites, resulting in 100% biotin saturation (100 BS) at all biotin binding sites. Due to the strongest known non-covalent binding interactions in nature and the very slow rate of biotin dissociation from streptavidin under normal physiological conditions and pH, the likelihood of 100 BS streptavidin binding to other biotin or conjugated biotin, such as biotinylated antibodies, proteins, oligomers, and antigens, in diagnostic tests is extremely low. When used herein, saturation refers to the blocking of biotin binding sites on streptavidin with biotin; this is not biotinylation, i.e., covalent attachment of biotin to streptavidin. Saturated streptavidin can bind to anti-streptavidin substances, but will not bind to or will cross-link with biotin-containing substances. Biotin-saturated streptavidin can also be referred to as quenched streptavidin (QSAv).

[0057] Streptavidin can be saturated with biotin (i.e., D-biotin) to prepare 100BS streptavidin for use as a blocker to mitigate or manage interference from anti-streptavidin substances. In other embodiments, streptavidin can be exposed to a dissolved biotinylated agent such as biotin-PEG(n)-COOH, biotin-PEG(n)-CH3, biotin-PEG(n)-OH, or other biotin-R- (non-reactive terminal chemical groups) to quench the active biotin binding site of streptavidin, where R is a carbon chain or ring structure. Saturation involves exposing streptavidin to a molar excess of biotin. In various embodiments, the molar ratio of biotin to streptavidin is in the range of 5:1 to 11:1, 7:1 to 11:1, or 7:1 to 8:1. In some embodiments, the molar ratio is 7.4:1. In some embodiments, streptavidin is exposed in a single batch to all the saturated biotin constituting the stated molar ratio. In other embodiments, saturation is performed through iterative batches, each containing a portion of the total biotin, the sum of which constitutes the stated molar ratio. For example, one could use three iterative batches with a biotin:streptavidin ratio of 2:1 instead of a single batch with a ratio of 6:1; the biotin:streptavidin ratio need not be identical in each of the iterative batches. In some embodiments, the biotin and streptavidin solutions are combined by metering via a Y-connector. In some embodiments, an in-line mixer is present in the tubing connected to the Y-connector outlet to ensure rapid, immediate, and complete mixing. The total interaction time during saturation can be determined using a combination of pump speed and tubing length. In some embodiments, 9 volumes of biotin solution are combined with 1 volume of streptavidin solution. In some embodiments, the streptavidin and biotin solutions are prepared in a Tris buffer at pH 8.5. In one case, the initial streptavidin concentration is in the range of 0.1 to 10.0 mg / mL, so that the resulting QSAv solution has a streptavidin concentration in the range of 0.01 to 1.0 mg / mL, but preferably 0.02 to 0.05 mg / mL. Such conditions promote the saturation of biotin binding sites and reduce nonspecific binding of biotin to streptavidin.

[0058] In some embodiments, the QSAv is then subjected to a series of hot buffer washes by repeated concentration and redispersion, for example, using percolation in a hollow fiber filter. These washes remove excess and non-specifically bound biotin, preventing it from becoming a source of interference when the QSAv is used as an interference blocking agent. In some embodiments, 4-6 or more washes are used, for example, 5 washes, followed by a final concentration step to reduce the volume to a desired concentration, for example, 0.1 to 30 mg / mL or 1 to 10 mg / mL. In one case, the volume can be reduced to 5-20%, for example, 10%, of the original volume of the QSAv solution. In some embodiments, the temperature of the hot wash is 15°C to 60°C, preferably 40°C to 55°C, but more preferably 45°C to 50°C. In some embodiments, the hot wash buffer has a pH in the range of 7.5 to 11 or 8 to 9, for example, 8.5. In some embodiments, the hot washing buffer has a NaCl concentration in the range of 10 to 500 mM, 20 to 150 mM, or 25 to 75 mM. In some embodiments, the buffer is 10 mM Tris, 50-150 mM NaCl. After the washing and final concentration steps, the concentration of free biotin should be <1200 pg / mL, for example <1000, <800, <700, or <600 pg / mL. In some embodiments, the washed QSAv solution contains 1-6 pg free biotin / μg streptavidin. In some embodiments, the effluent from the final percolation is combined with PBS by metered addition and suitably concentrated to provide QSAv in PBS. In some embodiments, to clean the sample, a volume of QSAv is added to 400 μl of sample such that the volume will contain <480 pg free biotin.

[0059] In some implementations, the effluent from the saturation process is collected for later cleaning; in other implementations, the effluent from the saturation process is fed directly into the hollow fiber filter. The effluent from the saturation process can be fed separately into multiple hollow fiber filters to increase capacity and avoid excessive back pressure.

[0060] These streptavidin solutions are relatively dilute and slightly prone to aggregation, a problem not encountered when using bead-conjugated streptavidin. This is because an alkaline buffer is used in the saturation and washing procedures to generate QSAv. (In contrast, water is used in a similar wash for bead-conjugated streptavidin.) Aggregation can be further mitigated by including 0.01-1% w / v TWEEN 20 or another surfactant in the washing buffer. Aggregation can be further mitigated by covalently modifying the solution with a preparative blocking agent prior to biotin saturation, for example, by PEGylation of streptavidin. Therefore, in some embodiments, the QSAv is a blocking, monomeric, biotin-saturated streptavidin. In some embodiments, the monomeric QSAv has <5% aggregates as measured by size exclusion chromatography HPLC; in other embodiments, the monomeric QSAv has <1% aggregates.

[0061] In one embodiment, 100BS streptavidin (QSAv) can be used as a blocking agent or protein blocker to target and deplete anti-streptavidin interferences in a sample. 100BS streptavidin can be added to assay buffers, blocking buffers, or test components (e.g., detection antibodies) used in a test formulation, or any combination thereof, to reduce the sensitivity of the test to anti-streptavidin interferences. In another embodiment, streptavidin is covalently coupled to a particle-binding surface and subsequently incubated with a molar excess of biotin to prepare 100BS streptavidin beads. These 100BS streptavidin beads can be used to pretreat samples to target and deplete anti-biotin interferences prior to diagnostic testing.

[0062] The interference cleaning agent disclosed herein is based on streptavidin-coupled beads. In some embodiments, the streptavidin is quenched (saturated) with biotin after attachment to a core particle. In some embodiments, the streptavidin is quenched with biotin before attachment to the core particle, for example, using QSAv as described herein. In some embodiments, the core particle is magnetic. In some embodiments, the core particle has a diameter ≥500 nm or about half a μm and can therefore be referred to as a nanoparticle or microparticle. In some embodiments, the core particle is covalently coupled to the streptavidin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chemistry. To remove passively adsorbed streptavidin and to prevent non-specific binding to the beads in further preparation steps and when used as an interference cleaning agent, the surface of the beads is modulated with a stripping agent (salt, detergent, low and high pH) and the beads are blocked using detergents and polymer blocking agents. This also promotes monodispersity and colloidal stability of the nanoparticles. To produce 100BS streptavidin beads, biotin quenching of streptavidin can be performed similarly to the production of 10BS streptavidin (QSAv) described above. Saturation involves exposing the streptavidin coupled to the beads to a molar excess of biotin. As mentioned above, saturation of the active biotin-binding sites of streptavidin can be achieved instead by exposing streptavidin to a dissolved biotinylated reagent such as biotin-Peg(n)-COOH, biotin-Peg(n)-CH3, biotin-Peg(n)-OH, or other biotin-R- (non-reactive terminal chemical groups), where R is a carbon chain or ring structure. In various embodiments, the molar ratio of biotin to streptavidin is in the range of 4:1 to 6:1, for example, 5:1. The effective molar excess of biotin is slightly higher than this formal ratio because some biotin-binding sites will be inaccessible due to the steric hindrance of the core particles. In some embodiments, the streptavidinized beads are suspended and the biotin solution is prepared in PBS at pH 6.8. In some embodiments, the biotin solution and bead suspension are combined in a container and mixed, for example, at room temperature for 1 hour. In some embodiments, the streptavidinized bead suspension and biotin solution are combined by metering, essentially as described above in the production of QSAv.

[0063] In some embodiments, the biotin-saturated streptavidinized beads are thermally washed to remove passively adsorbed biotin, preventing it from detaching and becoming a source of interference during use. Unlike the 100BS streptavidin wash described above, washing the 100BS streptavidin beads can utilize filtration, sedimentation, or magnetic separation as alternatives to percolation. The difference between washing with 100BS streptavidin can also be the use of hot alkaline (pH ≥ 7.5) water instead of a buffer solution. The washing suspension is also sonicated. In some embodiments, the free biotin concentration in the washed 100BS streptavidin bead suspension is <1200 pg / mL, for example <1000, <800, <600, <400, or <200 pg / mL. In some embodiments, the washed 100BS streptavidin bead suspension contains 5-30 pg free biotin / μg streptavidin. In some embodiments, to clean the sample, a volume of 100BS streptavidin beads is added to a 400μl sample, such that the volume will contain <480 pg of free biotin.

[0064] Free biotin (i.e., D-biotin) can also be added to 100BS streptavidin or 100BS streptavidin bead storage solutions, assay buffers, or test components to improve the stability of the 100BS streptavidin or 100BS streptavidin beads and ensure that the streptavidin remains 100% saturated with biotin over time. In one embodiment, 100BS streptavidin in a storage solution, assay buffer, or test component containing excess biotin can be used as a blocking agent to target both anti-streptavidin and anti-biotin interference mechanisms with a single blocking agent. In one embodiment, the blocking agent can be used to pretreat samples prior to testing to block anti-streptavidin and / or anti-biotin interference mechanisms. In one embodiment, the blocking agent can be used in diagnostic test or assay design, for example, in assay buffers or reagent buffers, to block and mitigate interference mechanisms during testing. In one embodiment, free biotin can be added to 100BS streptavidin or 100BS streptavidin beads at concentrations up to 1,100 pg / mL within the physiological range. In another embodiment, free biotin can be added to 100BS streptavidin or 100BS streptavidin beads at high concentrations, such as 100,000 pg / mL (100 ng / mL) or 1,000,000 pg / mL (1,000 ng / mL). If 100BS streptavidin or 100BS streptavidin beads are stored in a solution containing free biotin, it will remain 100BS streptavidin because any biotin dissociated from streptavidin will be immediately replaced by another biotin from the biotin added to the storage solution due to the very strong binding constant and rapid binding rate of streptavidin to biotin. Therefore, 100BS streptavidin or biotin-quenched streptavidin can be used as a blocking agent in streptavidin-based assays or immunoassays, wherein 100BS streptavidin is added to an assay buffer that also contains a subphysiological biotin concentration to achieve stability. If any biotin dissociates from the streptavidin blocking agent, it will be replaced by the biotin added to the assay buffer, and the amount of biotin subsequently added from the assay buffer to the sample or test reaction will be minimal and within the physiological biotin concentration range.

[0065] IVD companies provide test-specific biotin interference thresholds for each biotin-sensitive assay in their product instructions (PI) or instruction for use (IFU) [9, 14-15]. Assays with a biotin interference threshold < 51 ng / mL, such as the Ortho Clinical Diagnostics Vitros Cardiac TnI assay with a threshold of 2.4 ng / mL, are considered high-risk assays or susceptible immunoassays and competitive methods [9]. Although biotin can be added to 100 BS streptavidin or 100 BS streptavidin bead stock solutions to improve stability and ensure 100% saturation over time, the final free biotin concentration must be below the test-specific biotin interference threshold to mitigate test interference from biotin added to the stock solution. In one embodiment, 100 BS streptavidin or 100 BS streptavidin beads are stored in a biotin solution with a biotin concentration within the physiological range or < 1,100 pg / mL so that it does not interfere with the assay. In another embodiment, 100BS streptavidin beads are stored in a biotin solution containing >1,100 pg / mL biotin, for example, 2, 5, 10, 20, 30, 50, 100, 250, or 500 ng / mL biotin. The 100BS streptavidin beads are then separated from the sample by filtration, centrifugation, or magnetic methods, or a combination thereof, to remove the biotin storage solution from the 100BS streptavidin beads before adding the sample. The biotin storage solution is removed just before the 100BS streptavidin beads are used, reducing the free biotin concentration to below 1,100 pg / mL, below 500 pg / mL, or preferably below 100 pg / mL, and ensuring that the free biotin concentration is below the biotin interference threshold tested.

[0066] The primary amine (R-NH2) of 100BS streptavidin and 100BS streptavidin beads can be labeled using amine-reactive biotinylate reagents such as NHS-Biotin, NHS-LC-Biotin, NHS-LC-LC-Biotin, NHS-chromalink-Biotin, NHS-PEO4-Biotin, and NHS-(PEO). n -Biotin, TFP-(PEO) n- Biotin (amine-reactive method) is covalently coupled to biotin, and biotin coupling is performed with a molar excess of free biotin to reduce the binding and capture of biotin-labeled reagents by the biotin binding sites of streptavidin. In one embodiment, streptavidin is covalently coupled to a microparticle binding surface, which is modulated (blocked and stripped) to retain only the covalently attached streptavidin. The streptavidin-coupled microparticle binding surface is exposed to a molar excess of free biotin (D-biotin) to prepare 100BS streptavidin beads. A molar excess of free biotin is added to the 100BS streptavidin bead storage solution, such as PBS pH 7.4, and the primary amine of the 100BS streptavidin is coupled to NHS-PEO4-biotin to prepare biotinylated 100BS streptavidin beads. Biotinylation of streptavidin-coupled beads refers to the covalent coupling of biotin to a magnetic particle (or streptavidin on it) and should not be confused with or equated with saturation of the biotin-binding site of streptavidin. Biotinylated streptavidin beads can bind anti-biotin substances (in addition to anti-streptavidin substances). Biotin used for saturating streptavidin typically does not bind the most problematic anti-biotin substances because an essential portion of the biotin molecule binds to streptavidin. In another embodiment, 100BS streptavidin or 100BS streptavidin beads have a thiol group (mercapto or R-SH) introduced onto streptavidin by thiolation of streptavidin primary amine (R-NH2) using standard thiolation chemicals known in the art, such as trans-4-(maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 3-(2-pyridyldithio)propionic acid succinimide ester (SPDP), or SPDP-PEG. (4、6、8、12、24或36) -NHS ester, SPDP NHS ester, SPDP-C6-NHS ester, SPDP-C6-Sulfo-NHS ester, PC SPDP-NHS carbonate, and SPDP-C6-Gly-Leu-NHS ester (thiolization method). If SPDP is used, the SPDP coupled to streptavidin is cleaved using TCEP and EDTA, and the leaving group of the SPDP is washed, desalted, or dialyzed off, leaving only SH-R coupled 100BS streptavidin. After preparing 100BS thiolized streptavidin with a molar excess of biotin, a thiol-reactive or thiol-reactive biotin labeling reagent such as maleimide-PEO can be used. (2、3、6或11)-Biotin and Biotin-SPDP (thiol or thiol-reactive means) covalently couple the thiol (R-SH) of 100BS streptavidin and 100BS streptavidin beads to biotin. This thiol-reactive biotin labeling is performed in PBS pH 6.8 buffer containing EDTA (up to 2 mM), TCEP (< 1 mM), and a molar excess of free biotin to 1) reduce the thiol and decrease disulfide bonds or bridging, and 2) reduce the binding and capture of the biotin-labeled reagent by the biotin binding site of streptavidin. Since maleimide groups are 1000 times more reactive to free thiols than amines at pH 6.5 to 7.5, and maleimide groups prefer primary amines at pH > 8.5, maleimide coupling is performed at pH 6.8 to minimize reactivity with primary amines. As an alternative to SPDP, similar reagents based on N-succinimide-S-acetyl-thioacetate (SATA) can be used.

[0067] In another embodiment, the 100BS streptavidin or 100BS streptavidin beads have a standard ester-maleimide heterobifunctional crosslinking chemical known in the art, such as 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid maleimide ester (SMCC), maleimide-PEG-NHS ester, maleimide-PEO (1、2、3、4、5、6、8或12) -NHS ester or maleimide-PEG (1、2、3、4、5或6) -PFP (maleimide-based) is used to introduce a maleimide group onto the primary amine (R-NH2) of streptavidin. After preparing 100BS maleimide streptavidin using a molar excess of D-biotin, the maleimide of the 100BS streptavidin and 100BS streptavidin beads can be covalently coupled to biotin using a maleimide-reactive biotin labeling reagent such as biotin-PEG-SH or biotin-PEG-thiol, wherein the PEG... n or PEO n The lengths can vary, for example, n = 1, 2, 3, 4, 5, 6, 8, or 12. The maleimide-reactive biotin labeling is performed in a PBS pH 6.8 buffer containing EDTA (up to 2 mM), TCEP (< 1 mM), and a molar excess of free biotin to 1) reduce thiols and decrease disulfide bonds or bridging of the biotin-labeled reagent, and 2) reduce the binding and capture of the biotin-labeled reagent by the biotin binding site of streptavidin. Since maleimide groups are 1000 times more reactive to free thiols than amines at pH 6.5 to 7.5, and maleimide groups prefer primary amines at pH > 8.5, maleimide-biotin conjugation is performed at pH 6.8.

[0068] Similar ester, thiol, or maleimide chemistry is also applicable to embodiments in which streptavidin is not saturated with biotin. For example, ruthenium esters can be reacted with the primary amine of streptavidin.

[0069] In a specific embodiment, 1) streptavidin is covalently coupled to a microparticle binding surface; 2) the microparticle binding surface is modulated such that only covalently attached streptavidin remains on the microparticle binding surface, and the surface has very low nonspecific binding; 3) the streptavidin-coupled microparticle binding surface is exposed to a molar excess of free biotin (D-biotin) to prepare 100BS streptavidin beads; 4) in the presence of a molar excess of free biotin, the 100BS streptavidin beads are covalently coupled to biotin using a biotin-labeled reagent; 5) the biotinylated 100BS streptavidin beads are filtered, centrifuged, or magnetically separated to remove buffer solution and excess biotin; 6) the biotinylated 100BS streptavidin beads are washed multiple times with water at 50°C and resuspended in a storage solution to obtain the finished reagent.

[0070] In a particular embodiment, 1) the biotinylated 100BS streptavidin beads are filtered, centrifuged, or magnetically separated to remove the storage solution; 2) a sample containing anti-streptavidin interferon, anti-biotin interferon, or both interferons is added to the biotinylated 100BS streptavidin beads to pretreat the sample; 7) the sample interference is depleted or reduced below the assay blocking threshold (ABT) or test interference threshold; 8) the biotinylated 100BS streptavidin beads are filtered, centrifuged, or magnetically separated from the sample; and 9) the sample supernatant, which is substantially free of beads, is aspirated and tested using the diagnostic test to report accurate test results.

[0071] In one embodiment, prior to diagnostic testing, 100BS streptavidin beads are used to pretreat the sample to bind anti-streptavidin interfering agents and deplete the anti-streptavidin interfering agents below the assay blocking threshold (ABT) or below the test interference threshold. In another embodiment, prior to diagnostic testing, biotinylated 100BS streptavidin beads are used to pretreat the sample to bind anti-biotin interfering agents and deplete the anti-biotin interfering agents below the assay blocking threshold (ABT) or below the test interference threshold. In yet another embodiment, prior to diagnostic testing, biotinylated 100BS streptavidin beads are used to pretreat the sample to simultaneously bind both anti-streptavidin and anti-biotin interfering agents from the same sample and deplete both interfering agents below the assay blocking threshold (ABT) or the test interference threshold.

[0072] Currently, there is no rapid and easy-to-use product solution for detecting, characterizing, and reducing biotin, antibiotin, and antistreptavidin interfering substances in patient samples. In a specific embodiment, streptavidin beads (bead 1), 100BS streptavidin beads (bead 2), and biotinylated 100BS streptavidin beads (bead 3) can be used systematically or sequentially to detect and determine which interfering mechanisms(s) are present in a sample. Samples suspected of interfering are tested cleanly (without beads) as a control. Three different aliquots of the sample are treated with beads 1 (aliquot 1), beads 2 (aliquot 2), and beads 3 (aliquot 3), respectively. The three pretreated aliquots are retested, and the test results for each bead type are compared with the control test results (Table 1). If the control test results are similar to the test results from the bead 1, 2, and 3 pretreatments, sample interference is unlikely and can be ruled out. However, if the pretreatment results of bead 1 are significantly different from the control, biotin interference and / or anti-streptavidin interference are possible, and sample interference can be included. If the pretreatment results of bead 2 are significantly different from the control, anti-streptavidin interference is possible, and sample interference can be included. If the pretreatment results of bead 3 are significantly different from the control, anti-streptavidin interference and / or anti-biotin interference are possible, and sample interference can be included. If the pretreatment results of bead 1 are significantly different from the control, but the pretreatment results of beads 2 and 3 are similar to the control, biotin interference can be included. If the pretreatment results of beads 1 and 2 are similar to the control, but the results of bead 3 are significantly different from the control, anti-biotin interference can be included. If the pretreatment results of beads 1, 2, and 3 are all significantly different from the control, anti-streptavidin interference can be included.

[0073] Table 1.

[0074]

[0075] Possible results were obtained by using three different sample pretreatment reagents, beads 1, 2, and 3, and comparing the results with a control; similar (-) and different (+). Bead 1+, bead 2-, and bead 3+ are unlikely because the anti-biotin interferon will bind free biotin unless it only recognizes conjugated biotin. Bead 1-, bead 2+, and bead 3- are impossible because the anti-streptavidin interferon will be depleted by both beads 1 and 2. As shown in result 4, bead 3 may not deplete the anti-streptavidin interferon (-) if the conjugated biotin spatially blocks or interferes with the binding of the anti-streptavidin antibody or protein.

[0076] The production of free (or soluble) biotin-saturated streptavidin (quenched streptavidin; QSAv) is very similar to the production of biotin-saturated streptavidin-coupled beads. Furthermore, streptavidin can be coupled to additional portions to act as trapping portions or block sites that can promote streptavidin aggregation. Such coupling can be performed before or after quenching (unless the additional trapping portion is biotin, in which case it can only be performed after quenching). A biotin to streptavidin molar ratio of approximately 7 to 8 is used, slightly higher than the 5:1 ratio used in the minimum saturation procedure for beads. This is because some biotin-binding sites on the streptavidin coupled to the beads will be spatially blocked, resulting in an effective ratio slightly higher than the formal ratio.

[0077] The QSAv is preferably primarily a monomer. In various embodiments, the QSAv is at least 80, 90, 95, 97, 98, 99% monomer, or any range defined by these values. To ensure that the reagent is and remains a monomer and does not form aggregates, streptavidin can be blocked with a detergent or polymer blocking agent. Blocking can include PEGylation. A wide variety of commercially available PEGylation agents with various sizes and chemical modifications are available, for example from ThermoFisherScientific, Broadpharm, Quanta Biodesign, and Creative Pegworks. One example is NHS-ester-PEG(4)-OH. Other examples include TFP-(PEO)n-OH or TFP-(PEG)n-OH (Quanta Biodesign). They can be covalently attached to any exposed lysine residue on streptavidin via NHS-ester chemocovalent bonding. Alternatively, TFP-(PEG)n-COOH or NHS-(PEG)n-COOH can be attached to lysine residues via EDC chemistry. Many other alternatives are familiar to those skilled in the art. The preparation of monomer QSAv can also be achieved by biotin quenching in a buffer containing a hydrophilic reagent such as urea, imidazole, trehalose, etc. Detailed Implementation

[0078] The following non-limiting embodiments are provided for illustrative purposes only to provide a more complete understanding of the representative implementations currently contemplated. These embodiments should not be construed as limiting any implementations described herein.

[0079] Example 1

[0080] Preparation of biotinylated streptavidin-coated magnetic nanoparticles or biotinylated 100BS streptavidin Method of using beads

[0081] Magnetic carboxylic acid nanoparticles of 550–600 nm were covalently coupled to streptavidin using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) chemistry. The bead surface was modulated with stripping agents (salts, detergents, low and high pH) to remove passively adsorbed streptavidin. The beads were then blocked using detergents and polymer blocking agents to reduce nonspecific binding and promote nanoparticle monodispersion and colloidal stability. The total concentration of streptavidin covalently coupled to the beads was determined to be 17.69 µg / mg using a modified micro-BCA total protein assay. The final bead concentration was determined gravimetrically and adjusted to 10.0 mg / mL beads in pH 6.8 PBS containing 2 mM EDTA.

[0082] Prepare a 10.0 mg / mL stock solution of D-biotin (Sigma, part number B4601-100MG, lot number SLBS8478, MW 244.31) in PBS at pH 7.4 as follows: Prepare a concentrated stock solution of D-biotin in DMSO (Baker, part number 9224-01, lot number 0000217025) at a concentration of 100 mg / mL, or add 10 mg of D-biotin to 100 µL of DMSO and mix. After the D-biotin has completely and uniformly dissolved in the DMSO, add 900 µL of PBS at pH 7.4 and mix to prepare a 1.0 mL 90:10 (PBS:DMSO) 10.0 mg / mL D-biotin stock solution.

[0083] A total of 2.5 mL of streptavidin magnetic nanoparticles was divided into equal aliquots and dispensed into reaction tubes, corresponding to a total of 442.25 µg streptavidin: [(25 mg beads) x (17.69 µg streptavidin / mg beads)]. The total of 442.25 µg streptavidin corresponds to 0.00804 µM streptavidin: [(442.25 µg streptavidin) / (55,000 µg streptavidin / µM streptavidin)].

[0084] To prepare a 1000-fold molar excess of D-biotin compared to the total molar amount of streptavidin: 1,964.475 µg of D-biotin was added to streptavidin-coupled magnetic nanoparticles containing 25 mg of streptavidin at a concentration of 17.69 µg streptavidin / mg beads, or to 442.25 µg streptavidin: [(0.00804 µM x 1000) x (244.31 µg biotin / µM)]. To add a 1000-fold molar excess of D-biotin to 0.00804 µmol streptavidin, 200 µL of a 10.0 mg / mL D-biotin stock solution or 2,000 µg of D-biotin was added to streptavidin magnetic nanoparticles containing 17.69 µg streptavidin / mg beads in 25 mg of streptavidin at a concentration of 2 mM EDTA in pH 6.8 PBS. Streptavidin magnetic nanoparticles were mixed with D-biotin and incubated at room temperature for 1 hour to saturate the streptavidin-biotin binding sites with biotin and prepare 100BS streptavidin beads.

[0085] To covalently couple 100BS streptavidin beads to biotin in a molar excess of D-biotin, add 100 times molar excess of NHS-PEG4-biotin (Broadpharm, part number 20566, lot number B93-039, MW 588.7) or 500 µg of NHS-PEG4-biotin to 25 mg of streptavidin-coupled magnetic particles at a concentration of 17.69 µg streptavidin / mg beads, or 442.25 µg streptavidin, or 0.00804 µM streptavidin. Add 5 mg of NHS-PEG4-biotin to 100 µL of DMSO and mix to prepare a 50.0 mg / mL stock solution of NHS-PEG4-biotin in DMSO. The following steps were performed to add 100 molar excess of NHS-PEG4-biotin or 473.315 µg NHS-PEG4-biotin [(0.804 µM NHS-PEG4-biotin) x 100) x (588.7 µg biotin / µM)] to 100BS streptavidin beads: 10.0 µL of a 50.0 mg / mL stock solution of NHS-PEG4-biotin in DMSO was added to 25 mg of 17.69 µg streptavidin / mg beads in PBS containing 2 mM EDTA at pH 6.8, obtained from the saturation step, containing 1000 molar excess of D-biotin, and mixed at room temperature for 1 hour. The biotinylated 100BS streptavidin beads were then washed four times with PBS at pH 7.4 to remove excess NHS-PEG4-biotin.

[0086] To verify that the 100BS streptavidin beads were successfully coupled to biotin and that there was no bead aggregation caused by streptavidin-mediated binding (i.e., bead cross-linking) of biotin-coupled beads on different beads, the biotinylated 100BS streptavidin beads were analyzed by particle size measurement using an Anton Paar Litesizer 500 analyzer. The average size distribution was 883.9 nm ( Figure 1 ).

[0087] To confirm successful biotin coupling to streptavidin on 100BS streptavidin beads, a limited amount of natural streptavidin was added to biotinylated 100BS streptavidin beads to promote bead aggregation induced by streptavidin-mediated bead crosslinking. A total of 50 µg of streptavidin was added to 25 mg of biotinylated 100BS streptavidin beads and incubated at room temperature for 4 hours. Bead aggregation was observed 30 minutes after streptavidin addition, with peaks at 1,441.3 nm and 6,641 nm and a polydispersity index of 173.3%. Figure 2A The peaks are located at 1,512.6 nm and 14,536 nm, and the polydispersity index is 242.8%. Figure 2B ).

[0088] Similar bead aggregation was performed using monoclonal antibodies against biotin conjugates that recognize conjugated biotin with an affinity similar to streptavidin but recognize free biotin with an affinity millions of times lower than streptavidin, or antibodies that specifically bind biotin to biotin conjugates and have a higher affinity for biotin in biotin conjugates than for free biotin (WO2020 / 028776; VeraBind Biotin™, Veravas). These antibodies were added to biotinylated 100 BS streptavidin beads and incubated overnight at room temperature. The beads exhibited aggregation with a peak at 2,148 nm and a polydispersity index of 316.2% (…). Figure 3 ).

[0089] Example 2

[0090] Magnetic nanoparticles coated with biotinylated streptavidin or biotinylated 100BS streptavidin were used. Methods for reducing anti-biotin antibodies from samples using beads

[0091] To confirm successful biotinylation of 100% biotin-saturated streptavidin-conjugated magnetic nanoparticles or 100BS streptavidin beads, lyophilized mouse ascites containing monoclonal anti-biotin antibodies specific to the conjugated biotin was purified using a Melon gel purification kit (ThermoFisher, part number 45214) and ascites conditioning buffer (ThermoFisher, part number 45219, batch number TB263120). The ascites was then desalted in PBS at pH 7.2 using a Zeba Spin 40K MWCO column (ThermoFisher, part number 87770). The final concentration of the anti-biotin antibody was 0.205 mg / mL, and 50 µL or 10.25 µg of anti-biotin antibody was added to 950 µL of pH 7.4 PBS in a glass HPLV tube to prepare a 10.25 µg / mL anti-biotin antibody stock solution. 100 µL, 50 µL, 25 µL, and 10 µL of the described antibiotin antibody stock solution were sequentially injected onto a Phenomenex S4000 SEC HPLC column at a flow rate of 1.0 mL / min using PBS at pH 7.4 as the mobile phase. The peak area for each injected antibody concentration was determined to generate a calibration curve of peak area (Y-axis) versus antibody concentration (X-axis): y = 6.6466x + 21.4286, R² = 1.0000 (…). Figure 4A ).

[0092] Next, 750 µL of 0.205 mg / mL anti-biotin antibody was pretreated with biotinylated 100BS streptavidin beads using the following steps:

[0093] 1. Remove the biotinylated 100BS streptavidin bead test tube from the storage location and vortex at a medium speed for at least 10 seconds.

[0094] 2. Place an empty 2 mL Sarstedt Micro tube into the VeraMag 400™ Magnetic Separator until the neck of the tube contacts the magnet frame.

[0095] 3. Dispense 750 µL or 7.5 mg beads at a concentration of 10.0 mg / mL into a 2 mL Sarstedt Microtube.

[0096] 4. Wait at least 30 seconds, then carefully aspirate and discard all supernatant without disturbing the magnetic nanoparticle deposit.

[0097] 5. Dispense 750 µL of thoroughly mixed antibiotin antibody at a concentration of 0.205 mg / mL.

[0098] 6. Cover the tube and vortex the sample at a medium speed for at least 10 seconds.

[0099] 7. Place the tube on a rotary mixer running at medium speed and incubate at RT for 30 minutes.

[0100] 8. Unscrew the screw cap and place the tube in the VeraMag 400 until the neck of the tube contacts the magnet frame.

[0101] 9. Allow the nanoparticles to magnetically separate from the sample for 5 minutes.

[0102] 10. Carefully aspirate the sample without disturbing the magnetic nanoparticle deposit and dispense it into a clean tube. If this step is performed carefully, all the sample can be aspirated. Note: If any magnetic nanoparticles are accidentally aspirated, simply put the mixture back into the tube, cap it, and return to step 9.

[0103] 11. The prepared sample is now ready for analysis.

[0104] 12. Filter the sample using a 0.2-micron cellulose acetate injection filter. The average protein loss using this filter was 16.5 µg.

[0105] 13. Inject 100 µL of sample onto a Phenomenex s4000 SEC HPLC column at a flow rate of 1.0 mL / min. The mobile phase is (50 mM potassium phosphate, 250 mM potassium chloride, pH 6.8). Determine the peak area at retention times of ~9.6 to 9.9 minutes.

[0106] 14. Based on the calibration curve equation, use the peak area (y) of the antibody peak to solve for x (µg / mL antibody).

[0107] The peak area of ​​the 100 µL avidin Ab sample was 1,384 ( Figure 4B ), and the concentration corresponding to this peak area on the calibration curve is 205 µg / mL ( Figure 4A The peak area of ​​100 µL of pretreated and depleted antibiotin antibody sample was 318 ( ). Figure 4C ), and the concentration corresponding to this peak area on the calibration curve is 44.62 µg / mL ( Figure 4ASince the initial volume of antibody pretreated with the depletion reagent was 750 µL, this corresponds to 33.465 µg of antibody: [44.62 µg / mL x 0.750 mL]. Due to the loss of 16.5 µg of antibody on the 0.2 μm cellulose acetate syringe filter, the total remaining antibody after sample pretreatment was 49.965 µg of antibody: [33.465 µg + 16.5 µg]. The initial amount of pretreated antibiotin-containing antibody was 153.75 µg of antibody: [205 µg / mL x 0.750 mL]. The percentage of antibiotin-containing antibody captured and depleted by the biotinylated 100BS streptavidin beads was 67.5%: [((153.75 µg - 49.965 µg) / 153.75 µg) x 100%].

[0108] This study confirms that biotinylated 100BS streptavidin beads can deplete 103.785 µg of antibody: (153.75 µg - 49.965 µg). This corresponds to a binding capacity of 13.838 µg of antibiotin antibody per mg of biotinylated 100BS streptavidin beads: [(103.785 µg antibody) / (7.5 mg beads)].

[0109] Example 3

[0110] Biotinylated 100BS streptavidin beads were prepared using a low molar excess of biotin.

[0111] Biotin saturation of streptavidin-coated beads with a 1000-fold molar excess of free biotin can lead to nonspecific binding of biotin to streptavidin or the beads. In use, despite successful depletion of both anti-biotin and anti-streptavidin substances, the nonspecifically bound biotin can leach or dissociate from the 100 BS streptavidin beads, potentially causing biotin interference in assays. Several methods for removing or reducing this nonspecific biotin binding were investigated, including saturating the streptavidin before coupling to magnetic nanoparticles, various post-saturation washing procedures, using lower molar excesses of biotin, and various different biotin-linker molar excesses. Ultimately, a combination of low molar excess biotin and specific washing conditions yielded a product without the problem of free biotin leaching.

[0112] After streptavidin was coupled to magnetic nanoparticles but before biotinylation, the streptavidin beads were exposed to a 5-molar excess of free biotin (i.e., a 5:1 biotin:streptavidin molar ratio or a 5:4 biotin:biotin binding site ratio). The saturated beads were then washed with water at 50°C and ultrasonically. (The effective ratio of biotin to biotin binding sites is slightly higher because the coupling of streptavidin to the beads causes steric hindrance at some biotin binding sites).

[0113] Biotinylation was performed using 4, 25, and 50 molar excesses of the biotinylating agent (biotin-PEG4-NHS linker). A 50-molar excess was found to yield the best results.

[0114] The neutrality of the beads was tested according to the following protocol to confirm that the beads themselves do not cause interference when used for pretreatment of serum samples:

[0115] 1. Remove the biotinylated 100BS streptavidin bead test tube from the storage location and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent.

[0116] 2. Insert the reagent tubes into the foam tube holder.

[0117] 3. Insert the empty 2ml Microtube (SARSTEDT catalog number 72.694) into the VeraMag™ magnet (Veravas) until the neck of the tube contacts the magnet frame.

[0118] 4. Dispense 200 µL of the well-mixed reagent (beads) into an empty tube and separate the reagent on a magnet for >30 seconds to form a reagent deposit.

[0119] 5. Carefully aspirate and discard all storage buffer supernatant (~200 µL) without disturbing the reagent deposits.

[0120] 6. Dispense 400 µL of thoroughly mixed serum or plasma sample into a tube containing reagent deposits.

[0121] 7. Tighten the screw cap on the tube, remove the tube from the magnet, and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent in the sample.

[0122] 8. Place the tube on a laboratory mixer running at medium speed and incubate at room temperature for 10 minutes.

[0123] 9. Unscrew and remove the screw cap, insert the tube into the magnet until the neck of the tube contacts the magnet frame.

[0124] 10. Magnetic separation of reagents for >4 minutes to form reagent deposits.

[0125] 11. Carefully aspirate the sample supernatant without disturbing the reagent deposits and dispense the sample into the transfer tube for testing. Note: If this step is performed carefully, all sample supernatant (~400 µL) can be aspirated. If any reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and return to step 10.

[0126] 12. The sample is now ready for testing.

[0127] The pretreated samples were then used for the Roche Elecsys TSH assay, an example of a sandwich immunoassay, and the Roche Elecsys FT4 assay, an example of a competitive immunoassay (see Table 2). For all samples tested by both assays, there were no significant analytical or clinical differences between the treated and untreated results.

[0128] Table 2

[0129]

[0130] Example 4

[0131] Preparation of validation batches

[0132] Three batches of beads were prepared using streptavidin from different sources, essentially as described in Example 3, involving saturation with a 5:1 biotin to streptavidin molar ratio, 50-fold molar excess of biotin-PEG4-NHS linker, and ultrasonic cleaning at 50°C. One batch of FSAv used fresh streptavidin; one batch of RSAv used streptavidin recovered from a previous bead coating reaction; and one batch of MSAv used a mixture of 80% recovered streptavidin and 20% fresh streptavidin. The streptavidin content for the three batches was 35 μg / mg beads for the FSAv batch, 30 μg / mg beads for the RSAv batch, and 19 μg / mg beads for the MSAv batch. These three batches were then used in validation studies described in Examples 5-8 below.

[0133] The coupling of streptavidin to magnetic nanoparticles utilizes an excess of streptavidin. Unused reagent is not discarded but recovered through filtration, desalination, and concentration. It has been found that this recovered streptavidin can be incorporated into functional products without affecting stability or performance.

[0134] Example 5

[0135] Granularity as an indicator of aggregation in the manufacturing process

[0136] As initial quality control, dimensional analysis was performed on the finished beads to check for aggregation during the manufacturing process. 5 μL of beads were mixed with 1 mL of diH₂O in a disposable cuvette and read using an Anton Paar Litesizer™ 100 particle size analyzer after a brief vortexing (5–10 seconds), mixing (10 minutes on a mixer), and sonication for 30–60 seconds (if used). No aggregations from the production process were observed in any of the three batches before or after sonication (Tables 3 and 4). On average, the polydispersity of the aggregates was greater than that of the monomers.

[0137] Table 3. Particle size analysis - No ultrasonic treatment during preparation

[0138]

[0139] Table 4. Particle size analysis - Ultrasonic treatment used in preparation

[0140]

[0141] Example 6

[0142] Biotin leaching test

[0143] To test potential problem levels of biotin leaching, biotin-saturated and streptavidin-coated beads (biotinylated 100BS streptavidin beads) were suspended in serum with a biotin concentration below 100 pg / mL. 400 μL of serum was treated with 0.5 mg beads (200 μL - 2.5 mg / mL) on a mixer at RT for 10 min, followed by magnetic separation according to the following protocol:

[0144] 1. Remove the biotinylated 100BS streptavidin bead batch MSAv, FSAv, or RSAv reagent tube from the storage location and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent.

[0145] 2. Insert the reagent tubes into the foam tube holder.

[0146] 3. Insert the empty 2ml Microtube (SARSTEDT catalog number 72.694) into the VeraMag magnet until the neck of the tube contacts the magnet frame.

[0147] 4. Dispense 200 µL of the well-mixed reagent (beads) into an empty tube and separate the reagent on a magnet for >30 seconds to form a reagent deposit.

[0148] 5. Carefully aspirate and discard all storage buffer supernatant (~200 µL) without disturbing the reagent deposits.

[0149] 6. Dispense 400 µL of thoroughly mixed serum or plasma sample into a tube containing reagent deposits.

[0150] 7. Tighten the screw cap on the tube, remove the tube from the magnet, and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent in the sample.

[0151] 8. Place the tube on a laboratory mixer running at medium speed and incubate at room temperature for 10 minutes.

[0152] 9. Unscrew and remove the screw cap, insert the tube into the magnet until the neck of the tube contacts the magnet frame.

[0153] 10. Magnetic separation of reagents for >4 minutes to form reagent deposits.

[0154] 11. Carefully aspirate the sample supernatant without disturbing the reagent deposits and dispense the sample into the transfer tube for testing. Note: If this step is performed carefully, all sample supernatant (~400 µL) can be aspirated. If any reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and return to step 10.

[0155] 12. The sample is now ready for testing.

[0156] The treated serum was tested using the IDK Biotin ELISA assay (Immundiagnostik AG). The IDK Biotin ELISA is a competitive immunoassay; biotin in the sample will reduce the generated signal. Biotin concentration was determined by comparison with a calibration curve. The calibration curve was run using each of three test batches. In all cases, biotin was detected at levels below 1200 pg / ml, indicating that any leaching of biotin from the beads was at levels that would not cause heterophilic interference in standard immunoassays (Table 5).

[0157] Table 5. Quantification of biotin in treated serum

[0158]

[0159] Example 7

[0160] HPLC depletion assay

[0161] Three batches of biotin-saturated and streptavidin-coated beads were used in a depletion assay to evaluate their ability to specifically remove streptavidin and antibiotin interfering substances without removing other interfering substances. For this purpose, a series of four HPLC depletion studies were performed:

[0162] 1) An HPLC depletion assay was performed using affinity-purified goat IgG and biotin-conjugated affinity-purified goat IgG to assess the specificity of the product for anti-SAv and anti-Bt antibodies only.

[0163] 2) The binding capacity of the reagent was quantified by HPLC depletion assay using anti-streptavidin antibody.

[0164] 3) The binding capacity of the reagent was quantified by HPLC depletion assay using anti-biotin antibody.

[0165] 4) The multi-purpose capability and specificity of the reagents were confirmed by HPLC depletion assays using a mixture of A) anti-biotin antibody and anti-streptavidin antibody and B) anti-streptavidin antibody and affinity-purified goat IgG.

[0166] The concentrations of various antibodies (Abs) and antibody-biotin (Ab-Bt) conjugates in phosphate-buffered saline (PBS) at pH 7.4 were determined. For each sample, 200 μl of a biotin-saturated and streptavidin-coated bead suspension (2.5 mg / ml) was aliquoted into a tube, magnetically separated, and the storage buffer was removed. 400 μl of each Ab or Ab-Bt conjugate was added to the bead-containing tube, vortexed, and incubated on a mixer for 10 min. The beads were magnetically separated again, the supernatant of the treated sample was extracted, and loaded onto a microplate for HPLC for size exclusion chromatography (SEC) analysis. Detailed pretreatment protocols are as follows:

[0167] 1. Remove the biotinylated 100BS streptavidin bead batch MSAv, FSAv, or RSAv reagent tube from the storage location and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent.

[0168] 2. Insert the reagent tubes into the foam tube holder.

[0169] 3. Insert the empty 2ml Microtube (SARSTEDT catalog number 72.694) into the VeraMag magnet until the neck of the tube contacts the magnet frame.

[0170] 4. Dispense 200 µL of the well-mixed reagent (beads) into an empty tube and separate the reagent on a magnet for >30 seconds to form a reagent deposit.

[0171] 5. Carefully aspirate and discard all storage buffer supernatant (~200 µL) without disturbing the reagent deposits.

[0172] 6. Dispense 400 µL of thoroughly mixed serum or plasma sample into a tube containing reagent deposits.

[0173] 7. Tighten the screw cap on the tube, remove the tube from the magnet, and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent in the sample.

[0174] 8. Place the tube on a laboratory mixer running at medium speed and incubate at room temperature for 10 minutes.

[0175] 9. Unscrew and remove the screw cap, insert the tube into the magnet until the neck of the tube contacts the magnet frame.

[0176] 10. Magnetic separation of reagents for >4 minutes to form reagent deposits.

[0177] 11. Carefully aspirate the sample supernatant without disturbing the reagent deposits and dispense the sample into the transfer tube for testing. Note: If this step is performed carefully, all sample supernatant (~400 µL) can be aspirated. If any reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and return to step 10.

[0178] 12. The sample is now ready for testing.

[0179] Untreated antibodies were also run on the SEC buffer as a control. The SEC buffer was 50 mM potassium phosphate, 250 mM potassium chloride, pH 6.8, and was pumped at a flow rate of 1 ml / min on a G4000 column (7.8 mm x 30 cm) for 20 min, using an injection volume of 5 μg / 100 μl. Absorbance was monitored at 220 nm and 280 nm, and A280 was used for peak analysis. The results are shown in Table 6.

[0180] Table 6. Quantitative analysis of impoverishment and bead ability

[0181] ("α-" indicates "anti"; "AP" indicates "affinity-purified antibody"; "Bt" indicates "biotinylated antibody")

[0182]

[0183] 1 Anti-streptavidin antibody

[0184] 2 Anti-biotin antibody

[0185] 3 Affinity-purified goat IgG

[0186] 4 Affinity-purified goat IgG-Biotin conjugate

[0187] 5 A mixture of anti-streptavidin antibodies and anti-biotin antibodies

[0188] 6 A mixture of anti-streptavidin antibody and affinity-purified goat IgG

[0189] * Peak areas of each fraction of the multiplexed sample: 5.85 μg anti-biotin antibody - area 134; 6.05 μg affinity-purified goat IgG - area 289; 5.85 μg anti-streptavidin antibody - area 243.

[0190] Affinity-purified goat IgG was run as a control to establish background impairment, as the beads were intended to be neutral against nonspecific antibiotin or antistreptavidin antibodies. All three batches showed very little to no binding to this control (92.56%–96.78% of the antibody remained after treatment); Figure 5A The results using MSAv batches are depicted.

[0191] Affinity-purified goat IgG conjugated with biotin was run as another control to establish background impairment, as the beads were also intended to be neutral to nonspecific anti-biotin or anti-streptavidin antibodies, even when biotinylated. All three batches showed very little to no binding to this control (98.91%–100% of the antibody remained after treatment); Figure 5B The results using MSAv batches are depicted.

[0192] All three batches showed anti-biotin antibody impairment of more than 10 μg per mg of beads (12, 34.4 and 21 μg / mg impairment; see Table 6); Figure 5C Results using MSAv batches are depicted. All three batches showed anti-SAv antibody depletion greater than 20 μg per mg bead (31, 33.5, and 27.6 μg / mg depletion; see Table 6; exemplary curves are shown in... Figure 5D The results are depicted using MSAv batches.

[0193] A mixture of anti-streptavidin antibody and anti-biotin antibody was tested using beads from an MSAv batch to confirm the multiplexing capability of biotin-saturated and streptavidin-coated beads. A mixture of anti-streptavidin antibody and AP goat IgG antibody was also tested to confirm the binding specificity of the reagent. Data showed that biotin-saturated and streptavidin-coated beads depleted both anti-streptavidin and anti-biotin antibody (when they were present in tandem) (depleting 19.1 μg out of the 23.4 μg present), and that the product specifically removed only the anti-streptavidin antibody (when it was mixed with AP goat IgG) (depleting 10.9 μg out of the 23.8 μg present). Since previous data showed no binding to AP goat IgG alone (97% of the Abs remained after treatment), it can be safely inferred that the depletion of approximately half of the mixture (46%) indicates that only the anti-streptavidin Ab was depleted.

[0194] Example 8

[0195] Impact of treatment on analyte detection

[0196] Neutrality was tested in a commercially available assay for serum parathyroid hormone using biotin-saturated and streptavidin-coated beads (biotinylated 100BS streptavidin beads). The DRG PTH Intact ELISA (DRG International, Inc., part number EIA3645) is a sandwich ELISA assay that uses two different goat anti-PTH polyclonal antibodies that recognize different parts of the hormone. One antibody is biotinylated and acts as a capture reagent, while the other antibody is conjugated to horseradish peroxidase and acts as an detection reagent.

[0197] Two serum samples (QC1 and QC3), 400 μl each, were treated with 0.5 mg beads (200 μl, 2.5 mg / mL) on a mixer at RT for 10 minutes and magnetically separated.

[0198] 1. Remove the biotinylated 100BS streptavidin bead batch SAv, FSAv, or RSAv reagent tube from the storage location and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent.

[0199] 2. Insert the reagent tubes into the foam tube holder.

[0200] 3. Insert the empty 2ml Microtube (SARSTEDT catalog number 72.694) into the VeraMag magnet until the neck of the tube contacts the magnet frame.

[0201] 4. Dispense 200 µL of the well-mixed reagent (beads) into an empty tube and separate the reagent on a magnet for >30 seconds to form a reagent deposit.

[0202] 5. Carefully aspirate and discard all storage buffer supernatant (~200 µL) without disturbing the reagent deposits.

[0203] 6. Dispense 400 µL of thoroughly mixed serum or plasma sample into a tube containing reagent deposits.

[0204] 7. Tighten the screw cap on the tube, remove the tube from the magnet, and vortex at medium speed for at least 10 seconds to thoroughly mix and resuspend the reagent in the sample.

[0205] 8. Place the tube on a laboratory mixer running at medium speed and incubate at room temperature for 10 minutes.

[0206] 9. Unscrew and remove the screw cap, insert the tube into the magnet until the neck of the tube contacts the magnet frame.

[0207] 10. Magnetic separation of reagents for >4 minutes to form reagent deposits.

[0208] 11. Carefully aspirate the sample supernatant without disturbing the reagent deposits and dispense the sample into the transfer tube for testing. Note: If this step is performed carefully, all sample supernatant (~400 µL) can be aspirated. If any reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and return to step 10.

[0209] 12. The sample is now ready for testing.

[0210] The treated sera were then tested using the DRG PTH ELISA assay. QC1 was a homemade QC sample with less than 100 pg / mL biotin (which does not affect the PTH assay mechanism) and approximately 190 pg / mL PTH. QC3 was a homemade QC sample with approximately 250,000 pg / mL biotin (which will affect the PTH assay mechanism – the assay produces severely reduced results) and approximately 190 pg / mL PTH. No significant deviations were observed in the PTH results from QC1 sera treated with each of the different batches (100.6%, 101.2%, and 106.1% detection), and QC3 samples, as expected, all produced severely reduced results because the 100BS streptavidin beads do not bind free biotin (Table 7). These data confirm the neutrality of the 100BS streptavidin beads.

[0211] Table 7. Neutral data of PTH ELISA

[0212]

[0213] Analyte detection was determined after processing samples containing interfering anti-streptavidin and anti-biotin antibodies. This was achieved by incorporating affinity-purified anti-streptavidin and anti-biotin goat antibodies into serum QC1 and comparing analyte detection results from treated and untreated samples.

[0214] 400 μl aliquots of each serum sample (QC1 and QC1 incorporating antibiotin antibody to 16.5 μg / mL) were treated with varying amounts of beads (100 to 400 μl, 2.5 mg / mL) from all three batches on a mixer at RT for 10 minutes, magnetically separated, and the treated serum was tested using the DRG PTH ELISA assay. QC1 was a homemade QC sample containing less than 100 pg / mL of biotin (which does not affect the PTH assay mechanism) and approximately 190 pg / mL of PTH. The concentration of antibiotin antibody incorporated into QC1 interfered with the PTH assay mechanism, resulting in severely reduced results.

[0215] When 1 mg of beads was used per 200 μl sample (with an anti-Bt Aby concentration of 16.5 μg / mL), MSAv beads successfully depleted all anti-Bt antibodies (anti-Bt Aby) and restored correct PTH results. FSAv and RSAv achieved the same results with far fewer amounts: 0.25 mg for FSAv and 0.375 mg for RSAv. QC1 samples doped with anti-Bt Aby and not treated with biotinylated 100BS streptavidin beads produced severely reduced results, only 2.7% of the control, which was consistent with expectations (Tables 8 and 9). The lower results of the MSAv batches were consistent with those observed in Example 7 (above).

[0216] Table 8. Detection of analytes in serum containing avidin Ab and treated with beads from batch MSAv

[0217]

[0218] Table 9. Analytes in serum containing anti-Bt Aby and treated with beads from FSAv and RSAv batches. Test

[0219]

[0220] Similarly, 200 μl aliquots of each serum sample (QC1 and QC1 infused with anti-streptavidin Aby (anti-SAv Aby) to 16.5 μg / mL or anti-SAv Aby / anti-Bt Aby multiplex to 16.5 μg / mL (8.25 μg / mL each)) were treated with 100 μl of 2.5 mg / mL beads on a mixer at RT for 10 min, magnetically separated, and the treated serum was tested on a DRG PTH ELISA assay. As previously mentioned, QC1 is a homemade QC sample with less than 100 pg / mL of biotin (which does not affect the PTH assay mechanism) and approximately 190 pg / mL of PTH. Anti-SAv Aby and anti-Bt Aby interfere with the PTH assay mechanism, causing severely reduced results.

[0221] At the concentration used (16.5 μg / mL), anti-SAv Aby resulted in the expected severe reduction (29% of baseline). The same was true for multiplex mixtures (21% of baseline). Using 0.25 mg beads per 200 μl sample, beads from batch RSAv successfully depleted anti-SAv Aby and began to recover PTH detection, producing readings up to 82% of baseline. Increasing the amount of beads used in the treatment to 0.5 mg per 200 μl of the sample restored these values ​​to 104% of baseline. Using 0.25 mg beads per 200 μl of the sample from batch FSAv restored PTH levels to 91% of baseline. Using 0.375 mg beads per 200 μl of the sample from batch MSAv restored PTH values ​​to 92% of baseline. Using 0.5 mg beads, all batches were able to successfully restore the correct PTH results (RSAv-104%; FSAv-101%; MSAv-100%) (Table 10).

[0222] Table 10. Beads containing anti-SAv Aby or anti-SAv Aby and anti-Bt Aby from all three batches. Detection of analytes in processed serum

[0223]

[0224] The results of the PTH detection experiments using batch MSAv described above are summarized in the following... Figure 6 In the meantime, biotinylated 100BS streptavidin beads have no effect when no interfering agent is used or when biotin is an interfering agent, but effectively remove individual or mixed antibiotin and antistreptavidin interfering agents.

[0225] Specifically, in Figure 6In the leftmost group of bars marked "None," no interfering agent was added to the sample; that is, it was a rerun of the baseline sample, and the detected PTH concentration differed by only -0.1%. Treatment of the baseline sample (None) without any interfering agent with biotin-saturated and coupled streptavidin-coated beads resulted in a difference of only +1.3% from the baseline sample and only +1.4% from the rerun of the baseline sample (without interfering agent). These results are entirely within the precision curve of the PTH ELISA and confirm the neutrality of the reagents and that sample treatment did not introduce any dilution or matrix effects. Biotin doping caused significant interference in this PTH ELISA assay, leading to an 87% reduction in detection. When the biotin-doped sample was treated with beads, the results did not change significantly and differed by only -2.3%, also 88% lower than the baseline result. This is expected because biotinylated 100BS streptavidin beads do not bind free biotin and will not mitigate this interference mechanism. Anti-Bt Aby dopant caused significant interference in this PTH ELISA assay, resulting in a 97% reduction in detection. When treated with the anti-Bt Aby dopant, the results were significantly altered by +3,546%, but only -1.5% from the baseline. This is expected, as biotinylated 100BS streptavidin beads are designed to bind and deplete the anti-Bt Aby interferon, reporting accurate results close to the baseline without the anti-Bt Aby interferon. Anti-SAv Aby dopant also caused significant interference in this PTH ELISA assay, resulting in a 74% reduction in detection. When treated with the anti-SAv Aby dopant, the results were significantly altered by +253%, but only -8.2% from the baseline. This is expected, as biotinylated 100BS streptavidin beads are designed to bind and deplete the anti-SavAby interferon, reporting accurate results close to the baseline without the anti-SAv Aby interferon. Finally, a 1:1 mixture of anti-SAv Aby and anti-Bt Aby dopants caused significant interference in this PTH ELISA assay, resulting in an 81% reduction in detection. When treated with both anti-SAv Aby and anti-Bt Aby dopants, the results changed significantly by +379%, but differed from the baseline by only -9.9%. These results remain within the precision curve of this PTH ELISA. This is expected, as biotinylated 100BS streptavidin beads are designed to bind and deplete both anti-biotin and anti-streptavidin interfering agents, reporting accurate results close to the baseline without both anti-biotin and anti-streptavidin interfering agents. These data also confirm the ability of biotinylated 100BS streptavidin beads to simultaneously bind and deplete two interfering mechanisms from the same sample.

[0226] Example 9

[0227] Biotin saturation of soluble streptavidin

[0228] Prepare a dilute solution of streptavidin (SAv) (or modified SAv or blocking SAv) in tris-buffered saline (TBS) at pH 8.5 at a concentration of approximately 200 µg SAv / mL. Also prepare a dilute solution of biotin in TBS at a concentration of approximately 0.50–1.00 µg biotin / mL. Measure both solutions together and mix them online at a ratio of 1 volume SAv / TBS to 9 volumes biotin / TBS, e.g., by pumping through a silicone tube (e.g., PN 96440-13 (Cole Parmer)), converging at a Y-connector (e.g., Masterflex PN 30614-08 (Cole Parmer)), and immediately mixing using an online mixer (e.g., online mixer PN HT-40-3.18-12-PP (StaMixCo)) in the outlet tube. Figure 7 The SAv solution can be pumped at 2 mL / min, and the biotin solution can be pumped at 18 mL / min, using, for example, a peristaltic pump (e.g., Masterflex EZload2 07522-20 (Cole Parmer)). The solutions containing biotin and streptavidin are mixed for 30 to 120 minutes.

[0229] Other biotin and SAV concentrations, other buffer systems, and other pump rates can be used, but the biotin to SAV concentration ratio and the stoichiometric addition ratio should be maintained. For each mole of streptavidin in the SAV solution, 9 volumes of biotin solution should contain 7.4 moles of biotin. Note that this is a slightly higher biotin to streptavidin ratio than used in the minimum saturation procedure for SAV used in bead coupling. Taking a streptavidin molecular weight of 52,000, 300 mL of 200 μg / mL SAV solution contains 1.1538 μmoles of streptavidin. Taking a biotin molecular weight of 244.31, 8.53072 μmoles of biotin (for a 7.4:1 molar ratio) is 2084 μg of biotin, resulting in a biotin concentration of 772 ng / mL in the stated 9 volumes.

[0230] To remove unbound and non-specifically bound biotin, biotin-saturated streptavidin is percolated and washed. A hot water bath is filled with pure water and heated to 50°C. A second hot water bath is filled with a buffer solution of 10 mM Tris and 50-150 mM NaCl and heated to 50°C. (Alternatively, this buffer solution may contain 0.01-1% w / v TWEEN 20 or other surfactants). A reservoir containing biotin-saturated streptavidin is placed in the first hot water bath. Using a hollow fiber filter, such as a MiniKros Sampler hollow fiber filter with a molecular weight cutoff of 10 kD (Repligen; PN S04-E010-05-N; mPes; 0.5 mm), tubing is attached to the in-line flow port (top and bottom) and a side port. The second side port is capped. The side port tubing directs the filtrate to a waste container. The tubing from the reservoir is advanced to the hollow fiber filter via a peristaltic pump. The residual liquid is returned to the reservoir via a pipeline leading from the online flow outlet port. Figure 8 When the water bath and reservoir reach 50°C, turn on the peristaltic pump and apply clamps to the transpiration line to create back pressure, causing filtration to occur, reducing the volume of the biotin-saturated streptavidin solution and concentrating the protein. The transpiration flow rate is approximately 360 ml / min, and the filtrate flow rate is approximately 95 ml / min. When the reservoir reaches approximately 15% (or less) of its original volume, add buffer from the second water bath to the reservoir to restore the original volume. (Obtain a sample for quality control just before volume restoration). Repeat the filtration and volume restoration at least 5 times in total; additional wash cycles may be added if necessary. After the last volume restoration, concentrate the transpiration to approximately 10% of its original volume (an additional volume may be used if necessary). The final transpiration should contain less than 1200 pg / ml of free biotin. Filter the concentrated biotin-saturated streptavidin through a 0.2 μm filter.

[0231] In the presence of dissolved streptavidin, free biotin is considered evidence that the biotin quenching process is complete. However, free biotin itself can cause interference and is therefore undesirable in blocking agents used to block streptavidin and other interferences, and thus needs to be removed. The free biotin content in the effluent after each wash and the final effluent (in duplicate) was determined using the ELISA biotin assay (Immundiagnostik, PN KR8141) to obtain the results shown in Table 11.

[0232] Table 11. Quantification (and calibration) of free biotin in QSAv prepared quality control samples.

[0233]

[0234] The final dialysis fluid contained less than 700 pg / mL of free biotin, significantly below the requirement of 1200 pg / mL. The final dialysis fluid had a concentration of 201 μg streptavidin / mL, therefore it contained ~3.16-3.33 pg free biotin / μg streptavidin.

[0235] An additional step can be added to remove any incompletely quenched streptavidin. 2-Iminobiotin coupled to agarose (Sigma Aldrich PN I4507-5ML) can be used for this purpose. 2-Iminobiotin reversibly binds to SAv under alkaline conditions. Binding is strongest at pH 10 to 11. Under acidic conditions, such as pH 4.0, SAv will be released. SAv that has been quenched with biotin should not bind. Therefore, the flow-through from the alkaline 2-Iminobiotin-agarose column should contain only biotin-quenched SAv. Unquenched SAv should adhere to the column. The column can be regenerated with a pH 4 buffer for subsequent reuse.

[0236] Example 10

[0237] Goat anti-mouse antibodies were removed using streptavidin beads conjugated with mouse IgG.

[0238] Magnetic nanoparticles (beads) with a diameter of 500 to 600 nm were coated with streptavidin. Two affinity-purified mouse IgG preparations were covalently biotinylated with NHS ester-Peg(4)-biotin. Mouse IgG #1 was a polyclonal nonspecific mouse IgG. Mouse IgG #2 was a monoclonal mouse IgG. When the beads were exposed to the biotinylated mouse IgG, approximately 30 µg of IgG was attached to each mg of bead. The beads were made with polyclonal mouse IgG only, monoclonal mouse IgG only, and a mixture of the two mouse IgG preparations. These mouse antibodies acted as a capture fraction of any specific affinity-purified or heterophilic anti-mouse IgG antibodies to which they would be exposed. These mouse IgG-conjugated streptavidin beads were then used to clean samples (in this case, buffer) already doped with affinity-purified goat anti-mouse antibodies (Lampire Biological Laboratories). The IgG content was then determined by size exclusion chromatography (HPLC) of aliquots of the sample.

[0239] One potential concern with using biotinylation to anchor the capture reagent to streptavidin is that the affinity of biotin may be lower than that of free biotin, causing the capture reagent to dissociate from streptavidin during the cleaning procedure. To check this, the cleaning procedure was performed normally and in the presence of 20 μg / mL free biotin (>200 molar excess compared to streptavidin). If the biotinylated IgG dissociates from the streptavidin-coated beads, it will not be able to rebind in the presence of excess free biotin, thus reducing the amount of goat anti-mouse antibody removed using the beads. Alternatively, if the dissociation of the biotinylated IgG does not occur to a significant degree, the amount of goat anti-mouse IgG will not change significantly between samples containing and without free biotin.

[0240] Specifically, one aliquot of each of the three types of mouse IgG-conjugated streptavidin beads (containing IgG#1, IgG#2, and a mixture of both) was magnetically separated from storage buffer (TBS) and mixed with TBS containing 20 μg / ml biotin. Goat anti-mouse IgG was diluted to 200 μg / mL in a) TBS and b) TBS containing 20 μg / ml biotin. Two aliquots of each of the three types of mouse IgG-conjugated streptavidin beads (one exposed to biotin, the other unexposed) were magnetically separated from their buffers. Using 2.5 mg of beads per mL of goat anti-mouse IgG antibody, goat anti-mouse IgG antibody in TBS was added to the unexposed beads, and goat anti-mouse IgG antibody in TBS containing 20 μg / ml biotin was added to the biotin-exposed beads. These mixtures were vortexed for 10 seconds to suspend the beads and mixed overnight. The resulting mixture was subjected to magnetic separation using beads, and the supernatant sample was analyzed by HPLC-SEC using the area under the absorbance curve corresponding to the peak of goat IgG at 280 nm. The results are shown in Table 12.

[0241] Table 12. Removal of goat anti-mouse IgG antibodies

[0242]

[0243] These data confirm that streptavidin beads conjugated with mouse IgG can remove 26.0–60.1 μg of anti-mouse antibody per mg of beads. These data also confirm that the attachment of the biotinylated capture agent (mouse IgG) is stable under the conditions of the cleaning procedure, even in the presence of a large excess of free biotin. Finally, these data confirm the effectiveness of these procedures when using capture fractions as polyclonal and monoclonal antibodies, as well as mixtures of both.

[0244] Example 11. Biotin was removed using streptavidin beads conjugated with mouse IgG.

[0245] The ability of the three mouse IgG-conjugated streptavidin bead preparations (polyclonal mouse IgG, monoclonal mouse IgG, and a mixture of polyclonal and monoclonal mouse IgG) described in Example 10 above to remove free biotin from samples was also tested. Using essentially the same protocol as described above, 0.75 mg of beads were combined with 200 μL of a homemade QC sample containing approximately 250,000 pg biotin / mL and 50 ng biotin, incubated for 10 min, magnetically separated for 5 min, and the sample supernatant was collected. The treated samples were tested on the IDK biotin ELISA assay (Immundiagnostik AG; see Example 6). Under these conditions, all three bead preparations were able to remove at least 49.7 ng of biotin from 0.200 mL of 250 ng biotin / mL QC3 (total 50 ng biotin), or 66.3 ng biotin / mg beads, and reduce the biotin concentration from 250,000 pg / mL to below 300 pg / mL.

[0246] Finally, it should be understood that although various aspects of this specification are highlighted by reference to specific embodiments, those skilled in the art will readily recognize that these disclosed embodiments merely illustrate the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific methods, schemes, and / or reagents described herein. Consequently, various modifications, alterations, or alternatives can be made to the disclosed subject matter based on the teachings herein without departing from the spirit of this specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims. Therefore, the invention is not limited to exactly as shown and described.

[0247] This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art after reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend to practice the invention in ways other than those specifically described herein. Therefore, the invention includes all modifications and equivalents to the subject matter recited in the claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, any combination of all possible variations of the foregoing embodiments is included in this invention.

[0248] The grouping of optional embodiments, elements, or steps of the invention should not be construed as limiting. Each member of a group may be cited and claimed individually or in any combination with other members of the groups disclosed herein. For convenience and / or patentability reasons, it is contemplated that one or more members of a group may be included in a group or removed from a group. When any such inclusion or removal occurs, the specification is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the claims.

[0249] Unless otherwise stated, all figures used in this specification and claims to represent characteristics, entries, quantities, parameters, properties, items, etc., should be understood to be modified by the term "about" in all cases. When used herein, the term "about" means that the characteristic, entry, quantity, parameter, property, or item so defined includes a range of plus or minus 10% above or below the value of the stated characteristic, entry, quantity, parameter, property, or item. Therefore, unless stated to the contrary, the numerical parameters presented in this specification and claims are approximate values ​​that may vary. At least, and not at all, an attempt is made to limit the application of the equivalence principle to the scope of the claims, each numerical indication should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques. Although the numerical ranges and values ​​that set forth the broad scope of the invention are approximate, the numerical ranges and values ​​set forth in the specific embodiments are reported as accurately as possible. However, any numerical range or value inherently contains some error necessarily caused by the standard deviation present in their respective test measurements. The description of numerical ranges herein is intended only as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise stated herein, each individual value of the numerical range is incorporated into this specification as if it were described separately herein.

[0250] Unless otherwise stated or clearly contradicted by the context, the terms “a,” “the,” and similar designations used in the context of describing the invention (especially in the context of the claims below) should be interpreted to cover both the singular and the plural. Unless otherwise stated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all instances or exemplary language (such as “for example”) provided herein is intended merely to better illustrate the invention and not to limit the scope of the otherwise claimed invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0251] The specific embodiments disclosed herein may be further limited in the claims using the language of "consisting of" or "substantially consisting of". When used in the claims, whether filed or amended, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transitional term "substantially consisting of" limits the scope of the claims to the specified materials or steps and those that do not substantially affect the essential and novel features. The embodiments of the invention thus claimed are inherently or explicitly described and enabled herein.

[0252] All patents, patent publications, and other publications referenced and specified in this specification are individually and expressly incorporated herein by reference in their entirety for describing and disclosing, for example, compositions and methods that may be used in combination with the present invention as described in such publications. These publications are provided only because their disclosures predate the filing date of this application. In this regard, nothing should be construed as an admission that the inventor has no right to a prior disclosure by virtue of a prior invention or for any other reason. All statements regarding the dates of these documents or descriptions of their contents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0253] References:

[0254] 1. Society to Improve Diagnosis in Medicine (SIDM); https: / / betterdiagnosis.org / .

[0255] 2. Frequency that Laboratory Tests Influence Medical Decisions; http: / / jalm.aaccjnls.org / content / 1 / 4 / 410.

[0256] 3. Frame, IJ et al., (2019) Susceptibility of Cardiac Troponin Assays to Biotin Interference, Am J Clin Pathol. 151: 486-493.

[0257] 4. Wild, David. (2018) Biotin interference: answering questions, reducing the risk, http: / / captodayonline.com / biotin-interference-answering-questions-reducing-the-risk / .

[0258] 5. Katzman, BM et al. (2018) Prevalence of biotin supplement usage in outpatients and plasma biotin concentrations in patients presenting to the emergency department. Clinical Biochemistry. 60:11–16.

[0259] 6. Colon, PJ, Green, DN (2018) Biotin Interference in Clinical Immunoassays, JALM. 02(06): 941-951.

[0260] 7. Kirkwood, Julie. (2018) Meeting the Biotin Challenge, Clinical Laboratory News. January, 2018.

[0261] 8. Chun, Kelly Y. (2017) Biotin Interference in Diagnostic Tests, Clinical Chemistry. 63:619-620.

[0262] 9. Samarasinghe, S. et al. (2017), Biotin Interference with Routine Clinical Immunoassays: Understand the Causes and Mitigate the Risks, Endocr Pract. 23(8): 989-998.

[0263] 10. Lam L., Kyle CV (2017), A simple method to detect biotin interference on immunoassays, Clin Chem Lab Med. 55(6): e104-e106.

[0264] 11. Trambas C. et al. (2017), Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin-biotin immunoassays, Ann Clin Biochem. 55(2): 216-226.

[0265] 12. Piketty, ML et al. (2017), High-dose biotin therapy leading to false biochemical endocrine profiles: validation of a simple method to overcome biotin interference, Clin. Chem. Lab Med. 55(6):817–825.

[0266] 13. Barbesino, G. (2016), The Unintended Consequences of Biotin Supplementation: Spurious Immunoassay Results Lead to Misdiagnoses, Clinical Laboratory News, BenchMatters, December: 1–3.

[0267] 14. FDA warns that biotin may interfere with lab tests: FDA Safety Communication, https: / / www.fda.gov / medical-devices / safety-communications / fda-warns-biotin-may-interfere-lab-tests-fda-safety-communication.

[0268] 15. Testing for Biotin Interference in In Vitro Diagnostic Devices, https: / / www.fda.gov / regulatory-information / search-fda-guidance-documents / testing-biotin-interference-vitro-diagnostic-evices.

[0269] 16. Tytgat, HLP et al. (2015), Endogenous biotin binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection, Microb Biotechnol. 8(1): 164–168.

[0270] 17. Chen, T. et al. (2012), Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays. PLoS One . 7(8): e42376.

[0271] 18. Favresse, J. et al. (2018), Interferences with thyroid function immunoassays: clinical implications and detection algorithm, Endocrine Reviews. 39(5): 830–850.

[0272] 19. Favresse J. et al. (2018), Antistreptavidin antibodies mimicking heterophilic antibodies in thyroid function tests, Clin Chem Lab Med. 56(7): e160-e163.

[0273] 20. Lam, L. et al. (2018), Apparent Hyperthyroidism Caused by Biotin-Like Interference from IgM Anti-Streptavidin Antibodies, Thyroid. 28(8):1063-1067.

[0274] 21. Harsch IA et al., (2017), Implausible elevation of peripheral thyroid hormones during therapy with a protein supplement, Clin Chem Lab Med. 55(9): e197-e198.

[0275] 22. Peltier L. et al., (2016), Anti-streptavidin interferences in Roche thyroid immunoassays: a case report, Clinical Chemistry and Laboratory Medicine. 54(1): e11-14.

[0276] 23. Rulander NJ et al. (2013), Interference from antistreptavidin antibody, Archives of pathology & laboratory medicine. 137(8): 1141-1146.

[0277] 24. Wouters et al. (2020), Alarmed by misleading interference in free T3 and free T4 assays: a new case of anti-streptavidin antibodies, Clin Chem Lab Med. 58(3): e69–e71.

[0278] 25. Robier et al. (2020), Anti-streptavidin antibodies as a cause of false-positive results of streptavidin-based autoantibody assays, Clin Chem Lab Med. 58(1):e5-e7.

[0279] 26. Verugstaete et al., (2019), Interference of anti-streptavidin antibodies in immunoassays: a very rare phenomenon or a more common finding?, Clin Chem Lab Med. 20191064, eISSN 1437-4331, ISSN 1434-6621.

[0280] 27. U.S. Patent Publication No. 2019 / 0137484.

[0281] 28. Waner MJ et al. (2019), Streptavidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence, Biochem BiophysRep. 17: 127-131.

[0282] 29. Sano T., Cantor CR (1990), Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea, J Biol Chem. 265(6): 3369-3373.

[0283] 30. Song, J. et al., (2015), Functional loop dynamics of the streptavidin-biotin complex, Sci Rep. 5: 7906.

[0284] 31. Deng, L. et al. (2013), Dissociation kinetics of the streptavidin-biotin interaction measured using direct electrospray ionization mass spectrometry analysis, J Am Soc Mass Spectrom. 24(1): 49-56.

Claims

1. A biotin-saturated streptavidin (QSAv) prepared by a method comprising the step of exposing streptavidin to a 5:1 to 11:1 molar excess of biotin.

2. The QSAv according to claim 1, wherein the molar excess is 7:1 to 8:

1.

3. The QSAv according to claim 1 or 2, the method further comprising hot alkaline buffer washing to remove non-specifically bound biotin.

4. The QSAv according to any one of claims 1-3, the method further comprising the step of blocking the streptavidin to mitigate aggregation.

5. The QSAv of claim 4, wherein the blocking comprises incorporating a surfactant during hot alkaline buffer washing.

6. The QSAv according to claim 4 or 5, wherein blocking comprises covalent modification with a preparative blocking agent.

7. The QSAv of claim 6, wherein the blocking comprises PEGylation.

8. Biotin-saturated and streptavidin-coupled microparticles (QSAv beads) prepared by a method comprising the step of exposing streptavidin to a 4:1 to 6:1 molar excess of biotin.

9. The QSAv beads according to claim 8, further comprising hot water washing to remove non-specifically bound biotin.

10. The QSAv or QSAv bead according to any one of claims 1-9, the method further comprising coupling an additional capture portion to the QSAv or QSAv bead.

11. A microparticle coupled with streptavidin, wherein the streptavidin is coupled to a further capture portion.

12. The QSAv, QSAv beads, or streptavidin-coupled microparticles according to claim 10 or 11, wherein the additional capture portion is biotinylated and exposed to the streptavidin or streptavidin-coupled microparticles before or during the biotin saturation step.

13. The QSAv, QSAv beads, or streptavidin-coupled microparticles according to claim 10 or 11, wherein the additional capture portion is covalently coupled to the streptavidin or streptavidin-coupled microparticles before, during, or after the biotin saturation step.

14. The QSAv or QSAv beads according to any one of claims 1-9, the method further comprising storing the QSAv or QSAv beads in a buffer solution containing <1200 pg / mL free biotin.

15. The QSAv beads or microparticles coupled with streptavidin according to any one of claims 8-14, wherein the microparticles are magnetic.

16. A QSAv solution or QSAv bead suspension, wherein the ratio of free biotin to streptavidin does not exceed 1 ng free biotin: 50 μg streptavidin.

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