Composition and method for detecting and exhausting sample interference

Abstract

To provide a method for reducing sample interference in an assay using a streptoavidin-biotin bond.SOLUTION: A reagent specific to anti-streptoavidin interference and anti-biotin interference and a method for preparing a fine particles bond surface are provided, and also a method for blocking, exhausting or reducing the concentrations of the anti-streptoavidin interference and the anti-biotin interference in a sample to less than an assay blockage threshold value (ABT) using the reagent and the fine particle bond surface before a diagnostic test is disclosed.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 006,630, filed Apr. 7, 2020, and No. 62 / 866,318, filed Jun. 25, 2019, the entire contents of which are hereby incorporated by reference herein.

Background Art

[0002] Every nine minutes, someone dies due to misdiagnosis or delayed diagnosis [1]. Physicians rely on diagnostic tests to proceed with treatment, but more than 2% of tests may be inaccurate due to multiple interferences in blood or urine (e.g., biotin in blood tests) [2].

[0003] 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) nutritional supplements, multivitamins, and prenatal vitamins. Biotin is commercially available for health and beauty, including hair, skin, and nail growth, as well as weight loss. It is also administered to patients at high pharmaceutical doses to treat certain medical conditions such as multiple sclerosis. However, biotin can significantly interfere with certain clinical tests, resulting in inaccurate test results, which may go undetected or lead to misdiagnosis or delayed treatment [3 - 13].

[0004] In 2017, the FDA issued a safety warning because a number of adverse events were associated with inaccurate test results and biotin supplementation

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 On June 13, 2019, the FDA published a notice of a draft guidance on "Testing for Biotin Interference in In Vitro Diagnostic Devices"

[15] .

[0005] In vitro diagnostic (IVD) companies are actively working on redesigning or reconstructing tests so that they require much higher biotin concentrations to interfere with the test in order to reduce biotin interference or increase the biotin interference threshold. However, these biotin-based tests are still vulnerable to secondary interference mechanisms related to biotin or biotin use by patients, namely anti-biotin interference [16-17], as well as interference mechanisms related to the use of streptavidin in test design to capture biotin conjugated to antibodies, proteins or antigens, namely anti-streptavidin interference [18-26].

[0006] Anti-biotin and anti-streptavidin antibodies and proteins can significantly interfere with certain clinical tests and cause inaccurate test results. Depending on the assay design and format, anti-biotin interference and anti-streptavidin interference, like biotin interference which causes a decrease in the test signal and false low or false high patient results, also result in a decrease in the test signal, but through different mechanisms, so they may be mistaken for biotin interference [16-26].

[0007] Although the FDA recently issued guidelines, in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI) standards and reflecting current biotin consumption trends, that IVD companies should test for biotin interference at a concentration of up to 1200 ng / mL, the FDA has not yet issued a safety warning regarding adverse events related to inaccurate test results due to human anti-biotin interference or human anti-streptavidin interference

[15] . Human anti-streptavidin interference and human anti-biotin interference have been reported in the literature, but it is difficult to detect and confirm these specific interference mechanisms or to distinguish them from biotin interference.

[0008] Pretreatment of a sample using a binding surface (i.e., magnetic beads, non-magnetic beads, nanoparticles, microtiter plates / wells, cuvettes, slides, sensors, chips, rods, filters, membranes, tubes, or any other solid phase used to process a sample) that is immobilized or covalently attached to capture a partial or interfering specific target can be used to deplete, concentrate, and / or characterize sample interference or biomarkers prior to an assay to improve the accuracy of assay results

[27] .

SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

[0009] Most IVD assays utilize streptavidin-biotin binding. These assays are subject to heterophilic interference from free biotin and from agents that compete with or otherwise interfere with the binding between assay reagents and streptavidin or biotin, including anti-streptavidin and anti-biotin antibodies. Substances that interfere with the binding between assay reagents and streptavidin or biotin are referred to herein as anti-biotin or anti-streptavidin, whether the substance is an antibody or not. Reagents (beads) are disclosed herein that can be used 1) to detect or quantify these different types of interference and 2) to remove or deplete interfering substances that may be present in the assay reagents, sample, or reaction mixture so as to obtain more accurate assay results. Methods for making and using these reagents are also provided.

[0010] Some of the reagents disclosed herein include nanoparticles coated with streptavidin to form streptavidin beads. In some embodiments, some or all of the streptavidin is covalently bound to the nanoparticles. In some embodiments, the nanoparticles are magnetic to facilitate separation of the beads from the preservation solution and the processed sample, assay reagents, etc. In some embodiments, the nanoparticles may or may not be magnetic, and separation of the beads from the preservation solution or the processed sample is achieved by precipitation (such as centrifugation) or filtration. Other embodiments include free (or soluble) streptavidin. In embodiments of either type of free or coated beads, the streptavidin is saturated (or quenched) with a minimal excess of biotin so that the streptavidin cannot crosslink between biotinylated assay reagent molecules and become a heteroaffinity interference source. Saturation means that all accessible biotin-binding sites on the streptavidin are occupied by biotin. Free biotin-saturated streptavidin is suitable for use, for example, as an anti-streptavidin heteroaffinity interference blocking reagent that can be added (if present) to the assay reaction mixture. Biotin-saturated streptavidin-coated beads are suitable for use, for example, as an anti-streptavidin heteroaffinity interference washing reagent that can be added to the biological fluid or extract (sample) to be assayed and then removed before being added to the assay reaction mixture. In some applications, the washing reagent can be removed in addition to the assay reagent or partial assay reaction mixture before completing the assay reaction mixture and starting the assay reaction.

[0011] Embodiments using streptavidin are described throughout this disclosure. However, additional embodiments are contemplated that include alternatives such as avidin, deglycosylated avidin (neutravidin), CaptAvidin, monomeric avidin, etc. Natural and recombinant forms of streptavidin, as well as alternatives thereto, are also contemplated. These reagents can be referred to as means for binding biotin or means for binding anti-streptavidin interference.

[0012] Embodiments that utilize biotin to quench or saturate the streptavidin active biotin-binding site are described throughout this disclosure. However, biotinylated agents such as biotin-PEG n -COOH and biotin-PEG n -CH3 and biotin-PEG n -OH, or additional embodiments using other biotin-R-(non-reactive end chemistry) where R is a carbon chain or a ring structure are also contemplated. Biotin and these modified forms of biotin may be referred to as means for these reagents to bind to biotin or means for binding to anti-biotin interference.

[0013] In further embodiments, streptavidin-coated beads, biotin-saturated streptavidin-coated beads, streptavidin, or quenched streptavidin are modified by conjugating to one or more additional capture moieties (in addition to anti-streptavidin interference) to remove other heteroaffinity or cross-reactivity interferences. In one aspect of these embodiments, the additional capture moiety is conjugated to the biotin-saturated streptavidin-coated beads and is further suitable for use as an anti-biotin heteroaffinity interference wash reagent. In a further aspect of these embodiments, the additional capture moiety is a protein such as ruthenium (element), luminol, acridinium ester, ABEI or cyclic ABEI (organic small molecule such as biotin), or signal-generating enzyme (e.g., alkaline phosphatase or horseradish peroxidase), streptavidin, antibody (e.g., antibody from a non-human species), or antigen. In still further aspects, the capture moiety may be any non-antibody peptide or protein. In all of these examples, the additional capture moiety renders the streptavidin-coated beads or biotin-saturated streptavidin-coated beads suitable for use as a wash reagent to remove or deplete heteroaffinity or cross-reactivity interferences associated with the conjugate molecule. Some embodiments include one or more capture moieties. Some embodiments do not include one or more capture moieties. For example, in some embodiments, the additional capture moiety is not biotin.

[0014] To achieve conjugation to streptavidin (soluble, or bead-bound, biotin-saturated or unsaturated), several chemicals are available. Conjugation can proceed using amine-reactive reagents to form a bond with streptavidin, typically a primary amine such as an ester, for example, an NHS-modified compound or protein. Alternatively, the primary amines of streptavidin may be thiolated. Standard thiolation reagents are known in the art and include succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC) and succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Next, conjugation can be carried out using a thiol or sulfhydryl-reactive reagent, such as a maleimide-modified compound or protein. In another alternative, the primary amines of streptavidin are reacted with maleimide using a standard ester-maleimide heterobifunctional crosslinker. Next, conjugation can be carried out using a thiol or sulfhydryl-modified (or containing) compound or protein. These chemical reactions and related reagents may be referred to as means of conjugation, and the reaction itself may be referred to as a step of conjugation.

[0015] Typically, conjugation of an additional capture moiety to streptavidin-coated beads, biotin-saturated streptavidin-coated beads, streptavidin, or quenched streptavidin involves the use of a heterobifunctional linker. The functional group at one end of the linker forms a covalent bond to streptavidin, and the functional group at the other end forms a covalent bond to the additional capture moiety. The chemical nature of specific functional groups is described below. In some embodiments, the capture moiety attached to the linker may be commercially available. Some embodiments specifically include a particular functional group or set of functional groups. Some embodiments do not specifically include a particular functional group or set of functional groups. In some embodiments, the central portion of the heterobifunctional linker includes polyethylene glycol (PEG) or polyethylene oxide (PEO). In aspects of this embodiment, the linker is, for example, PEG n or PEO nIt may contain multiple units of PEO or PEG such as, and n is an arbitrary integer from 1 to 36. In a further aspect, instead of being a linear monofunctional PEG, the PEG linker may be branched or dendritic, such as monodisperse PEG, trifunctional PEG, 4-arm PEG, 8-arm PEG, heterobifunctional PEG, homobifunctional PEG, etc. Other linkers are disclosed below.

[0016] Various methods of conjugating biotin to biotin-saturated streptavidin are disclosed herein below. However, any other capture reagent may be similarly conjugated to streptavidin (biotin-saturated or non-biotin-saturated, bead-bound or non-bead-bound).

[0017] In an alternative embodiment, the additional capture moiety is not covalently bound but is bound using a biotin linker. In some embodiments, the additional capture moiety is biotin and the linker has biotin molecules at each end, for example, biotin-PEG n -biotin. In such embodiments, the bis-biotin linker is added as a small proportion of the biotin used in the streptavidin saturation procedure (to avoid cross-linking between beads). Thus, the biotin at one end binds to streptavidin and the biotin at the other end is free to serve as a capture moiety. In other embodiments, a linker having biotin at one end and any other capture moiety at the other end, for example, biotin-(PEO)n-ruthenium, is used. In further embodiments, two or more different capture moieties can be introduced via this approach, such as co-coating streptavidin with, for example, biotin-(PEO)n-ruthenium and biotin-(PEO)n-alkaline phosphatase. In these embodiments, cross-linking between beads should not be a problem, so a potentially larger proportion of the biotin used in the streptavidin saturation procedure can be the biotin linked to the capture moiety. However, steric considerations based on the size of the capture moiety and the length of the linker can be limiting factors.

[0018] One embodiment is a method of reducing interference in a liquid biological sample. Other embodiments are methods of reducing interference in a diagnostic assay. In some embodiments, biotinylated streptavidin (biotin quenched streptavidin, QSAv) is combined with a liquid biological sample and mixed to form a mixture that promotes the binding of interference to streptavidin to block or reduce interference. Some embodiments further include performing a diagnostic assay. In some embodiments, the combining and mixing are performed prior to the analysis stage of the assay. As used herein, the term "analysis stage of the assay" begins when the sample is mixed with reagents to obtain or detect an analyte and / or generate a signal that indicates or quantifies the presence of the analyte, and continues throughout the measurement of the signal.

[0019] In other embodiments (for reducing or minimizing interference), particles comprising streptavidin (whether or not biotin quenched) are combined with a liquid biological sample and mixed to form a mixture that promotes the binding of interference to streptavidin, and the particles are separated from the sample to remove or reduce interference. Some embodiments further include performing a diagnostic assay. In some embodiments, the combining, mixing, and separation are performed prior to the analysis stage of the assay. In one aspect of these methods, the particles are magnetic, and separating the particles from the sample comprises exposing the mixture to a magnet and recovering the liquid sample. In a further aspect, the sample is not diluted and there is little or no loss of the sample.

[0020] In some embodiments of these methods for reducing or minimizing interference, the sample is used in a sandwich immunoassay. In other embodiments, the sample is used in a competitive immunoassay.

[0021] One embodiment is a method of making quenched streptavidin. Some of these embodiments include exposing streptavidin to a minimal molar excess of free biotin. In one aspect, this can 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 aspect, this can include diafiltration. Some embodiments include blocking streptavidin to avoid aggregate formation. Some embodiments include conjugating an additional capture moiety to the quenched streptavidin. Some embodiments include quenched streptavidin made by any of these methods.

[0022] Some embodiments are methods of making particle-conjugated streptavidin. Some of these embodiments include exposing the particle-conjugated streptavidin to a minimal molar excess of free biotin. In one aspect, this can include metered addition to combine a biotin solution with a particle-conjugated streptavidin suspension. Some of these embodiments include washing the quenched streptavidin with hot water. In one aspect, this can include magnetic separation of the particles. In other aspects, this can include separation of the particles by filtration or precipitation. Some embodiments include conjugating an additional capture moiety to the particle-conjugated streptavidin, whether or not biotin quenched. Some embodiments include particle-conjugated streptavidin made by any of these methods, whether or not biotin quenched. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [Figure 1] FIG. 1 shows the size distribution of biotinylated 100BS streptavidin beads. This data shows a single peak of uniform size at 883.9 nm with a polydispersity index of 12.2%.

[0024] [Figure 2A]Figures 2A - B show the size distribution of biotinylated 100BS streptavidin beads after incubation with streptavidin for 30 minutes (2A). Bead aggregation occurred at peaks of 1,441.3 nm and 6,641 nm, and the polydispersity index was 173.3%. Also shown is (2B) the size distribution of biotinylated 100BS streptavidin beads after incubation with streptavidin for 4 hours. Bead aggregation occurred at peaks of 1,512.6 nm and 14,536 nm, and the polydispersity index was 242.8%. [Figure 2B] Figures 2A - B show the size distribution of biotinylated 100BS streptavidin beads after incubation with streptavidin for 30 minutes (2A). Bead aggregation occurred at peaks of 1,441.3 nm and 6,641 nm, and the polydispersity index was 173.3%. Also shown is (2B) the size distribution of biotinylated 100BS streptavidin beads after incubation with streptavidin for 4 hours. Bead aggregation occurred at peaks of 1,512.6 nm and 14,536 nm, and the polydispersity index was 242.8%.

[0025] [Diagram 3] Figure 3 shows the size distribution of biotinylated 100BS streptavidin beads after incubation with monoclonal anti - biotin conjugated antibody overnight. Bead aggregation occurred at a peak of 2,148 nm, and the polydispersity index was 316.2%.

[0026] [Figure 4A]Figures 4A - C show the (4A) SEC - HPLC standard curve of the anti - biotin antibody. The data points indicated by the arrows correspond to the peak area and the remaining amount of anti - biotin antibody (μg / mL) in the sample after pretreatment with biotinylated 100BS streptavidin beads to deplete the anti - biotin antibody. 4B - C show the SEC - HPLC analysis of the anti - biotin antibody (4B) before depletion and (4C) after depletion using biotinylated 100BS streptavidin beads. After depletion of the anti - biotin antibody, the peak area decreased from 1,384 to 318, and the anti - biotin concentration decreased from 205 μg / mL to 44.62 μg / mL. [Figure 4B] Figures 4A - C show the (4A) SEC - HPLC standard curve of the anti - biotin antibody. The data points indicated by the arrows correspond to the peak area and the remaining amount of anti - biotin antibody (μg / mL) in the sample after pretreatment with biotinylated 100BS streptavidin beads to deplete the anti - biotin antibody. 4B - C show the SEC - HPLC analysis of the anti - biotin antibody (4B) before depletion and (4C) after depletion using biotinylated 100BS streptavidin beads. After depletion of the anti - biotin antibody, the peak area decreased from 1,384 to 318, and the anti - biotin concentration decreased from 205 μg / mL to 44.62 μg / mL. [Figure 4C] Figures 4A - C show the (4A) SEC - HPLC standard curve of the anti - biotin antibody. The data points indicated by the arrows correspond to the peak area and the remaining amount of anti - biotin antibody (μg / mL) in the sample after pretreatment with biotinylated 100BS streptavidin beads to deplete the anti - biotin antibody. 4B - C show the SEC - HPLC analysis of the anti - biotin antibody (4B) before depletion and (4C) after depletion using biotinylated 100BS streptavidin beads. After depletion of the anti - biotin antibody, the peak area decreased from 1,384 to 318, and the anti - biotin concentration decreased from 205 μg / mL to 44.62 μg / mL.

[0027] [Figure 5-1]Figures 5A - D are chromatograms before and after the HPLC - SEC depletion assay using biotinylated 100BS streptavidin beads. 5A shows the absence of depletion of affinity - purified goat IgG by the beads. 5B shows the absence of depletion of biotinylated affinity - purified goat IgG by the beads. 5C shows the depletion of goat anti - biotin Ab by the beads. 5D shows the depletion of goat anti - streptavidin by the beads. In all cases, the profiles for untreated and treated are indicated by labeled arrows. [Figure 5-2] Figures 5A - D are chromatograms before and after the HPLC - SEC depletion assay using biotinylated 100BS streptavidin beads. 5A shows the absence of depletion of affinity - purified goat IgG by the beads. 5B shows the absence of depletion of biotinylated affinity - purified goat IgG by the beads. 5C shows the depletion of goat anti - biotin Ab by the beads. 5D shows the depletion of goat anti - streptavidin by the beads. In all cases, the profiles for untreated and treated are indicated by labeled arrows.

[0028] [Figure 6] Figure 6 shows the amounts of serum parathyroid hormone detected by ELISA using various interfering substances and treated and untreated with biotinylated 100BS streptavidin beads. None - no interfering substance; Biotin -> 250 ng / mL; Anti - biotin IgG - 16.5 μg / mL of Ab; Anti - SAv IgG 16.5 μg / mL of Ab; Anti - biotin IgG / SAv IgG - 8.25 μg / mL of each Ab.

[0029] [Figure 7] Figure 7 shows an apparatus for quantitatively adding biotin to streptavidin in a saturation procedure.

[0030] [Figure 8] Figure 8 shows an apparatus for diafiltration, washing, and concentrating biotin - saturated streptavidin.

Best Mode for Carrying Out the Invention

[0031] Despite existing approaches to the identification or depletion of compounds that cause assay interference, there remains a clinical need for a rapid and easy-to-use product solution for detecting and reducing anti-biotin and anti-streptavidin interference in patient samples. Such a product solution would also facilitate prevalence studies and help clinicians and laboratory medicine specialists better understand which patients and patient populations are most at risk from these interferences.

[0032] There is also a clinical need to reduce anti-streptavidin interference in patient samples by using a blocking reagent specific for anti-streptavidin interference in assay formulations. This product solution would also minimize the impact on laboratory workflows as diagnostic test designs would result in reduced anti-streptavidin interference.

[0033] Immunoassays are susceptible to the effects of interferences that can lead to false high or low level reporting of the analyte being assayed. One type of interference is associated with 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 special reagents. The embodiments disclosed herein do not address such signal generation / observation interferences and the general reference to interference herein does not include such interferences.

[0034] Another type of immunoassay interference relates to analyte capture and physical detection. These include interferences that inhibit the interaction between the analyte and the capture or detection reagent, or that result in the association of the capture and detection reagents regardless of the presence (or absence) of the analyte. This type of interference is referred to as heteroaffinity interference, and as used herein, "interference" should be understood to mean heteroaffinity interference unless the context indicates otherwise. The embodiments disclosed herein address various specific heteroaffinity interferences. Generally, heteroaffinity interferences cannot be directly observed, nor can their presence be easily demonstrated with standard assay reagents. Some of the embodiments disclosed herein include things that can be used to indicate or quantify the presence of specific heteroaffinity interferences. Heteroaffinity interferences include, in addition to biotin, antibiotin, antistreptavidin, and anti - heterologous antibody interferences, interferences that bind to components of the assay signal - generating system (enzymes, phosphors, etc.). Anti - heterologous antibody interferences include human antibodies that recognize immunoglobulins of mouse, rat, rabbit, sheep, cow, and / or goat.

[0035] Another type of immunoassay interference is related to cross - reactive antibodies. Cross - reactivity between antigens occurs when an antibody directed against a particular antigen succeeds in binding to another, 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, for example, in immunoassays aimed at detecting antibodies that recognize antigens derived from a particular bacterial or viral strain. Such assays are commonly used to determine whether a subject has been exposed to (infected with) a problematic pathogen or drug. If the subject has been previously exposed to a related strain, there is a possibility that the subject has antibodies that cross - react with the antigen from the strain that the assay is intended to detect, thus producing a false - positive result.

[0036] As used herein, "immunoassay" generally refers to an assay in which the detection or quantification of an analyte uses an antibody (or an antigen-binding fragment or derivative thereof) that specifically binds to the analyte. However, it is also possible to design an assay in which a non-antibody agent that can specifically bind to the analyte is used in a manner similar to an anti-analyte antibody. In some embodiments, the non-antibody agent that can specifically bind to the analyte is an aptamer or a molecularly imprinted polymer. Thus, in some embodiments, "immunoassay" can include an assay in which a non-antibody agent provides analyte-specific binding activity typically provided by an antibody. Various embodiments specifically include or do not include an antibody or a non-antibody agent as analyte-specific binding activity. Some embodiments specifically include or do not include an aptamer or a molecularly imprinted polymer. Immunoassays can be classified into classes based on the technical and physical arrangement of the components and the assay format. In one class, it involves combining a detection and / or signal generation reagent in a container, such as a microtube or microtiter plate, where the assay reaction occurs, with a sample (which may contain the analyte). The reagent can be added to or removed from the container during the course of the assay. (In some variations, a portion of the assay components is removed from the first container and added to a second container where the assay proceeds.) Such assays are referred to herein as "pot" assays. In another class, a portion of the detection and / or signal generation reagent is immobilized in a specific region of a solid substrate or matrix (e.g., a membrane). The sample (potentially containing the analyte) is applied to a specific location on a device containing the solid substrate or matrix and, for example, by lateral flow, often passes through and encounters the immobilized reagent within the region where the reagent is immobilized. Additional assay reagents move with the mobile phase. Such assays are referred to herein as "zone" assays.From the point when a sample is added to a container in which an assay reaction for a pot assay is carried out, or from the point when a sample is added to a specific position of a solid substrate for a zone assay or a device containing a substrate, the entire measurement of the generated signal is referred to as the analysis stage of the assay. In many embodiments, the interference washing or blocking reagents disclosed herein are added to the sample and removed from the sample before the analysis stage for the washing reagent. That is, in these embodiments, the interference reduction reagent is used as a "pretreatment".

[0037] Patients who consume high doses of biotin for health and beauty (5,000 - 20,000 mcg per day) or therapeutically (100,000 - 300,000 mcg per day) may have circulating biotin levels of up to 1,000 ng / mL or more in the blood, depending on the elapsed time since biotin intake, the patient's individual biotin clearance time, and if the patient has a kidney disease or renal dysfunction that may impair biotin clearance and increase biotin's circulating levels. If the patient's free biotin has not yet been removed below the assay-specific biotin interference threshold before a blood, serum, or plasma sample is taken, or before a urine sample is collected, biotin in the sample that exceeds the assay-specific biotin interference threshold competes for and binds to an anti-biotin capture moiety (i.e., streptavidin, avidin, neutravidin, monomeric avidin, CaptAvidin, or an antibody / antibody fragment / Fab / F(ab)'2, aptamer, and molecularly imprinted polymer specific for biotin), which then interferes with the binding of a biotinylated antibody, protein, or antigen used in the assay formulation. This results in a false low assay signal and, depending on the assay format, a false low dose (sandwich assay) or false high dose (competitive inhibition assay).

[0038] Streptavidin is a protein of approximately 52,000 - 55,000 KDa and is composed of four identical polypeptide chains. As used herein, monomeric streptavidin refers to the non-aggregating streptavidin protein and not to the dissociable streptavidin polypeptide chains. The binding of biotin to streptavidin (10 -14 or 10 -15 mol / L as variously reported in the literature) is one of the strongest non-covalent interaction known in nature. Recombinant streptavidin is a suitable tool for enabling universal assay systems in immunology and molecular diagnostics and is commonly used in diagnostic assays such as immunoassays for capturing biotinylated antibodies, proteins, and antigens, or for attaching various biomolecules to each other or onto solid supports such as microplates, beads, and microarrays. The use of streptavidin allows assay developers to utilize the proven anti-biotin delayed capture assay format for improving assay kinetics, accuracy, and sensitivity, while facilitating the shortening of assay incubation times and turnaround time (TAT) for STAT assays.

[0039] When the sample contains interference with specificity for streptavidin, anti-streptavidin interference can sterically inhibit or weaken the ability of a conjugate biotin to bind to the biotin-binding site of streptavidin by binding to streptavidin or its polypeptide chain. If streptavidin can no longer freely bind to a biotinylated antibody, protein, or antigen used in the test design or assay format, similar to biotin interference, anti-streptavidin interference will result in a false low assay signal and can result in a false low dose (sandwich assay) or false high dose (competitive inhibition assay). Similarly, when the sample contains interference with specificity for biotin, anti-biotin interference can sterically inhibit or weaken the ability of a conjugate biotin to bind to the biotin-binding site of streptavidin by binding to biotin. If the biotin is derived from, for example, a biotinylated antibody, protein, or antigen used in the test design or assay format, anti-biotin interference will result in a false low assay signal and can result in a false low dose (sandwich assay) or false high dose (competitive inhibition assay). Some embodiments address anti-streptavidin interference. Some embodiments address both anti-streptavidin interference and anti-biotin interference.

[0040] The biotin-streptavidin interaction is commonly used in the capture portion of immunoassay mechanisms, whereas heteroaffinity interference can also occur through interaction with common detection components. Such components can include fluorophores such as fluorescein or elemental ruthenium, chemiluminescent agents such as luminol, acridinium esters, ABEI, and cyclic ABEI, bioluminescent agents such as luciferin, and enzymes such as alkaline phosphatase or horseradish peroxidase. Interference that binds to these signal-generating molecules can cause cross-linking between the detection antibody (or other detection reagent) bound to the analyte and the detection antibody (or other detection reagent) that does not result in a false high signal. Some embodiments address both anti-streptavidin interference and anti-signal generating molecule interference.

[0041] Immunoassays typically utilize antisera, polyclonal antibodies, or monoclonal antibodies derived from non-human species. Serum or other assay samples may contain interference that recognizes these heterologous antibodies (sometimes referred to as human anti-animal antibodies (HAAA)). In particular, humans produce antibodies against a wide variety of animal species. Generally, these are the most interacting species such as mice, cows, horses, dogs, cats, goats, rabbits, and sheep. Among them, antibodies derived from mice, goats, rabbits, and sheep, especially IgG, are very commonly used in clinical immunoassay systems. However, in samples from non-human subjects, similar heteroaffinity interferences may occur. Such anti-antibody interferences can cause cross-linking between the capture antibody and the detection antibody in the absence of the bound analyte, or between the detection antibody bound to the analyte and the detection antibody not so bound, resulting in false high or false low signals. Some embodiments address both anti-streptavidin interference and anti-heterologous antibody interference.

[0042] There are two modes for dealing with interference, blocking, and washing in immunoassays. As used herein, a blocking reagent is present during the assay reaction and by its interaction with interfering substances, prevents or reduces interference. Some embodiments include or utilize soluble biotinylated streptavidin and are suitable for blocking anti-streptavidin interference. In some embodiments, the soluble biotinylated streptavidin is conjugated with a second molecule that is the target to be bound by an interfering substance such as, for example, biotin, a signal generating molecule, or a heterologous antibody. Embodiments that include or utilize soluble biotinylated streptavidin conjugated with a second interfering target molecule are suitable for blocking both anti-streptavidin interference and anti-second molecule interference. Some embodiments specifically include one or more genera or species of the second interfering target molecule. Some embodiments do not specifically include one or more genera or species of the second interfering target molecule. The blocking reagent may be added during the analysis stage of the assay or added at a pre-analysis stage and remain during the analysis stage. In some embodiments, the blocking reagent may also be encountered during the analysis stage of a zone assay such as a lateral flow assay or may be retained in a particular zone of such an assay. As used herein, the analysis stage of an assay refers to the temporal and / or physical part of the assay or assay system where capture, detection, and quantification of the analyte occur.

[0043] It should be noted that the terms "block" and "blocking" are conceptually related but are used in more than one sense in this specification. In the preparation of the reagents disclosed herein, "blocking", etc., is used to account for the inhibition, or reduction in another way, of the reactivity of chemical reaction sites and the effective affinity of specific and / or non-specific binding sites. This may be referred to as preparation blocking. Thus, the surfactants and polymeric blocking reagents used herein, which are used to prevent reaction and / or non-specific binding to streptavidin-coated beads or the core nanoparticles of protein assemblies disclosed herein, are related to "block" and "blocking" in this sense. The saturation of streptavidin with biotin can also be considered a form of preparation blocking, where biotin is considered a blocking reagent. Preparation blocking should not be confused with blocking that prevents or reduces assay interference, which is a separate function.

[0044] As used herein, a wash reagent is added to serum or other biological samples (or other components of an immunoassay reaction mixture), and then removed from the sample or other components before the components of the immunoassay reaction mixture are combined and mixed. That is, the wash reagent is used and removed during the pre-analytical stage of the assay and is not present during the analytical stage. Interference is prevented or reduced by depleting or removing interfering substances from the sample and / or other assay reagent interferences. Some embodiments include or utilize biotin-saturated streptavidin beads, i.e., biotin-saturated streptavidin coated on magnetic nanoparticles. Such biotin-saturated streptavidin beads are suitable for washing streptavidin interference. In some embodiments, the biotin-saturated streptavidin of the beads is conjugated with a second molecule that is bound by an interfering substance such as, for example, biotin, a signal generating molecule, a heterologous antibody, or an antigen. Embodiments that include or utilize biotin-saturated streptavidin beads in which streptavidin is conjugated with a second interfering target molecule are suitable for washing both anti-streptavidin interference and anti-second molecule interference. Some embodiments specifically include one or more genera or species of the second interfering target molecule. Some embodiments do not specifically include one or more genera or species of the second interfering target molecule.

[0045] The biotin-saturated streptavidin beads are magnetically separated from their storage buffer, and after the storage buffer is removed, the sample or reagent can be washed and added to the beads so that the sample or reagent is not diluted during the washing process, unlike the use of a soluble blocking reagent. In other embodiments, the beads are separated from the fluid phase by filtration or precipitation.

[0046] Assays that use streptavidin in the assay design, format, or formulation cannot simply use native streptavidin as a specific blocker, additive, or component in the assay buffer to reduce anti-streptavidin interference in the sample. When used as a blocker in the test, streptavidin can also compete with and bind to biotinylated antibodies, proteins, oligomers, or antigens, generating false low assay signals and resulting in false low doses (sandwich assays) or false high doses (competitive inhibition assays). This is a particular concern with streptavidin because it has a very strong binding constant and affinity for biotin. Some assays can reduce lower titers or concentrations of anti-streptavidin interference by increasing the total amount or concentration of streptavidin used in the assay, but this is assay-specific and assay format-specific and increases the cost of the assay. This may not work if the sample contains high titers or high levels of anti-streptavidin interference that exceed the assay-specific streptavidin interference threshold.

[0047] The interference blocking reagent disclosed herein is based on biotinylated streptavidin, also known as quenched streptavidin (QSAv). QSAv should be mainly non-aggregating, i.e., it should be based on monomeric streptavidin protein. In some embodiments, the mainly non-aggregating QSAv has <1% dimer or <5% aggregation by size exclusion chromatography HPLC, and the observed average molecular weight of the monomer peak is 52 - 55 KD. In other embodiments, QSAv is at least 80, 90, 95, 97, 98, 99% monomer, or any range bounded by those values. In some embodiments, streptavidin is blocked with, for example, a surfactant or a polymeric blocking reagent to maintain it in a monomeric non-aggregating state. Anti-streptavidin interference can be blocked using QSAv. In some embodiments, streptavidin may be modified with one or more additional capture moieties before, during, or after biotinylation. The capture moiety may be covalently attached to streptavidin before or after the biotinylation process. Alternatively, the capture moiety may be biotinylated and attached to streptavidin via a biotin-avidin bond before or during the biotinylation process. However, if the additional capture moiety is biotin (e.g., achieved through the use of a bis-biotin linker), this must bind to streptavidin in the presence of excess free biotin, i.e., during saturation. Embodiments containing streptavidin modified with one or more additional capture moieties can be used to block anti-streptavidin interference by an agent that binds to the one or more capture moieties.

[0048] The interference washing reagent disclosed in this specification is based on streptavidin being conjugated to microparticles (or nanoparticles) to form streptavidinylated beads. The use of beads, particularly magnetic beads, facilitates the washing of samples or assay reagents without loss or dilution. In some embodiments, streptavidin is saturated with biotin, while in other embodiments it is not. Embodiments containing biotin-saturated streptavidin can be used as a washing reagent to remove or reduce anti-streptavidin interference. Embodiments containing streptavidin that is not saturated with biotin can be used as a washing reagent to remove or reduce both biotin interference and anti-streptavidin interference. In some embodiments, streptavidin may be modified with one or more additional capture moieties before, during, or after biotin saturation. The capture moiety may be covalently attached to streptavidin before or after the biotin saturation process. Alternatively, the capture moiety may be biotinylated and attached to streptavidin via a biotin-avidin bond before or during the biotin saturation process. Embodiments containing streptavidin modified with one or more additional capture moieties can be used to block anti-streptavidin interference by agents that bind to the one or more capture moieties. The one or more additional capture moieties may include biotin in those embodiments that utilize biotin-saturated streptavidin.

[0049] The additional capture moiety may be any substance that causes interference with heteroaffinity or cross-reactivity, except when streptavidin is not saturated with biotin, in which case the additional capture moiety cannot be biotin. In some embodiments, the additional capture moiety is ruthenium (element), luminol, acridinium ester, ABEI or cyclic ABEI (organic small molecule such as biotin), or a signal generating enzyme (e.g., alkaline phosphatase or horseradish peroxidase), streptavidin, an antibody (e.g., an antibody from a non-human species), or a protein such as an antigen. In some embodiments, the antigen is a plateau that can be recognized by an antibody that can cross-react with the antigen used as the capture moiety in an immunoassay. In various embodiments, the washing or blocking reagent, or the antigen used as the capture moiety in the assay, is an allergen, an antigen derived from a pathogen, or an antigen associated with a disease or disorder, a herpes simplex virus antigen, and an autoimmune substance such as cardiac troponin I or TSH that has known autoantibody interference problems. In some embodiments, the antigen derived from a pathogen is a viral antigen, a bacterial antigen, or a protozoal antigen. In some embodiments, the capture moiety removes cross-reactive antibodies to coronaviruses other than MERS virus, SARS virus, or SARS-CoV-2. Some embodiments specifically include one or more of these genera or species of capture moieties. Some embodiments specifically exclude one or more of these genera or species of capture moieties.

[0050] Biotin linkers, or conjugated biotins, can be constructed or purchased using different linker types and lengths, as well as different functional groups for the covalent attachment of biotin to antibodies, antibody fragments, peptides, oligomers, antigens, and small molecules (conjugated biotin). Common linkers and functional groups used with biotin (e.g., NHS ester, TFP ester, hydrazide, maleimide, thiol, etc.) include NHS-biotin, NHS-LC-biotin, TFP-LC-biotin, NHS-chromalink-biotin, NHS-PEO4-biotin, NHS-(PEO) n -biotin, TFP-(PEO)n - Biotin, hydrazide-biotin, hydrazide-LC-biotin, hydrazide-PEO4-biotin, maleimide-(PEO) n - Biotin, and SH-(PEO) n - Biotin. Biotin labeling 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, desthiobiotin, sulfhydryl-reactive, tetrazine ligation, biotin alcohol, bis-biotin-PEG, and D-biotin-PEG-salidomide.

[0051] If the sample contains interference with the specificity for biotin, anti-biotin interference can bind to the conjugated biotin used in the assay design or assay format and sterically inhibit or impede the accessibility of the conjugated biotin, and can bind to the streptavidin solid phase or other anti-biotin capture moieties. When the conjugated biotin cannot bind freely to the anti-biotin capture moiety, similar to biotin interference and anti-streptavidin interference, anti-biotin interference can generate false low assay signals and result in false low doses (sandwich assays) or false high doses (competitive inhibition assays).

[0052] Assays that use biotin conjugates in the assay design, format, or formulation cannot simply use biotin or conjugated biotin as a specific blocker, additive, or component in the assay buffer to reduce avidin interference in the sample. When used as a blocker in an assay, biotin can also compete with and bind to streptavidin used in the assay, generating a false low assay signal and resulting in a false low dose (sandwich assay) or false high dose (competitive inhibition assay). Low concentrations of biotin below the test-specific biotin interference threshold can be used to block avidin interference, but this can be problematic when the patient sample contains a combination or sum of sample biotin (endogenous biotin) and test biotin (biotin as a blocker) that exceeds the test-specific biotin interference threshold and includes biotin interference close to the interference threshold, which can result in a false low assay signal and false low dose (sandwich assay) or false high dose (competitive inhibition assay).

[0053] Streptavidin binds biotin very rapidly and strongly (the binding constant is reported in the literature as 10 -14 or 10 -15mol / L, as variously reported). Some studies indicate the protein structural changes or cooperative binding of biotin to four binding sites [28-29], while other studies conclude that there is no cooperative binding to biotin bound to the four subunits of the tetramer [30-31]. When streptavidin is exposed to a molar excess of free biotin, a very rapid and strong binding interaction of biotin to all four binding sites occurs, resulting in 100% biotin saturation (100BS) at all biotin binding sites. Due to having the strongest non-covalent binding interaction known in nature and a very slow off-rate of biotin from streptavidin under normal physiological conditions and pH, 100BS streptavidin has a very low potential to bind additional biotin or conjugated biotin such as biotinylated antibodies, proteins, oligomers, and antigens in diagnostic assays. As used herein, saturation refers to the blocking of biotin binding sites on streptavidin by biotin. This is a covalent bond of biotin to streptavidin rather than biotinylation. Saturated streptavidin can bind anti-streptavidin substances but does not bind or cross-link biotin-containing substances. Biotin-saturated streptavidin may also be referred to as quenched streptavidin (QSAv).

[0054] Streptavidin can be saturated with biotin (i.e., D-biotin) to prepare 100BS streptavidin as a blocking agent, and anti-streptavidin interference can be reduced and managed. In other embodiments, quenching of the streptavidin active biotin-binding site can alternatively be achieved by exposing streptavidin to a soluble biotinylating agent such as biotin-PEG(n)-COOH or biotin-PEG(n)-CH3 or biotin-PEG(n)-OH, or other biotin-R-(non-reactive end chemical reaction) where R is a carbon chain or a cyclic structure. Saturation involves exposure of 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, or 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 to all of the saturated biotin constituting the described molar ratio in a single batch. In other embodiments, saturation proceeds through repeated batches, each containing a fraction of the total biotin, and the sum of the batches constitutes the described molar ratio. For example, instead of a single batch with a ratio of 6:1, three repeated batches with a biotin:streptavidin ratio of 2:1 can be used, and the biotin:streptavidin ratio does not need to be the same in each repeated batch. In some embodiments, the biotin solution and the streptavidin solution are combined by quantitative addition via a Y-shaped connector. In some embodiments, there is an in-line mixer within the tube connected to the outlet of the Y-shaped connector to ensure rapid, immediate, and complete mixing. The combination of pump speed and tube length can be used to determine the total interaction time in the saturation process. In some embodiments, 9 volumes of the biotin solution are combined with 1 volume of the streptavidin solution. In some embodiments, the streptavidin and biotin solutions are prepared in Tris-buffered saline, pH 8.5.In one embodiment, the initial streptavidin concentration is in the range of 0.1 to 10.0 mg / mL, and the resulting QSAv solution has a streptavidin concentration in the range of 0.01 to 1.0 mg / mL, preferably 0.02 to 0.05 mg / mL. Such conditions promote saturation of the biotin binding sites and reduce non-specific binding of biotin to streptavidin.

[0055] In some embodiments, the QSAv is then washed with a series of hot buffers by repeating the concentration and re-dilution of the QSAv, for example, using diafiltration in a hollow fiber filter. The wash is used to remove excess and non-specifically bound biotin so that it does not act as an interfering source when the QSAv is used as an interference blocking reagent. In some embodiments, 4 to 6 or more washes, for example 5 washes, are used, followed by a final concentration step to reduce the volume to reach the desired concentration, for example 0.1 to 30 mg / mL, or 1 to 10 mg / mL. In one aspect, the volume can be reduced to 5 to 20% of the original volume of the QSAv solution, for example 10%. In some embodiments, the temperature of the hot wash is 15°C to 60°C, preferably 40°C to 55°C, 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 wash buffer has an NaCl concentration in the range of 10 to 500 mM, or 20 to 150 mM, or 25 to 75 mM. In some embodiments, the buffer is 10 mM Tris, 50 to 150 mM NaCl. After the wash and final concentration steps, the free biotin concentration should be <1200 pg / mL, for example, <1000, <800, <700, or <600 pg / mL. In some embodiments, the washed QSAv solution contains 1 to 6 pg of free biotin / μg of streptavidin. In some embodiments, the effluent from the final diafiltration is combined by quantitative addition with PBS and appropriately concentrated to provide QSAv in PBS. In some embodiments for washing the sample, the volume of QSAv is added to a 400 μl sample such that the volume contains <480 pg of free biotin.

[0056] In some embodiments, the effluent from the saturation process is collected for later washing, and in other embodiments, the effluent from the saturation process is fed directly to a hollow fiber filter device. The effluent from the saturation process can be split and fed to multiple hollow fiber filters to increase the capacity and avoid excessive backpressure.

[0057] These streptavidin solutions are relatively dilute and somewhat prone to aggregation, which is a problem not encountered with bead-bound streptavidin. This is the reason for using an alkaline buffer in the saturation and washing procedures for generating QSAv. (In contrast, water is used for similar washing of bead-bound streptavidin.) Aggregation can be further mitigated by including 0.01 - 1% w / v TWEEN® 20, or another surfactant, in the wash buffer. Aggregation can be further mitigated by covalent modification with a blocking reagent for preparation, for example, by PEGylation of streptavidin, prior to saturation with biotin. Thus, in some embodiments, QSAv is blocked monomeric biotin-saturated streptavidin. In some embodiments, monomeric QSAv is <5% aggregates by size exclusion chromatography HPLC, and in other embodiments, monomeric QSAv is <1% aggregates.

[0058] In one embodiment, 100BS streptavidin (QSAv) can be used as a blocking reagent or protein blocker to target and deplete anti-streptavidin interference in a sample. 100BS streptavidin can be added to an assay buffer, a blocking buffer, or an assay component used in an assay formulation such as a detection antibody, or any combination thereof, to reduce the sensitivity of the assay to anti-streptavidin interference. In another embodiment, streptavidin is covalently attached to a microparticle-binding surface and then incubated with a molar excess of biotin to prepare 100BS streptavidin beads. 100BS streptavidin beads can be used to pretreat a sample to target and deplete anti-biotin interference prior to a diagnostic assay.

[0059] The interference cleaning reagent disclosed in this specification is based on streptavidin-conjugated beads. In some embodiments, streptavidin is quenched (saturated) with biotin after binding to the core particles. In some embodiments, streptavidin is quenched with biotin before attaching to the core particles, for example, using QSAv as described herein. In some embodiments, the core particles are magnetic. In some embodiments, the core particles have a diameter ≧500 nm, or about 0.5 micrometers, and thus may be referred to as either nanoparticles or microparticles. In some embodiments, the core particles are covalently bound to streptavidin using a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chemical reaction. To remove passively adsorbed streptavidin, prevent non-specific binding to the beads in further preparation steps, and when used as an interference cleaning reagent, the bead surface is conditioned with beads blocked using stripping reagents (salts, surfactants, low pH and high pH) and surfactant and polymer blocking reagents. This also promotes monodispersity and colloidal stability of the nanoparticles. To generate 100BS streptavidin beads, biotin quenching of streptavidin can be performed similar to the production of the above-described 10BS streptavidin (QSAv). Saturation involves exposure of bead-bound streptavidin to a molar excess of biotin. As described above, saturation of the streptavidin active biotin-binding sites can alternatively be achieved by exposing streptavidin to a soluble biotinylating agent such as biotin-Peg(n)-COOH or biotin-Peg(n)-CH3 or biotin-Peg(n)-OH, or other biotin-R-(non-reactive end chemistry) 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. Since some of the biotin-binding sites become inaccessible due to steric hindrance by the core particles, the effective molar excess of biotin is somewhat higher than this formal ratio. In some embodiments, the streptavidinylated beads are suspended and the biotin solution is composed of PBS, pH 6.8.In some embodiments, the biotin solution and the bead suspension are combined in a container and, for example, mixed at room temperature for 1 hour. In some embodiments, the streptavidinylated bead suspension and the biotin solution are combined by quantitative addition and essentially carried out as described above in the production of QSAv.

[0060] In some embodiments, the biotin-saturated streptavidinylated beads are washed at a high temperature to remove passively adsorbed biotin so that they do not leach out during use and become a source of interference. Different from the above-mentioned 100BS streptavidin, the washing of 100BS streptavidin beads can utilize filtration, precipitation, or magnetic separation as an alternative to diafiltration. The washing may also be different from that of 100BS streptavidin, which uses high-temperature alkaline (pH≧7.5) water instead of buffer for washing. The washing suspension is also sonicated. In some embodiments, the free biotin concentration in the suspension of the washed 100BS streptavidin beads is <1200 pg / mL, for example, <1000, <800, <600, <400, or <200 pg / mL. In some embodiments, the suspension of the washed 100BS streptavidin beads contains 5 - 30 pg of free biotin / μg of streptavidin. In some embodiments for washing a sample, a certain volume of 100BS streptavidin beads is added to a 400 μl sample so that it contains <480 pg of free biotin.

[0061] Free biotin (i.e., D-biotin) can be added to 100BS streptavidin or 100BS streptavidin bead storage solutions, assay buffers, or test components to improve the stability of 100BS streptavidin or 100BS streptavidin beads and ensure that 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 reagent that targets both anti-streptavidin and anti-biotin interference mechanisms using a single blocking reagent. In one embodiment, the blocking reagent can be used to pretreat a sample prior to testing to thereby block anti-streptavidin and / or anti-biotin interference mechanisms. In one embodiment, the blocking reagent can be used to block and reduce interference mechanisms during testing in a diagnostic test or assay design such as an assay buffer or reagent buffer. In one embodiment, free biotin can be added to 100BS streptavidin or 100BS streptavidin beads at a concentration within the physiological range of up to 1,100 pg / mL. In another embodiment, free biotin can be added to 100BS streptavidin or 100BS streptavidin beads at a high concentration such as 100,000 pg / mL (100 ng / mL) or 1,000,000 pg / mL (1,000 ng / mL). When 100BS streptavidin or 100BS streptavidin beads are stored in a solution containing free biotin, if there is biotin dissociating from streptavidin, due to the very strong binding constant and fast on-rate for biotin, it will be immediately replaced by another biotin from the biotin added to the storage solution, so 100BS streptavidin remains. Thus, 100BS streptavidin, or biotin-quenched streptavidin, can be used as a blocking reagent for streptavidin-based tests or immunoassays, where 100BS streptavidin is added to an assay buffer containing less than the physiological biotin concentration for stability.If any biotin dissociates from the streptavidin blocker, that biotin is replaced with the biotin added to the assay buffer, and the amount of biotin added from this assay buffer to the sample or test reaction thereafter is minimal and within the physiological biotin concentration range.

[0062] IVD companies have tested and provided in their package inserts (PIs) or instructions for use (IFUs) the biotin interference thresholds specific to the test for each assay that is susceptible to the effects of biotin interference [9, 14 - 15]. Assays with a biotin interference threshold of <51 ng / mL are considered high - risk assays, such as the Ortho Clinical Diagnostics Vitros cardiac TnI assay with a threshold of 2.4 ng / mL, or vulnerable immunoassays and competitive methods [9]. To improve stability and ensure 100% saturation over time, biotin can be added to 100BS streptavidin or 100BS streptavidin bead storage solutions. However, to reduce assay interference from the biotin added to the storage solution, the final free biotin concentration must be below the test - specific biotin interference threshold. In one embodiment, 100BS streptavidin or 100BS streptavidin beads are stored in a biotin solution with a biotin concentration within the physiological range (i.e., <1,100 pg / mL) so as not to interfere with the test. In another embodiment, 100BS streptavidin beads are stored in a biotin solution containing >1,100 pg / mL of biotin, such as 2, 5, 10, 20, 30, 50, 100, 250, or 500 ng / mL of biotin. The 100BS streptavidin beads are then separated from the sample by filtration, centrifugation, or magnetically, or a combination thereof, such that the biotin storage solution is removed from the 100BS streptavidin beads before adding the sample to the 100BS streptavidin beads. Removing the biotin storage solution immediately prior to using the 100BS streptavidin beads reduces the free biotin concentration to less than 1,100 pg / mL, less than 500 pg / mL, or preferably less than 100 pg / mL, ensuring that the free biotin concentration is below the biotin interference threshold of the test.

[0063] The primary amines (R-NH2) of 100BS streptavidin and 100BS streptavidin beads react with NHS-biotin, NHS-LC-biotin, NHS-LC-LC-biotin, NHS-chromalink-biotin, NHS-PEO 4- -biotin, and NHS-(PEO) n -biotin, TFP-(PEO) n- By using an amine-reactive biotin labeling reagent such as biotin (amine-reactive means), and by biotin conjugating in molar excess of free biotin, the binding and capture of the biotin labeling reagent by the streptavidin biotin binding site is reduced, and thus it can be covalently bonded to biotin. In one embodiment, streptavidin is covalently bonded to the microparticle binding surface, and the microparticle binding surface is adjusted (blocked and removed) so that only the covalently bonded streptavidin remains. The streptavidin-conjugated microparticle binding surface is exposed to a molar excess of free biotin (D-biotin), 100BS streptavidin beads are prepared, 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 100BS streptavidin is conjugated to NHS-PEO4-biotin to prepare biotinylated 100BS streptavidin beads. The biotinylation of streptavidin-binding beads refers to the covalent bonding of biotin to magnetic particles (or streptavidin thereon), and should not be confused or identified with the saturation of the biotin binding site of streptavidin. Biotinylated streptavidin beads can bind an anti-biotin substance (in addition to an anti-streptavidin substance). The biotin used to saturate streptavidin generally does not bind to the most problematic anti-biotin substances because the necessary part of the biotin molecule engages with streptavidin. In another embodiment, 100BS streptavidin or 100BS streptavidin beads are thiolated through the primary amine of streptavidin (R-NH2) with succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), SPDP-PEG (4、6、8、12、24、または36)-NHS ester, SPDP NHS ester, SPDP-C6-NHS ester, SPDP-C6-Sulfo-NHS ester, PC SPDP-NHS carbonate ester, and SPDP-C6-Gly-Leu-NHS ester (means for thiolation), etc., which are standard thiolation chemical reactions known in the art, are used to introduce a thiol group (sulfhydryl group, or R-SH) onto streptavidin. When using SPDP, the SPDP conjugated to streptavidin is cleaved using TCEP and EDTA, and the SPDP leaving group is washed, desalted, or dialyzed, leaving only SH-R conjugated 100BS streptavidin. After preparing 100BS thiolated streptavidin using a molar excess of biotin, the thiol (R-SH) of 100BS streptavidin and 100BS streptavidin beads is maleimide-PEO (2、3、6、または11) -Biotin-labeled reagents that are thiol-reactive or sulfhydryl-reactive, such as -biotin and biotin-SPDP (thiol or sulfhydryl-reactive means), can be covalently bound to biotin. The sulfhydryl-reactive biotin label is carried out 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 thiols and mitigate disulfide bonds or crosslinking, and 2) reduce the binding and capture of the biotin-labeled reagent by the streptavidin biotin-binding site. The maleimide group has a reactivity 1000 times higher for free sulfhydryl than for amine at pH 6.5 - 7.5, and at pH > 8.5 the maleimide group reacts preferentially with primary amines, so the conjugation of maleimide is carried out at pH 6.8 to minimize the reaction with primary amines. As an alternative to SPDP, similar reagents based on N-succinimidyl-S-acetyl-thioacetate (SATA) can be used.

[0064] In another embodiment, 100BS streptavidin or 100BS streptavidin beads have maleimide groups introduced onto the streptavidin primary amine (R-NH2) using standard ester-maleimide heterobifunctional crosslinking chemistries known in the art such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimide-PEG-NHS ester, maleimide-PEO (1,2,3,4,5,6,8または12) -NHS ester, or maleimide-PEG (1,2,3,4,5または6) -PFP (means of maleimidation). After preparing 100BS maleimide streptavidin with a molar excess of D-biotin, the maleimides of 100BS streptavidin and 100BS streptavidin beads can be covalently bound to biotin using a maleimide-reactive biotin labeling reagent such as biotin-PEG-SH or biotin-PEG-thiol, where PEG n or PEO n may be of different lengths, such as n = 1, 2, 3, 4, 5, 6, 8, or 12. The maleimide-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 thiols and mitigate disulfide bond formation or crosslinking of the biotin labeling reagent, and 2) reduce binding and capture of the biotin labeling reagent by the streptavidin biotin binding site. The maleimide group has a reactivity 1000-fold higher for free sulfhydryl than for amine at pH 6.5 - 7.5, and at pH > 8.5 the maleimide group reacts preferentially with the primary amine, so maleimide biotin conjugation is performed at pH 6.8.

[0065] Similar ester, thiol, or maleimide chemical reactions are also applicable in embodiments where streptavidin is not biotin-saturated. For example, ruthenium esters can be reacted with the primary amine of streptavidin.

[0066] In certain embodiments: 1) streptavidin is covalently attached to the microparticle binding surface; 2) the microparticle binding surface is conditioned such that only streptavidin covalently attached on the microparticle binding surface remains and the surface has extremely low non-specific binding; 3) the streptavidin-conjugated microparticle binding surface is exposed to a molar excess of free biotin (D-biotin) to prepare 100BS streptavidin beads; 4) the 100BS streptavidin beads are covalently attached to biotin in the presence of a molar excess of free biotin using a biotinylated reagent; 5) the biotinylated 100BS streptavidin beads are filtered, centrifuged, or magnetically separated to remove buffer 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 final reagent.

[0067] In certain embodiments: 1) the biotinylated 100BS streptavidin beads are filtered, centrifuged, or magnetically separated to remove the storage solution; 2) a sample containing anti-streptavidin interference, anti-biotin interference, or both interferences is added to the biotinylated 100BS streptavidin beads to pretreat the sample; 7) the sample interference is depleted or reduced below an assay blocking threshold (ABT) or a test interference threshold; 8) the biotinylated 100BS streptavidin beads are filtered, centrifuged, or magnetically separated from the sample; and 9) the sample supernatant essentially free of beads is aspirated and tested by a diagnostic assay to report an accurate test result.

[0068] In certain embodiments, 100BS streptavidin beads are used to pretreat a sample to bind anti-streptavidin interference and deplete anti-streptavidin interference until it falls below an assay blocking threshold (ABT) or a test interference threshold prior to a diagnostic test. In another embodiment, biotinylated 100BS streptavidin beads are used to pretreat a sample to bind anti-biotin interference and deplete anti-biotin interference until it falls below an assay blocking threshold (ABT) or a test interference threshold prior to a diagnostic test. In another embodiment, biotinylated 100BS streptavidin beads are used to pretreat a sample to simultaneously bind both anti-streptavidin interference and anti-biotin interference from the same sample and deplete anti-biotin interference until it falls below an assay blocking threshold (ABT) or a test interference threshold prior to a diagnostic test.

[0069] At present, there is no rapid and easy-to-use product solution for detecting, characterizing, and reducing biotin, antibiotin, and antistreptavidin interferences in patient samples. In certain embodiments, streptavidin beads (Bead 1), 100BS streptavidin beads (Bead 2), and biotinylated 100BS streptavidin beads (Bead 3) are used systematically or sequentially to detect and determine which interference mechanism or mechanisms are present in the sample. Samples with suspected interference are assayed neat (no beads added) as a control result. Three different aliquots of the sample are each treated with Bead 1 (aliquot 1), Bead 2 (aliquot 2), and Bead 3 (aliquot 3). The three pretreated aliquots are retested, and the test results for each bead type are compared to the control test results (Table 1). If the control test results are similar to the test results from the pretreatment with Beads 1, 2, and 3, the likelihood of sample interference is low and may be determined to be absent. However, if the pretreatment result for Bead 1 is significantly different from the control result, the likelihood of biotin interference and / or antistreptavidin interference is high, and sample interference may be determined to be present. If the pretreatment result for Bead 2 is significantly different from the control result, the likelihood of antistreptavidin interference is high, and sample interference may be determined to be present. If the pretreatment result for Bead 3 is significantly different from the control result, the likelihood of antistreptavidin interference and / or antibiotin interference is high, and sample interference may be determined to be present. If the pretreatment result for Bead 1 is significantly different from the control result, but the pretreatment results for Beads 2 and 3 are similar to the control, biotin interference may be determined to be present. If the pretreatment results for Beads 1 and 2 are similar to the control result, but the result for Bead 3 is significantly different from the control result, antibiotin interference may be determined to be present. If the pretreatment results for Beads 1, 2, and 3 are all significantly different from the control result, antistreptavidin interference may be determined to be present.

Table 1

[0070] The production of free (or soluble) biotin-saturated streptavidin (quenched streptavidin, QSAv) is generally similar to the production of biotin-saturated streptavidin-conjugated beads. Furthermore, streptavidin can be conjugated to additional moieties to function as a capture moiety or to block sites that can promote streptavidin aggregation. Such conjugation can be performed before or after quenching (except when the additional capture moiety is biotin, in which case it can only be performed after quenching). A molar ratio of biotin to streptavidin of approximately 7 - 8 is used, which is slightly higher than the 5:1 ratio used in the minimal saturation procedure for beads. This is because some of the biotin-binding sites on bead-conjugated streptavidin are sterically hindered, resulting in an effective ratio that is somewhat higher than the formal ratio.

[0071] QSAv is preferably mainly monomeric. In various embodiments, QSAv is at least 80, 90, 95, 97, 98, 99% monomeric, or any range bounded by those values. To ensure that the reagent is monomeric and persists as a monomer without forming aggregates, streptavidin can be blocked with a detergent or a polymeric blocking reagent. Blocking can include PEGylation. There are a variety of commercially available PEGylation reagents of various sizes and chemical modifications, such as those from ThermoFisher Scientific, 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). These can covalently bind to any exposed lysine residues on streptavidin via the chemistry of the NHS-ester. Alternatively, TFP-(PEG)n-COOH or NHS-(PEG)n-COOH can bind to lysine residues through the chemistry of EDC. Many other alternatives will be well known to those skilled in the art. The preparation of monomeric QSAv can also be achieved by biotin quenching in a buffer containing a chaotropic reagent such as urea, imidazole, trehalose, or others.

Example

[0072] The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of representative embodiments currently contemplated. These examples should not be construed as limiting any of the embodiments described herein.

[0073] Example 1 Methods for preparing biotinylated streptavidin coated magnetic nanoparticles or biotinylated 100BS streptavidin beads. Magnetic carboxylic acid nanoparticles with a wavelength range of 550 - 600 nm were conjugated to streptavidin via covalent bonds using the chemical action of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). The bead surface was adjusted with stripping reagents (salts, detergents, low pH, and high pH) to remove passively absorbed streptavidin. The beads were blocked using detergents and polymer blocking reagents to reduce non-specific binding and promote the monodispersity and colloidal stability of the nanoparticles. The total concentration of streptavidin covalently conjugated to the beads was determined to be 17.69 micrograms per milligram (μg / mg) using a modified micro BCA total protein assay. The final bead concentration was determined by weight measurement and adjusted to 10.0 milligrams per milliliter (mg / mL) of beads in PBS with 2 mM EDTA, pH 6.8.

[0074] A 10.0 mg / mL stock solution of D-biotin in PBS, pH 7.4 (Sigma, product number B4601-100MG, lot SLBS8478, molecular weight 244.31) was prepared by making a 100 mg / mL concentrated stock solution of D-biotin in DMSO (Baker, product number 9224-01, lot 0000217025), or by adding 10 mg of D-biotin to 100 μL of DMSO and mixing. Once the D-biotin was completely and homogeneously dissolved in the DMSO, 900 μL of PBS, pH 7.4 was added and mixed to prepare 1.0 mL of a 90:10 (PBS:DMSO) 10.0 mg / mL D-biotin stock solution.

[0075] A total of 2.5 mL of streptavidin magnetic nanoparticles was aliquoted and dispensed into reaction vials. This corresponded to a total of 442.25 μg of streptavidin: [(25 mg of beads) × (17.69 μg of streptavidin / 1 mg of beads)]. A total of 442.25 μg of streptavidin corresponds to 0.00804 μM of streptavidin: [(442.25 μg of streptavidin) / (55,000 μg of streptavidin / 1 μM of streptavidin)].

[0076] 1,964.475 μg of D-biotin was added to 25 mg of streptavidin-conjugated magnetic particles with 17.69 μg of streptavidin per 1 mg of beads, or to 442.25 μg of streptavidin, to prepare a 1000-fold molar excess of D-biotin relative to the total moles of streptavidin: [(0.00804 μM × 1000) × (244.31 μg / μM of biotin)]. To add a 1000-fold molar excess of D-biotin to 0.00804 μmol of streptavidin, 200 μL of a 10.0 mg / mL stock solution of D-biotin, or 2,000 μg of D-biotin, was added to 25 mg of streptavidin magnetic nanoparticles with 17.69 μg of streptavidin per 1 mg of beads in PBS with 2 mM EDTA, pH 6.8. The streptavidin magnetic nanoparticles were incubated with D-biotin for 1 hour with mixing at room temperature to 100% saturate the streptavidin biotin-binding sites with biotin, preparing 100BS streptavidin beads.

[0077] 100BS streptavidin-beads were added to a 100-fold molar excess of NHS-PEG4-biotin (Broadpharm, product number 20566, lot B93-039, molecular weight 588.7), or 500 μg of NHS-PEG4-biotin, to covalently conjugate the 100BS streptavidin-beads to biotin in a molar excess. 25 mg of streptavidin-conjugated magnetic particles with 17.69 μg of streptavidin per 1 mg of beads, or 442.25 μg of streptavidin, or 0.00804 μM of streptavidin. 5 mg of NHS-PEG4-biotin was added to 100 μL of DMSO and mixed to prepare a stock solution of NHS-PEG4-biotin at 50.0 mg / mL in DMSO. 10.0 μL of the stock solution of NHS-PEG4-biotin at 50.0 mg / mL in DMSO was added to 25 mg of 100BS streptavidin-beads with 17.69 μg of streptavidin per 1 mg of beads in PBS with 2 mM EDTA, pH 6.8, together with a 1000-fold molar excess of D-biotin from the saturation step, and mixed at room temperature for 1 hour to add a 100-fold molar excess of NHS-PEG4-biotin, or 473.315 μg of NHS-PEG4-biotin [(0.804 μM of NHS-PEG4-biotin)×100]×(588.7 μg of biotin / μM] to the 100BS streptavidin beads. The biotinylated 100BS streptavidin-beads were washed 4 times with PBS at pH 7.4 to wash away the excess NHS-PEG4-biotin.

[0078] To confirm that the 100BS streptavidin-beads were successfully conjugated to biotin without any bead aggregation from streptavidin-mediated binding of conjugated biotin on different beads (i.e., bead cross-linking), 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).

[0079] To demonstrate that biotin was successfully conjugated to streptavidin on 100BS streptavidin beads, a limiting amount of native streptavidin was added to biotinylated 100BS streptavidin beads to promote bead aggregation from streptavidin-mediated cross-linking of the beads. 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. The beads showed aggregation 30 minutes after the addition of streptavidin, with peaks at 1,441.3 nm and 6,641 nm, a polydispersity index of 173.3% (Figure 2A), and peaks at 1,512.6 nm and 14,536 nm, a polydispersity index of 242.8% (Figure 2B).

[0080] Similar bead aggregation was performed using an anti-biotin conjugated monoclonal antibody that recognizes biotinylated biotin with an affinity similar to streptavidin but recognizes free biotin with an affinity 1 million times lower than streptavidin, or an antibody that specifically binds to the biotin of a biotin conjugate and has a higher affinity for the biotin of the biotin conjugate than for free biotin (WO2020 / 028776; VeraBind Biotin™, Veravas). The antibody was added to biotinylated 100BS streptavidin-beads and incubated overnight at room temperature. The beads showed aggregation with a peak at 2,148 nm and a polydispersity index of 316.2% (Figure 3).

[0081] Example 2 A method to deplete anti-biotin antibodies from samples using biotinylated streptavidin-coated magnetic nanoparticles or biotinylated 100BS streptavidin-beads. To demonstrate successful biotinylation of 100% biotin-saturated streptavidin-conjugated magnetic nanoparticles, or 100BS streptavidin-beads, freeze-dried mouse ascites containing monoclonal anti-biotin antibodies specific for conjugated biotin was purified using the Melon Gel purification kit (ThermoFisher, product number 45214), and ascites conditioning buffer (ThermoFisher, product number 45219, lot TB263120), and desalted in PBS at pH 7.2 using Zeba Spin Desalting Columns, 40K MWCO (ThermoFisher, product number 87770). The final concentration of the anti-biotin antibody was 0.205 mg / mL, and 50 μL of the anti-biotin antibody, or 10.25 μg of the anti-biotin antibody, was added to 950 μL of PBS at pH 7.4 in a glass HPLV vial to prepare a 10.25 μg / mL anti-biotin stock solution. 100 μL, 50 μL, 25 μL, and 10 μL of the anti-biotin stock solution were sequentially injected into a Phenomenex s4000 SEC HPLC column with a flow rate of 1.0 mL / min and mobile phase PBS pH 7.4, and the peak area was determined for each concentration of the injected antibody to generate a calibration curve of peak area (Y-axis) vs. antibody concentration (X-axis): y = 6.6466x + 21.4286, R 2 = 1.0000 (Figure 4A).

[0082] Next, 750 μL of the 0.205 mg / mL anti-biotin antibody was pretreated with biotinylated 100BS streptavidin-beads as follows. 1. Remove the biotinylated 100BS streptavidin-beads reagent vial from the storage and vortex at medium speed for at least 10 seconds. 2. Place an empty 2 mL Sarstedt Micro tube into a VeraMag 400™ magnetic separator until the tube collar touches the magnet frame. 3. Dispense 750 μL of the well-mixed reagent or 7.5 mg of beads at 10.0 mg / mL into the 2 mL Sarstedt Micro tube. 4. Wait for at least 30 seconds and carefully aspirate and discard all of the supernatant without disturbing the pellet of magnetic nanoparticles. 5. Dispense 750 μL of well-mixed anti-biotin antibody at 0.205 mg / mL. 6. Cap the tube and vortex the sample at medium speed for at least 10 seconds. 7. Place the tube on a medium-speed rotary mixer and incubate at room temperature for 30 minutes. 8. Loosen the screw cap and place the tube into VeraMag 400 until the rim of the tube touches the magnet frame. 9. Magnetically separate the nanoparticles from the sample for 5 minutes. 10. Carefully aspirate the sample without disturbing the pellet of magnetic nanoparticles and dispense it into a clean tube. If this step is performed carefully, all of the sample can be aspirated. Note: If magnetic nanoparticles are accidentally aspirated, simply return the mixture to the tube, cap the tube, and return to step 9. 11. Now the adjusted sample is ready for analysis. 12. Filter the sample using a 0.2 micron cellulose acetate syringe filter. The average protein loss using this filter was 16.5 μg). 13. Inject 100 μL of the sample into a Phenomenex s4000 SEC HPLC column and determine the peak area with a retention time of approximately 9.6 - 9.9 minutes at a flow rate of 1.0 mL / min and mobile phase (50 mM potassium phosphate, 250 mM potassium chloride, pH 6.8). 14. Based on the calibration curve equation, use the peak area of the antibody peak (y) to determine x (antibody μg / mL).

[0083] The peak area of 100 μL of the anti-biotin Ab sample was 1,384 (Figure 4B), and the concentration corresponding to this peak area on the calibration curve was 205 μg / mL (Figure 4A). The peak area of 100 μL of the pretreated and depleted anti-biotin sample was 318 (Figure 4C), and the concentration corresponding to this peak area on the calibration curve was 44.62 μg / mL (Figure 4A). Since the starting volume of the antibody pretreated with the depletion reagent was 750 μL, this corresponded to 33.465 μg of antibody [44.62 μg / mL × 0.750 mL]. Since 16.5 μg of antibody was lost in the 0.2 micron cellulose acetate syringe filter, the total remaining antibody after sample pretreatment was 49.965 μg of antibody [33.465 μg + 16.5 μg]. The starting amount of the pretreated anti-biotin antibody was 153.75 μg of antibody: [205 μg / mL x 0.750 mL]. The percentage of anti-biotin antibody captured and depleted by biotinylated 100BS streptavidin-beads was 67.5%: [((153.75 μg - 49.965 μg) / 153.75 μg) × 100%].

[0084] This study demonstrated that biotinylated 100BS streptavidin-beads were able to deplete 103.785 μg of antibody (153.75 μg - 49.965 μg). This corresponded to a binding capacity of 13.838 μg of anti-biotin antibody per mg of biotinylated 100BS streptavidin-beads: [(103.785 μg of antibody) / (7.5 mg of beads)].

[0085] Example 3 Preparation of biotinylated 100BS streptavidin-beads using a low molar excess of biotin. The use of a 1000-fold molar excess of free biotin for biotin saturation of streptavidin-coated beads can lead to non-specific association of biotin with streptavidin or the beads. Even if successful in depleting anti-biotin and anti-streptavidin during use, non-specifically associated biotin can leach or dissociate from 100BS streptavidin-beads and cause biotin interference in the assay. Several approaches were investigated to remove or mitigate this non-specific biotin binding. This included saturating streptavidin prior to conjugation to magnetic nanoparticles, various washing procedures after saturation, use of a lower molar excess of biotin, and use of various biotin linker molar excesses. Ultimately, a combination of a low molar excess of biotin and specific washing conditions resulted in the production of the product without issues of free biotin leaching.

[0086] After conjugating streptavidin to magnetic nanoparticles and prior to biotinylation, streptavidin beads were exposed to a 5-fold molar excess of free biotin (i.e., a 5:1 molar ratio of biotin:streptavidin or a 5:4 ratio of biotin:biotin binding sites). The saturated beads were then washed with water at 50 °C using sonication. (Since the conjugation of streptavidin to the beads leads to steric hindrance of some biotin binding sites, the effective ratio of biotin to biotin binding sites is somewhat higher.)

[0087] Biotinylation was carried out with 4-fold, 25-fold, and 50-fold molar excesses of the biotinylation reagent (biotin-PEG4-NHS linker). It was found that a 50-fold molar excess gave the optimal results.

[0088] The finished beads were tested for neutrality and shown not to cause interference themselves when the beads were used to pretreat serum samples according to the following protocol: 1. Remove the biotinylated 100BS streptavidin-bead reagent vial from the storage and vortex at medium speed for at least 10 seconds to mix well and resuspend the reagent. 2. Insert the reagent vial into the foaming vial holder. 3. Insert an empty 2 ml microtube (SARSTEDT order number 72.694) into the VeraMag (trademark) magnet (Veravas) until the collar of the tube touches the magnet frame. 4. Dispense 200 μL of the well - mixed reagent (beads) into the empty tube and separate the reagent on the magnet for more than 30 seconds to form a reagent pellet. 5. Carefully aspirate and discard all of the storage buffer supernatant (about 200 μL) without disturbing the reagent pellet. 6. Dispense 400 μL of the well - mixed serum or plasma sample into the tube containing the reagent pellet. 7. Tightly screw on the tube's screw cap, remove the tube from the magnet, and vortex at medium speed for at least 10 seconds to mix well and resuspend the reagent in the sample. 8. Place the tube on a lab mixer at medium speed and incubate at room temperature for 10 minutes. 9. Loosen and remove the screw cap, and insert the tube into the magnet until the color of the tube touches the magnet frame. 10. Magnetically separate the reagent for more than 4 minutes to form a reagent pellet. 11. Carefully aspirate the sample supernatant without disturbing the reagent pellet and dispense the sample into the test transport tube. Note: By performing this step carefully, all of the sample supernatant (about 400 μL) can be aspirated. If any of the reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and go back to step 10. 12. Thus, the sample is ready for testing.

[0089] Subsequently, in the Roche Elecsys TSH assay as an example of a sandwich immunoassay and the Roche Elecsys FT4 assay as an example of a competitive immunoassay, the pretreated samples were used (see Table 2). For all samples tested by both assays, there were no significant analytical or clinical differences in the results between the treated and untreated samples.

Table 2

[0090] Example 4 Preparation of validation lots Three lots of beads were prepared as described in Example 3 as a deceleration. That is, for saturation, a 5:1 molar ratio of biotin to streptavidin, a 50-fold molar excess of biotin-PEG4-NHS linker, and sonication using streptavidin from different sources were used for washing at 50 °C. For one lot, FSAv, fresh streptavidin was used. For one lot, RSAv, streptavidin recovered from a previous bead coating reaction was used. Further, for another lot, MSAv, a mixture of 80% recovered streptavidin and 20% fresh streptavidin was used. The streptavidin content of the three lots was 35 μg / mg beads for lot FSAv, 30 μg / mg beads for RSAv, and 19 μg / mg beads for MSAv. The three lots were then used in the validation tests described in Examples 5 to 8 below.

[0091] For the conjugation of streptavidin to magnetic nanoparticles, an excess amount of streptavidin is used. Instead of discarding the unconsumed reagent, it can be recovered by filtration, desalting, and concentration. It has been found that streptavidin recovered in this way can be incorporated into the functional product without affecting its stability or performance.

[0092] Example 5 Particle size as an indicator of agglomeration during manufacturing As an initial quality control, the size of the finished beads was measured and aggregation during production was checked. 5 uL of beads were mixed with 1 mL of diH2O in a disposable cuvette, and after a short vortex (5 - 10 seconds), mixing (on a mixer for 10 minutes), and sonication (if used) for 30 - 60 seconds, they were read by an Anton Paar Litesizer™ 100 particle size analyzer. None of the three lots showed aggregation from the products generated before and after sonication (Tables 3 and 4). On average, the aggregated polydispersity is larger than that of the monomeric polydispersity.

Table 3

Table 4

[0093] Example 6 Biotin leaching test To test for potentially problematic levels of biotin leaching, biotin-saturated, conjugated streptavidin-coated beads (biotinylated 100BS streptavidin beads) were suspended in serum at a biotin concentration of less than 100 pg / ml. 400 μL of serum was treated with 0.5 mg of beads (200 μL - 2.5 mg / mL) for 10 minutes at room temperature on a mixer and magnetically separated according to the following protocol. 1. Remove the MSAv, FSAv, or RSAv reagent vial of the biotinylated 100BS streptavidin-bead lot from stock, vortex at medium speed for at least 10 seconds to mix well, and resuspend the reagent. 2. Insert the reagent vial into the foaming vial holder. 3. Insert an empty 2 ml microtube (SARSTEDT order number 72.694) into the VeraMag magnet until the color of the tube touches the magnet frame. 4. Dispense 200 μL of the well-mixed reagent (beads) into the empty tube, separate the reagent on the magnet for more than 30 seconds to form a reagent pellet. 5. Without disturbing the reagent pellet, carefully aspirate and discard all of the storage buffer supernatant (about 200 μL). 6. Dispense 400 μL of well - mixed serum or plasma sample into the tube containing the reagent pellet. 7. Tightly screw the tube cap, remove the tube from the magnet, vortex at medium speed for at least 10 seconds to mix well, and resuspend the reagent in the sample. 8. Place the tube on a lab - mixer at medium speed and incubate at room temperature for 10 minutes. 9. Loosen and remove the screw cap, insert the tube into the magnet until the tube color touches the magnet frame. 10. Magnetically separate the reagent for more than 4 minutes to form a reagent pellet. 11. Without disturbing the reagent pellet, carefully aspirate the sample supernatant and dispense the sample into the test transport tube. Note: By performing this step carefully, all of the sample supernatant (about 400 μL) can be aspirated. If any of the reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and go back to step 10. 12. The sample is now ready for testing.

[0094] The treated serum was tested with the IDK BIOTIN ELISA assay (Immundiagnostik AG). The IDK BIOTIN ELISA is a competitive immunoassay where biotin in the sample decreases the generated signal. The concentration of biotin is determined by comparing to a calibration curve. A calibration curve was created using each of three test lots. In all cases, biotin was detected at levels below 1200 pg / ml. This indicates that any leaching of biotin from the beads was at a level that did not cause heterophilic interference in a standard immunoassay (Table 5).

Table 5

[0095] Example 7 HPLC depletion assay Three lots of biotin-saturated, conjugated streptavidin-coated beads were used in depletion assays to evaluate their ability to specifically remove anti-streptavidin interference and anti-biotin interference, but not other interferences. For this purpose, a series of four HPLC depletion tests were conducted. 1) HPLC depletion assay using affinity-purified goat IgG and affinity-purified goat IgG conjugated to biotin to evaluate the specificity of the product for anti-SAv antibody and anti-Bt antibody only. 2) HPLC depletion assay using anti-streptavidin antibody to quantify the binding ability of the reagent. 3) Perform an HPLC depletion assay using anti-biotin antibody to quantify the binding ability of the reagent. 4) A) HPLC depletion assay using a mixture of anti-biotin antibody and anti-streptavidin antibody, and B) anti-streptavidin antibody and affinity-purified goat IgG to demonstrate the multiplexing ability and specificity of the reagent.

[0096] The concentrations of various antibodies (Ab) 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, conjugated streptavidin-coated bead suspension (2.5 mg / ml) was dispensed 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 minutes. The beads were magnetically separated again, the supernatant of the treated sample was aspirated, and loaded into a microtube insert of an HPLC tube for size exclusion chromatography (SEC) analysis. The detailed protocol for the pretreatment was as follows: 1. Remove the MSAv, FSAv, or RSAv reagent vial of the biotinylated 100BS streptavidin-bead lot from stock, vortex at medium speed for at least 10 seconds to mix well, and resuspend the reagent. 2. Insert the reagent vial into the foaming vial holder. 3. Insert an empty 2 ml microtube (SARSTEDT order number 72.694) into the VeraMag magnet until the tube color touches the magnet frame. 4. Dispense 200 μL of the well - mixed reagent (beads) into the empty tube, and separate the reagent on the magnet for more than 30 seconds to form a reagent pellet. 5. Carefully aspirate and discard all of the storage buffer supernatant (about 200 μL) without disturbing the reagent pellet. 6. Dispense 400 μL of the well - mixed serum or plasma sample into the tube containing the reagent pellet. 7. Tightly screw on the tube's screw cap, remove the tube from the magnet, and vortex at medium speed for at least 10 seconds to mix well and resuspend the reagent in the sample. 8. Place the tube on a lab mixer at medium speed and incubate at room temperature for 10 minutes. 9. Loosen and remove the screw cap, and insert the tube into the magnet until the tube color touches the magnet frame. 10. Magnetically separate the reagent for more than 4 minutes to form a reagent pellet. 11. Carefully aspirate the sample supernatant without disturbing the reagent pellet and dispense the sample into the test transport tube. Note: By performing this step carefully, all of the sample supernatant (about 400 μL) can be aspirated. If any of the reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and go back to step 10. 12. Thus, the sample is ready for testing.

[0097] SEC was also performed on the untreated antibody as a control. The SEC buffer was 50 mM potassium phosphate, 250 mM potassium chloride, pH 6.8, and was pump - injected at 1 ml / min for 20 minutes using a 5 μg / 100 μl injection on a G4000 column 7.8 mm×30 cm. Absorbance at 220 nm and 280 nm was monitored, and A280 was used for peak analysis. The results are shown in Table 6.

Table 6

[0098] Since the beads are intended to be neutral with respect to antibodies that are not specific for anti-biotin or anti-streptavidin, affinity-purified goat IgG was used as a control to establish background depletion. All three lots showed minimal or no binding of this control (92.56% - 96.78% of the antibody remained after treatment. Figure 5A shows the results for the MSAv lot).

[0099] Again, even if biotinylated, since the beads are intended to be neutral with respect to antibodies that are not specific for anti-biotin or anti-streptavidin, affinity-purified goat IgG conjugated to biotin was used as an additional control to establish background depletion. All three lots showed minimal or no binding of this control (98.91% - 100% of the antibody remained after treatment. Figure 5B shows the results for the MSAv lot).

[0100] All three lots showed depletion of more than 10 μg per mg of beads for anti-biotin antibody (12, 34.4, and 21 μg / mg depletion; see Table 6; Figure 5C shows the results for the MSAv lot). All three lots showed depletion of more than 20 μg per mg of beads for anti-SAv antibody (31, 33.5, and 27.6 μg / mg depletion; see Table 6; an exemplary profile is shown in Figure 5D. This figure shows the results for the MSAv lot). A mixture of anti-streptavidin antibody and anti-biotin antibody was tested using beads from an MSAv lot, demonstrating the multiplexing ability of biotin-saturated, conjugated streptavidin-coated beads. Also, a mixture of anti-streptavidin and AP goat IgG antibody was tested to demonstrate the specificity of binding by the reagent. The data show that biotin-saturated, conjugated streptavidin-coated beads deplete both anti-streptavidin antibody and anti-biotin antibody when present tandemly (19.1 μg out of 23.4 μg present was depleted), and that the product specifically removes only the anti-streptavidin antibody when mixed with AP goat IgG (10.9 μg out of 23.8 μg present was depleted). Previous data showed no binding of individual AP goat IgG (97% of the Ab was still present after treatment), so it can be reasonably inferred that depletion of approximately half (46%) of the mixture indicates that only the anti-streptavidin Ab was depleted.

[0101] Example 8 Effect of processing on analyte detection The neutrality of biotin-saturated, conjugated streptavidin-coated beads (biotinylated 100BS streptavidin beads) was tested in a commercial assay for serum parathyroid hormone. The DRG PTH Intact ELISA (DRG International, Part Number EIA3645) is a sandwich ELISA assay that uses two different goat anti-PTH polyclonal antibodies that recognize distinct portions of the hormone. One of the antibodies is biotinylated and functions as the capture reagent, and the other antibody is conjugated to horseradish peroxidase and functions as the detection reagent.

[0102] Each 400 μl of two serum samples (QC1 and QC3) was processed on a mixer for 10 minutes at room temperature using 0.5 mg of beads (200 ul, 2.5 mg / mL) according to the following protocol and magnetically separated. 1. Remove the SAv, FSAv, or RSAv reagent vial of biotinylated 100BS streptavidin-beads lot from the stock, vortex at medium speed for at least 10 seconds to mix well, and resuspend the reagent. 2. Insert the reagent vial into the foaming vial holder. 3. Insert an empty 2 ml microtube (SARSTEDT order number 72.694) into the VeraMag magnet until the tube color touches the magnet frame. 4. Pipette 200 μL of the well-mixed reagent (beads) into the empty tube, separate the reagent on the magnet for more than 30 seconds to form a reagent pellet. 5. Carefully aspirate and discard all of the storage buffer supernatant (about 200 μL) without disturbing the reagent pellet. 6. Pipette 400 μL of well-mixed serum or plasma sample into the tube containing the reagent pellet. 7. Tightly screw on the tube cap, remove the tube from the magnet, vortex at medium speed for at least 10 seconds to mix well, and resuspend the reagent in the sample. 8. Place the tube on a lab mixer at medium speed and incubate at room temperature for 10 minutes. 9. Loosen and remove the screw cap, insert the tube into the magnet until the tube color touches the magnet frame. 10. Magnetically separate the reagent for more than 4 minutes to form a reagent pellet. 11. Carefully aspirate the sample supernatant without disturbing the reagent pellet and pipette the sample into the test transport tube. Note: By performing this step carefully, all of the sample supernatant (about 400 μL) can be aspirated. If any of the reagent is accidentally aspirated, simply return the sample / reagent mixture to the tube and go back to step 10. 12. Thus, the sample is ready for testing.

[0103] Next, the processed sera were tested in the DRG PTH ELISA assay. QC1 is an in - house QC sample containing less than 100 pg / mL biotin (which does not affect the mechanism of the PTH assay) and approximately 190 pg / mL PTH. QC3 is an in - house QC sample containing approximately 250,000 pg biotin / mL (which affects the mechanism of the PTH assay - the assay gives extremely low results) and approximately 190 pg / mL PTH. The QC1 sera processed in each of the different lots showed no significant deviation in the PTH results (detected at 100.6, 101.2, and 106.1%), and as predicted from the fact that 100BS streptavidin beads do not bind free biotin, all the QC3 samples gave extremely low results (Table 7). These data demonstrate the bead neutrality of the 100BS streptavidin beads.

Table 7

[0104] The analyte detection after treatment of samples containing interfering substances of anti - streptavidin antibody and anti - biotin antibody was determined. It was determined by adding affinity - purified anti - streptavidin antibody and anti - biotin goat antibody to serum QC1 and comparing the analyte detection results from the treated and untreated samples.

[0105] Four - hundred - microliter aliquots of each serum sample (QC1 and QC1 with 16.5 μg / mL anti - biotin antibody) were treated on a mixer at room temperature for 10 minutes with various amounts of beads (100 - 400 μl, 2.5 mg / mL) from all 3 lots, magnetically separated, and the processed sera were tested in the DRG PTH ELISA assay. QC1 is an in - house QC sample containing less than 100 pg / mL biotin (which does not affect the mechanism of the PTH assay) and approximately 190 pg / mL PTH. The concentration of anti - biotin antibody added to QC1 interfered with the PTH assay mechanism and resulted in extremely decreased results.

[0106] The MSAv beads successfully depleted all anti-Bt antibodies (anti-Bt Aby) and were able to restore the results of corrected PTH at a bead concentration of 1 mg per 200 μl of sample (16.5 μg / mL anti-Bt Aby concentration). FSAv and RSAv were able to achieve the same results with much smaller amounts: 0.25 mg for FSAv and 0.375 mg for RSAv. QC1 samples to which anti-Bt Aby was added and which were not treated with biotinylated 100BS streptavidin-beads had extremely low results, only 2.7% of the control, as expected (Tables 8 and 9). The low capacity of the MSAv lot is consistent with the results observed in Example 7 (above).

Table 8

Table 9

[0107] Similarly, 200 μl aliquots of each serum sample (QC1, and QC1 samples with anti-streptavidin Aby (anti-SAv Aby) added at 16.5 μg / mL, or an anti-SAv Aby / anti-Bt Aby multiplex mixture at 16.5 μg / mL and 8.25 μg / mL respectively) were treated with 100 μl of 2.5 mg / mL beads for 10 minutes at room temperature on a mixer, magnetically separated, and the treated serum was tested in the DRG PTH ELISA assay. As described above, QC1 is an in-laboratory QC sample containing less than 100 pg / mL biotin (which does not affect the mechanism of the PTH assay) and approximately 190 pg / mL PTH. Anti-SAv Aby and anti-Bt Aby interfere with the PTH assay mechanism and result in extremely low results.

[0108] At the concentration used (16.5 μg / mL), anti-SAv Abys gave extremely low results as expected (29% of the baseline). The same was true for the multiplex mixture (21% of the baseline). The beads of lot RSAv successfully depleted anti-SAv Abys and began to recover PTH detection with 0.25 mg of beads per 200 μl of sample, yielding reading results up to 82% of the baseline value. By increasing the amount of beads used in the treatment to 0.5 mg per 200 μl of the sample, the value recovered to 104% of the baseline. With 0.25 mg of beads per 200 μl of sample, the beads of lot FSAv recovered the PTH level to 91% of the baseline. With 0.375 mg of beads per 200 μl of the sample, the beads of lot MSAv recovered the PTH value to 92% of the baseline. All lots were able to restore corrected PTH results with 0.5 mg of beads (RSAv - 104%, FSAv - 101%, MSAv 100%) (Table 10).

Table 10

[0109] An overview of the results of the above PTH detection experiment using lot MSAv is shown in Figure 6. Biotinylated 100BS streptavidin-beads had no effect when no interferents were used or when biotin was the interferent, but were effective in removing anti-biotin interferents and anti-streptavidin interferents, either individually or mixed together.

[0110] Specifically, in the bar group at the left end of Fig. 6 labeled "none", there was no interfering substance added to the interfering substance-added sample. That is, it was a retest of the reference sample, and the detected PTH concentration had a difference of only -0.1%. When the reference sample was treated with biotin-saturated and conjugated streptavidin-coated beads without any interference (none), it differed from the reference sample by only +1.3% and from the reference sample retest by only +1.4% (no interference addition). These results are good in the accuracy profile of this PTH ELISA, indicating the neutrality of the reagent, and demonstrating that sample processing introduced no dilution or matrix effects. Biotin addition caused significant interference in this PTH ELISA assay, resulting in an 87% decrease in detection. When the biotin-added sample was treated with beads, the results did not change significantly, with a difference of only -2.3% and 88% lower than the reference result. This result was predicted because biotinylated 100BS streptavidin-beads do not bind free biotin and do not mitigate this interference mechanism. Anti-Bt Aby addition caused significant interference in this PTH ELISA assay, resulting in a 97% decrease in detection. When treated with anti-Bt Aby addition, the results changed significantly, differing by +3,546%, but the difference from the reference result was only -1.5%. This result was predicted because biotinylated 100BS streptavidin-beads are designed to bind and deplete anti-biotin interference and report accurate results similar to the reference result without anti-biotin interference. Anti-SAv Aby addition also caused significant interference in this PTH ELISA assay, resulting in a 74% decrease in detection. When treated with anti-SAv Aby addition, the results changed significantly, differing by +253%, but the difference from the reference result was only -8.2%. This result was predicted because biotinylated 100BS streptavidin-beads are designed to bind and deplete anti-streptavidin interference and report accurate results similar to the reference result without anti-SAv Aby interference.Finally, the addition of a 1:1 mixture of anti-SAv Aby and anti-Bt Aby caused significant interference in this PTH ELISA assay, resulting in an 81% decrease in detection. When anti-SAv Aby and anti-Bt Aby additions were processed, the results changed significantly and were +379% different, but the difference from the reference result was only -9.9%. Even with these results, it is within the accuracy profile of this PTH ELISA. This result was predicted because biotinylated 100BS streptavidin-beads are designed to bind and deplete both anti-biotin and anti-streptavidin interferences and report accurate results similar to the reference results without anti-biotin and anti-streptavidin interferences. These data also demonstrate the ability of biotinylated 100BS streptavidin-beads to bind and deplete both interference mechanisms in the same sample simultaneously.

[0111] Example 9 Biotin saturation of soluble streptavidin Prepare a solution of diluted streptavidin (SAv) (or modified SAv or blocked SAv) in Tris-buffered saline, pH 8.5 (TBS) at approximately 200 μg SAv / mL. A TBS dilution solution of biotin is also prepared at approximately 0.50 - 1.00 μg biotin / mL. The two solutions are metered together and pumped through a silicon tube (e.g., PN 96440-13 (Cole Parmer)) meeting at a Y connector (e.g., Masterflex PN 30614-08 (Cole Parmer)) and mixed inline by immediately mixing in an inline mixer (e.g., PPN HT-40-3.18-12-PP (StaMixCo)) in the discharge tube (Figure 7). The SAv solution may be pumped at 2 mL / min, and the biotin solution may be pumped at 18 mL / min, for example, using a peristaltic pump (e.g., Masterflex EZload2 model 07522-20 (Cole Parmer)). The solution containing biotin and streptavidin is mixed for 30 - 120 minutes.

[0112] Other concentrations of biotin and SAv, other buffer systems, and other pump speeds may be used, but the ratio of biotin concentration to SAv concentration and the metered addition ratio must be maintained. The 9-volume biotin solution must contain 7.4 moles of biotin per mole of streptavidin in the SAv solution. Note that the ratio of biotin to streptavidin is slightly higher than that used in the minimum saturation procedure of bead-conjugated SAv. Assuming a molecular weight of streptavidin of 52,000, a 300 mL solution of 200 μg / mL SAv contains 1.1538 moles of streptavidin. Assuming a molecular weight of biotin of 244.31, 8.53072 μmol of biotin (a molar ratio of 7.4:1) is 2084 μg of biotin, and as a result, at 9 volumes the biotin concentration is 772 ng / mL.

[0113] To remove unbound biotin and non-specifically bound biotin, dialysis filtration and washing were performed on biotin-saturated streptavidin. A water bath was filled with purified water and heated to 50 °C. A second water bath was filled with a buffer of 10 mM Tris, 50 - 150 mM NaCl and heated to 50 °C. (Alternatively, this buffer may contain 0.01 - 1% w / v TWEEN® 20 or other surfactants). A storage unit containing biotin-saturated streptavidin was placed in the first water bath. Tubes were attached to the in-line flow ports (upper and lower) and the side port using a hollow fiber filter such as a MiniKros Sampler Hollow Fiber Filter with a molecular weight cut-off of 10 kD (Repligen; PN S04-E010-05-N; mPes; 0.5 mm). The second side port was capped. The tube at the side port led the filtrate to a waste container. The tube led from the storage unit proceeded through a peristaltic pump to the hollow fiber filter. The tube led from the in-line flow discharge port returned the retentate to the storage unit (Figure 8). When the water bath and the storage unit reached 50 °C, the peristaltic pump was turned on, the retentate tube was clamped to generate back pressure for filtration, the volume of the biotin-saturated streptavidin solution was reduced, and the protein was concentrated. The flow rate of the retentate was approximately 360 ml / min and the flow rate of the filtrate was approximately 95 ml / min. When the storage unit reached approximately 15% (or less) of its original volume, buffer from the second water bath was added to the storage unit to restore the original volume. (Samples for quality control were taken immediately before volume restoration). Filtration and volume restoration were repeated at least 5 times in total. Additional washing cycles may be added as needed. After the final volume restoration, the retentate was concentrated to approximately 10% of its original volume (other volumes may be used as needed). The free biotin in the final retentate must be less than 1200 pg / ml. The concentrated biotin-saturated streptavidin was filtered through a 0.2 μm filter.

[0114] Free biotin in the presence of solubilized streptavidin is considered evidence of completion of the biotin quenching process. However, since free biotin itself can cause interference, free biotin is not desirable in blocking reagents that block streptavidin and other interferents and needs to be removed. The holding solution after each wash and the final holding solution (double) were assayed for free biotin content using the ELISA Biotin Assay (Immundiagnostik, PN KR8141), and the results shown in Table 11 were obtained.

Table 11

[0115] The final holding solution contained less than 700 pg / mL of free biotin and was substantially below the requirement of less than 1200 pg / mL. The final holding solution had a concentration of 201 μg of streptavidin / mL such that it contained approximately 3.16 - 3.33 pg of free biotin / μg of streptavidin.

[0116] An additional step may be added to remove incompletely quenched streptavidin. 2-Iminobiotin conjugated to agarose (Sigma Aldrich PN I4507-5ML) can be used for this purpose. 2-Iminobiotin binds reversibly to SAv under alkaline conditions. The binding is strongest at pH 10 - 11. SAv is released under acidic conditions such as pH 4.0. SAv that has already been quenched with biotin should not bind. Therefore, the flow-through of quenched SAv from the column of alkaline 2-iminobiotin-agarose should contain only SAv quenched with biotin. SAv not quenched with biotin should adhere to the column. The column can be regenerated using a pH 4 buffer and then reused.

[0117] Example 10 Removal of goat anti-mouse antibodies using streptavidin beads conjugated with mouse IgG Magnetic nanoparticles (beads) with a diameter of 500 - 600 nm were coated with streptavidin. Two affinity-purified mouse IgG preparations were covalently conjugated with NHS ester-Peg(4)-Biotin to be biotinylated. Mouse IgG #1 was polyclonal non-specific mouse IgG. Mouse IgG #2 was monoclonal mouse IgG. When the beads were exposed to biotinylated mouse IgG, approximately 30 μg of IgG adhered to each 1 mg of the beads. The beads were prepared using only polyclonal mouse IgG, only monoclonal mouse IgG, and a mixture of the two mouse IgG preparations. These mouse antibodies function as capture moieties for any anti-mouse IgG antibody to which they are exposed, either by specific affinity purification or avidity. These mouse IgG-conjugated streptavidin beads were then used to wash samples (in this case buffer) to which affinity-purified goat anti-mouse antibody (Lampire Biological Laboratories) was added. Aliquots of the samples were then analyzed by HPLC size exclusion chromatography for IgG content.

[0118] A concern regarding the use of biotinylation to anchor capture reagents onto streptavidin is that, during the course of the washing procedure, the affinity of biotin may be lower than that of free biotin to the extent that the capture reagent dissociates from streptavidin. To check this, the washing procedure was carried out as normal and in the presence of 20 μg / mL of free biotin (more than 200-fold molar excess over streptavidin). If biotinylated IgG dissociates from streptavidin-coated beads, it cannot rebind in the presence of excess free biotin, and as a result, the amount of goat anti-mouse antibody removed by the beads decreases. Alternatively, if dissociation of biotinylated IgG does not occur to a significant extent, the amount of goat anti-mouse IgG will not change significantly between samples with and without free biotin.

[0119] Specifically, one aliquot of each of three mouse IgG-conjugated streptavidin beads (including IgG#1, IgG#2, and a mixture of the two) was magnetically separated from the 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 mouse IgG-conjugated streptavidin beads, one with biotin exposure and the other without biotin exposure, were magnetically separated from their respective buffers. Using 2.5 mg of beads per mL of goat anti-mouse IgG, goat anti-mouse IgG in TBS was added to the beads without biotin exposure, and goat anti-mouse IgG 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 mixtures had the beads magnetically separated, and then the supernatant samples were analyzed by HPLC-SEC using the area under the curve of the absorbance at 280 nm of the peak corresponding to goat IgG. The results are shown in Table 12.

Table 12

[0120] These data demonstrated that mouse IgG-conjugated streptavidin beads can remove 26.0 - 60.1 μg of anti-mouse antibody per mg of beads. These data further demonstrated that the attachment of the biotinylated capture reagent (mouse IgG) is stable under the conditions of the washing procedure even in the presence of a large excess of free biotin. Finally, these data demonstrated that these procedures are effective with polyclonal and monoclonal antibodies and the capture moiety that is a mixture of the two.

[0121] Example 11. Removal of biotin using streptavidin beads conjugated with mouse IgG The three mouse IgG-conjugated streptavidin bead preparations (polyclonal mouse IgG, monoclonal mouse IgG, and a mixture of polyclonal mouse IgG and monoclonal mouse IgG) described above in Example 10 were tested for their ability to remove free biotin from samples. Using essentially the same protocol as described above, 0.75 mg of beads were mixed with 200 μL of QC3, an in-house QC sample containing approximately 250,000 pg biotin / mL and 50 ng biotin, incubated for 10 minutes, magnetically separated for 5 minutes, and the sample supernatant was collected. The treated samples were tested in an IDK BIOTIN ELISA assay (Immundiagnostik AG, see Example 6). All three bead preparations were able to remove at least 49.7 ng of biotin from 0.200 mL of QC3 (a total of 50 ng of biotin) at 250 ng biotin / mL under these conditions, or 66.3 ng biotin / mg beads, and were able to reduce the biotin concentration from 250,000 pg / mL to less than 300 pg / mL.

[0122] In summary, it is understood that the aspects of this specification are emphasized by reference to specific embodiments, but those skilled in the art will readily recognize that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it is understood that the disclosed subject matter is not limited to the specific methodologies, protocols, and / or reagents, etc. described herein. Thus, various modifications or alterations, or alternative configurations, of the subject matter of this disclosure can be made in accordance with 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 only by the claims. Accordingly, the invention is not limited to being precisely as illustrated and described.

[0123] For the purpose of carrying out the present invention, specific embodiments of the present invention, including the best mode known to the inventor, are described herein. Of course, variations of these described embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventor expects those skilled in the art to appropriately adopt such variations, and the inventors intend that the present invention be practiced in ways other than those specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, unless otherwise stated herein or clearly contradicted by context, any combination of the above-described embodiments is included in the present invention in all possible variations.

[0124] Groupings of alternative embodiments, elements, or steps of the present invention should not be construed as limitations. The components of each group may be referred to and claimed individually or in any combination with the components of other groups disclosed herein. It is contemplated that one or more components of a group may be included in or excluded from the group for reasons of convenience and / or patentability. If any such inclusion or exclusion occurs, the specification is considered to contain the group as thus modified and is therefore considered to satisfy the written description of all Markush groups used in the appended claims.

[0125] Unless otherwise indicated, all numbers expressing characteristics, items, quantities, parameters, properties, periods, etc. used in this specification and the claims are to be understood as being modified in all instances by the term "about." As used herein, the term "about" means that the characteristic, item, quantity, parameter, property, or period so modified encompasses a range of plus or minus 10 percent above and below the value of the stated characteristic, item, quantity, parameter, property, or period. Accordingly, unless the contrary is indicated, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical indication is to be construed in light of the reported significant digits and in reference to the normal rounding procedure. Although the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Nevertheless, any numerical range or value inherently contains certain errors resulting necessarily from the standard deviation found in the respective testing measurements. The recitation of numerical ranges of values herein is merely intended to serve as a convenient method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value within a numerical range is incorporated herein as if it were individually recited herein.

[0126] Unless the context clearly dictates otherwise or there is a contrary indication elsewhere in this specification, (especially in the context of the following claims), the use of the terms "a," "an," "the," and similar referential terms in the context of describing the present invention is to be construed as encompassing both the singular and the plural. Unless the context clearly dictates otherwise or there is a contrary indication elsewhere in this specification, all methods described herein can be performed in any suitable order. The use of any and all examples presented herein, or exemplary language (such as "for example"), is merely intended to illustrate the invention in more detail and is not intended to limit the scope of the invention as claimed. No language in the specification of the invention should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0127] The specific embodiments disclosed herein may be further limited in the claims by use of language such as "consisting of" or "consisting essentially of." When used in the claims, whether applied or added pursuant to amendment, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transitional term "consisting essentially of" limits the claim to the specified material or step and those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are exemplified and enabled as described herein, either explicitly or inherently.

[0128] All patents, patent publications, and other publications referred to and identified in this specification are hereby incorporated by reference in their entirety, individually and expressly, for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are presented only with respect to their disclosure prior to the filing date of the present application. No admission is made that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. Any opinion as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the accuracy of the date or contents of these documents. 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Dissociation kinetics of the streptavidin - biotin interaction measured using direct electrospray ionization mass spectrometry analysis. J Am Soc Mass Spectrom. 24(1): 49 - 56. The present invention provides, for example, the following items. (Item 1) 1. A method for reducing interference from a liquid biological sample, comprising: a) combining the sample with particles comprising streptavidin to provide a mixture; b) mixing the mixture to promote binding of the interference to the streptavidin; c) separating the particles from the sample; thereby eliminating or reducing the amount of said interference. (Item 2) 2. The method of claim 1, wherein the biotin binding sites on the streptavidin are unoccupied, thereby eliminating or reducing interference by anti-streptavidin, biotin, or both. (Item 3) 2. The method of claim 1, wherein the biotin binding sites on the streptavidin are saturated with biotin, thereby eliminating or reducing interference from anti-streptavidin. (Item 4) 4. The method of claim 3, wherein the streptavidin is biotinylated, thereby eliminating or reducing interference from anti-streptavidin, anti-biotin, or both. (Item 5) 5. The method according to any one of claims 1 to 4, wherein the streptavidin is conjugated to an additional non-biotin capture moiety, such that interference from substances that bind to the capture moiety is also eliminated or reduced. (Item 6) The method according to any one of items 1 to 5, wherein the particles are magnetic. (Item 7) The method according to item 6, wherein separating the particles from the sample comprises exposing the mixture to a magnet and recovering the liquid sample. (Item 8) A method for reducing interference from a liquid biological sample, comprising: a) combining the sample with biotinylated streptavidin (QSAv) to provide a mixture; and b) mixing the mixture to promote binding of the interference to the streptavidin, thereby blocking or reducing the amount of anti-streptavidin interference. A method for reducing interference from a liquid biological sample, comprising: (Item 9) The method according to item 8, wherein the QSAv is biotinylated, thereby blocking or reducing interference by anti-streptavidin, anti-biotin, or both. (Item 10) The method according to item 8 or 9, wherein the QSAv is conjugated with an additional non-biotin capture moiety, thereby also blocking or reducing interference by substances that bind to the capture moiety. (Item 11) The method according to any one of items 4, 5, 9, or 10, wherein the biotinylation or conjugation is covalent. (Item 12) The method according to item 11, wherein the biotinylation comprises the use of ester-derivatized biotin. (Item 13) The method according to item 12, wherein the ester-derivatized biotin is selected from the group consisting of NHS-biotin, NHS-LC-biotin, NHS-LC-LC-biotin, TFP-LC-biotin, NHS-chromalink-biotin, NHS-PEO4-biotin, TFP-(PEO)n-biotin, and NHS-(PEO)n-biotin. (Item 14) The method according to any one of items 4, 5, 9, or 10, wherein the biotinylation or conjugation is mediated by a biotin linker non-covalently bound to the biotin-binding site on the streptavidin. (Item 15) The method according to item 6 or 7, wherein the QSAv is primarily monomeric. (Item 16) The method according to any one of items 1 to 15, wherein particles comprising the QSAv or streptavidin are blocked. (Item 17) The method according to item 16, wherein particles comprising the QSAv or streptavidin are blocked by PEGylation. (Item 18) A method for reducing interference in a diagnostic assay, comprising: a) Combining a liquid biological sample with streptavidin saturated with biotin to provide a mixture; b) Mixing the mixture to promote binding of the interference to the streptavidin; c) Performing the diagnostic assay, thereby blocking or reducing the amount of interference in the diagnostic assay, a method. (Item 19) A method for reducing interference in a diagnostic assay, a) Combining the sample with particles containing streptavidin to provide a mixture; b) Mixing the mixture to promote binding of the interference to the streptavidin; c) Separating the particles from the sample; d) Performing the diagnostic assay, thereby removing or reducing the amount of interference, a method. (Item 20) The method according to item 18 or item 19, wherein the steps of combination, mixing, and separation if present occur prior to the analysis stage of the diagnostic assay. (Item 21) The method according to any one of items 18 to 20, wherein the diagnostic assay is a sandwich immunoassay. (Item 22) The method according to any one of items 18 to 20, wherein the diagnostic assay is a competitive immunoassay. (Item 23) The method according to item 5 or item 10, wherein the additional capture moiety blocks, removes, or reduces additional heterophilic interference. (Item 24) The method according to item 23, wherein the additional capture moiety is a heterologous antibody and the heterophilic interference is human anti-animal antibody interference. (Item 25) The method according to item 5 or item 10, wherein the additional capture moiety blocks, removes, or reduces cross-reactivity interference. (Item 26) The method according to item 23, wherein the additional capture moiety is a viral antigen or an antigenic portion thereof. (Item 27) The method according to item 26, wherein the viral antigen or an antigenic portion thereof comprises a coronavirus epitope from a coronavirus other than SARS-CoV-2. (Item 28) Biotin-saturated streptavidin (QSAv) produced by a process comprising exposing the streptavidin to a molar excess of biotin in the range of 5:1 to 11:1. (Item 29) The QSAv according to item 28, wherein the molar excess is 7:1 to 8:1. (Item 30) The QSAv according to item 28 or item 29, further comprising a hot alkaline buffer wash to remove non-specifically bound biotin. (Item 31) The QSAv according to any one of items 28 to 30, further comprising a step of blocking the streptavidin to reduce aggregation. (Item 32) The QSAv according to item 31, wherein the blocking includes including a surfactant in the hot alkaline buffer wash. (Item 33) The QSAv according to item 31 or item 32, wherein the blocking includes covalent modification with a blocking reagent for preparation. (Item 34) The QSAv according to item 33, wherein the blocking includes PEGylation. (Item 35) Biotin-saturated streptavidin-conjugated microparticles (QSAv beads) produced by a process including exposing the streptavidin to a molar excess of biotin in the range of 4:1 to 6:1. (Item 36) The QSAv beads according to item 35, further comprising a warm water wash to remove non-specifically bound biotin. (Item 37) The QSAv or QSAv beads according to any one of items 28 to 36, further comprising conjugating an additional capture moiety to the QSAv or QSAv beads. (Item 38) Streptavidin-conjugated microparticles in which the streptavidin is conjugated to an additional capture moiety. (Item 39) The QSAv, QSAv beads, or streptavidin-conjugated microparticles according to item 37 or item 38, wherein the additional capture moiety is biotinylated and is exposed to the streptavidin or streptavidin-conjugated microparticles before or during the biotin saturation step. (Item 40) The QSAv, QSAv beads, or streptavidin-conjugated microparticles according to item 37 or item 38, wherein the additional capture moiety is covalently conjugated to the streptavidin or streptavidin-conjugated microparticles before, during, or after the biotin saturation step. (Item 41) The QSAv or QSAv beads according to any one of items 28 to 36, further comprising storing the QSAv or QSAv beads in a buffer containing less than 1200 pg / mL of free biotin. (Item 42) The QSAV beads or streptavidin-conjugated microparticles according to any one of items 35 to 41, wherein the microparticles are magnetic. (Item 43) A QSAv solution or a QSAv bead suspension in which the ratio of free biotin to streptavidin does not exceed 1 ng of free biotin: 50 μg of streptavidin.

Claims

【Claim 1】 The invention described in this specification.

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