Blocking method of interfering substances in immunoassays and use thereof

Abstract

This application relates to a method for blocking interfering substances in immunoassay and its application. The method includes: a reaction system for detecting a test sample containing a blocking reagent capable of disrupting the structure of interfering substances in the test sample. The solution provided in this application can weaken or eliminate the signal or signal shielding caused by the binding of interfering substances to the test sample or marker, reducing the incidence of false positives and false negatives, and improving the accuracy of immunoassay.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to a method for blocking interfering substances in immunoassay and its application. Background Technology

[0002] In immune detection, substances that interfere with the immune response, such as heterophilic antibodies, anti-animal antibodies, and rheumatoid factor, may bind nonspecifically to the test substance or marker, interfering with the normal immune response and thus leading to false positives or false negatives in the test results. Summary of the Invention

[0003] To address or partially address the problems existing in related technologies, this application provides a method for blocking interfering substances in immunoassay and its application. This method can weaken or eliminate the interference of interfering substances in the immunoassay system by destroying the structure of the interfering substances, thereby reducing the incidence of false positives or false negatives in the test results and improving the accuracy of immunoassay.

[0004] The first aspect of this application provides a method for blocking interfering substances in an immunoassay, wherein the reaction system for detecting a sample to be tested includes a blocking reagent, which is capable of destroying the structure of interfering substances in the sample to be tested.

[0005] In some embodiments of this application, The blocking agent includes a single blocking agent or a combination blocking agent; Preferably, the single blocking agent includes a biological blocking agent or a chemical blocking agent.

[0006] In some embodiments of this application, the chemical blocking agent is selected from chemical reagents that can destroy antibody structures; preferably selected from at least one of strong acids, strong bases, or reducing agents; more preferably, the reducing agent includes at least one of SDS, cysteine, reduced glutathione, DTT, TCEP, and β-mercaptoethanol.

[0007] In some embodiments of this application, the biological blocking agent is selected from proteolytic enzymes; preferably selected from cysteine ​​proteases, serine proteases, aspartic proteases, or metalloproteinases; more preferably selected from at least one of papain, pepsin, trypsin, subtilisin, bromelain, endonuclease IdeS, endonuclease Lys-C, and endonuclease Lys-N.

[0008] In some embodiments of this application, the composite blocker comprises a proteolytic enzyme and an enzyme-catalyzing reaction promoter, wherein the enzyme-catalyzing reaction promoter provides optimal reaction conditions for the proteolytic enzyme, and / or, the enzyme-catalyzing reaction promoter is an activator of the proteolytic enzyme; preferably, the enzyme-catalyzing reaction promoter comprises at least one of an acid, a base, a metal salt, and an oligopeptide.

[0009] In some embodiments of this application, the content of the proteolytic enzyme in the blocking reagent is 0.05~5 mg / mL, and the content of the enzyme-catalyzed reaction promoter in the composite blocking agent is 0.5~500 mM.

[0010] In some embodiments of this application, the blocking agent comprises pepsin and hydrochloric acid. The pepsin content in the blocking agent is 0.1–1 mg / mL, and the hydrochloric acid content is 1–500 mM; more preferably, the pepsin content is 0.1–0.2 mg / mL, and the hydrochloric acid content is 10–100 mM.

[0011] In some embodiments of this application, the blocking agent comprises papain and reduced glutathione. The papain content in the blocking agent is 0.1–5 mg / mL, and the reduced glutathione content is 1–50 mM; more preferably, the papain content is 1–3 mg / mL, and the reduced glutathione content is 5–20 mM.

[0012] In some embodiments of this application, the blocking agent comprises trypsin, calcium chloride, and sodium hydroxide. The trypsin content in the blocking agent is 0.05~5 mg / mL, and the calcium chloride content is 1~500 mM; more preferably, the trypsin content is 0.1~2 mg / mL, the calcium chloride content is 50~200 mM, and the sodium hydroxide content is 0.005~0.05 mM.

[0013] In some embodiments of this application, the blocking agent further includes a solvent; preferably, the solvent is selected from physiological saline.

[0014] In some embodiments of this application, the sample to be tested does not require additional diluent treatment.

[0015] A second aspect of this application provides a diagnostic product or diagnostic reagent comprising the blocking reagent A described in the first aspect of this application; the diagnostic product includes a reagent kit, a chip, or a test strip.

[0016] In some embodiments of this application, the blocking agent is stored separately; or, diagnostic products or diagnostic reagents containing the blocking agent A are stored separately.

[0017] The third aspect of this application provides the use of a blocking agent as described in the first aspect of this application or a diagnostic product or diagnostic reagent as described in the second aspect of this application in an indirect method, a double-antibody sandwich method, or a competitive method.

[0018] The fourth aspect of this application provides the use of a blocking reagent as described in the first aspect of this application or a diagnostic product or reagent as described in the second aspect of this application in a chemiluminescence detection system, an enzyme-linked immunosorbent assay (ELISA) system, or an immunochromatographic detection system.

[0019] The fifth aspect of this application provides the use of a blocking reagent as described in the first aspect of this application or a diagnostic product or reagent as described in the second aspect of this application in the qualitative detection of antigens or antibodies.

[0020] The technical solution provided in this application may include the following beneficial effects: The blocking reagent of this application reduces or eliminates the signal or shielding signal caused by the binding of interfering substances to the test substance or marker by changing the structure of interfering substances such as heterophilic antibodies, anti-animal antibodies, rheumatoid factor, etc. in the test sample, thereby reducing the incidence of false positives and false negatives and improving the accuracy of immune detection.

[0021] Furthermore, the method for blocking interfering substances in immunoassay provided in this application is applicable to a variety of immunoassay analysis platforms and various immunoassay modes, and has wide applicability, which can meet the needs of most antigen or antibody detection applications in the field of in vitro diagnostics.

[0022] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0023] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0024] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0025] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0026] I. Terminology The term "sample to be tested" as used in this application refers to a mixture that may contain the analyte. Typical samples to be tested that can be used in this application include bodily fluids, including blood, plasma, serum, urine, semen, saliva, etc.

[0027] The term "antibody" as used herein is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin.

[0028] As used in this application, the term "antigen" refers to a substance capable of stimulating an immune response in a matrix and binding with immune response products, antibodies and sensitized lymphocytes, in vivo and in vitro to produce an immune effect. The antigen may be a fusion antigen, and where necessary, the antigen may be further conjugated with other parts, such as specific binding pairing members, for example, biotin or avidin.

[0029] The term "heterophilic anti-body (HA)" as used in this application refers to a class of multispecific immunoglobulins produced by the human body in response to stimulation by known or unknown antigens, which can bind relatively weakly to immunoglobulins from multiple species.

[0030] The term “human anti-animal anti-bodies (HAAA)” as used in this application refers to a class of endogenous interfering antibodies that have a high affinity for target antigens and are induced by the entry of animal proteins into the human body due to iatrogenic and non-iatrogenic factors. Among them, human anti-mouse anti-body (HAAA) is the most common.

[0031] The term "rheumatoid factors (RF)" as used in this application refers to autoantibodies that target the Fc fragment of deformed IgG and are a type of heterophilic antibody.

[0032] The terms “bonding” or “connection” used in this application refer to the direct union between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges.

[0033] The term "specific binding" used in this application refers to a reaction in which two substances mutually distinguish and selectively bind to each other. From a stereostructural perspective, it refers to the conformational correspondence between the reactants in the response.

[0034] The term "specifically binding pair" as used in this application refers to a pair of molecules that can specifically bind to each other, such as enzyme-substrate, antigen-antibody, or ligand-receptor pairs. A specific example of a specific binding pair is the biotin-avidin system, where "biotin" is widely found in animal and plant tissues and has two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the main site of binding to avidin, which is a protein secreted by Streptomyces with a molecular weight of 65 kDa.

[0035] The term "reactive oxygen species" as used in this application refers to a general term for oxygen-containing and reactive substances in the body or natural environment, primarily excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH), as well as nitric oxide and reactive oxygen species). 1 O2), etc.

[0036] As used in this application, the term "microparticle" refers to a small, localized object attributable to physical properties such as volume, mass, or size. Microparticles can be symmetrical, spherical, or irregular, asymmetrical in shape; they can be of any size. Microparticles applicable to this application can be spherical, such as those with diameters in the nanometer or micrometer range. Microparticles can be solids (such as polymers, metals, glass, organic or inorganic materials such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and silica fluids), vesicles (such as synthetic phospholipids, or natural materials such as cells and organelles). Microparticles can be organic or inorganic, such as latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, small oil droplets, silica particles, metal sols, cells, and microcrystalline dyes. Microparticles can be expandable or non-expandable, porous or non-porous, have any density, but preferably have a density close to that of water, are preferably buoyant in water, and are composed of transparent, partially transparent, or opaque materials. The particles may or may not have an electric charge, and when they do, they are preferably negatively charged. The particles are typically multifunctional or capable of binding to donors or acceptors through specific or non-specific covalent or non-covalent interactions. Typical functional groups include carboxyl, acetaldehyde, amino, cyano, vinyl, hydroxyl, and mercapto groups. A non-limiting example of the particles suitable for use in this application is polystyrene latex microspheres. The particles described in this application have a diameter of 50 nm to 20 μm; in some embodiments, the particles have a diameter of 100 nm to 10 μm; in some embodiments, the particles have a diameter of 200 nm to 5 μm; and in some embodiments, the particles have a diameter of 750 nm to 1 μm.

[0037] As used herein, the term "blocking reagent" refers to a substance or reagent that can reduce or eliminate interfering antibodies in immunoassays. In this document, the blocking reagent includes single blocking agents or compound blocking agents; specifically, a single blocking agent is a reagent containing only one substance that has a blocking effect, preferably including biological blocking agents (e.g., proteolytic enzymes) and chemical blocking agents (e.g., strong acids, strong bases, or reducing agents); a compound blocking agent is a reagent containing at least two substances that work together to have a blocking effect, such as a proteolytic enzyme and an enzyme reaction promoter, wherein the enzyme reaction promoter can promote and / or activate the enzyme reaction.

[0038] As used in this article, the term "enzyme reaction promoter" refers to a substance or reagent that can provide an optimal environment for an enzymatic reaction or activate it. In this article, enzyme reaction promoters include chemical reagents that disrupt antibody structures and promote and / or activate enzymatic reactions, such as acids, bases, metal salts, oligopeptides (e.g., reduced glutathione), etc.

[0039] II. Specific Implementation Plan This application will now be described in more detail.

[0040] The method for blocking interfering substances in immunoassay according to the first aspect of this application involves adding a blocking reagent to the system for detecting the sample to be tested, thereby destroying the structure of the interfering substances in the sample to be tested, thereby reducing or eliminating the possibility of non-specific binding between the interfering substances and the sample or marker in the immunoassay system, and reducing the possibility of false positive and false negative results.

[0041] The interfering substances described in this application are endogenous interfering antibodies commonly used in immunoassay. Endogenous interfering antibodies refer to antibodies that can bind non-specifically or specifically to the analyte or marker, interfering with the specific reaction between antigen and antibody, and are present in human blood or other body fluids, such as heterophilic antibodies, anti-animal antibodies, rheumatoid factor, etc.

[0042] Among them, the interference mechanism of heterophilic antibodies in immunoassay mainly occurs in double-antibody sandwich immunoassay and competitive immunoassay.

[0043] For example, in the sandwich ELISA assay, the test antigen binds simultaneously to both the capture antibody and the labeled antibody, forming a normal solid-phase capture antibody-test antigen-labeled antibody complex. In the Fc-Fc binding mechanism, the Fc terminus of the heterophile antibody binds to the Fc terminus of the capture antibody, while the Fab region of the heterophile antibody binds to the Fab region of the labeled antibody, leading to false positive results. In the Fab-Fab binding mechanism, the Fab region of the heterophile antibody, by binding to both the Fab regions of the capture antibody and the labeled antibody, blocks the binding of the capture antibody and the labeled antibody to the test antigen, thus leading to false negatives. The F(ab')2 region of the heterophile antibody, by simultaneously binding to both the Fab regions of the capture antibody and the Fab region of the labeled antibody, forms a solid-phase capture antibody-heterophile antibody-labeled antibody complex. If the test antigen is not present in the sample, the heterophile antibody will cause a false positive result.

[0044] For example, in competitive immunoassays, the test antigen and the labeled antigen compete to bind to the capture antibody attached to the carrier, forming a normal reaction complex. When heterophilic antibodies bind to both the capture antibody and the labeled antigen simultaneously, forming an abnormal reaction complex, they interfere with the capture antibody's binding to the detection antigen and consume the capture antibody that can bind to the test antigen, thus leading to a false negative. Heterophilic antibodies can also directly bind to the site where the capture antibody binds to the antigen, forming an abnormal complex that occupies the binding space of the capture antibody to the test antigen or labeled antigen, consuming the capture antibody that can bind to the test antigen or labeled antigen, leading to a false positive.

[0045] In sandwich immunoassays, HAMA in the sample binds between mouse immunoglobulin capture antibodies and mouse immunoglobulin labeled antibodies, resulting in false positive results. HAMA binds to solid-phase antibodies or labeled antibodies, thus hindering the binding of analytical reagents to analytes. In competitive immunoassays, HAMA interferes by blocking capture antibodies and binding sites, resulting in false negative results.

[0046] In the double-antibody sandwich immunoassay, when RF binds to the Fc fragment of both the capture antibody and the labeled antibody simultaneously, it bridges the capture antibody and the labeled antibody, forming a capture antibody-RF-signal antibody complex, thus producing a false positive result. When RF can only bind to one of the capture antibody or the labeled antibody, it forms a steric hindrance (shield), preventing the formation of the capture antibody-antigen-labeled antibody complex, thus producing a false negative result.

[0047] To address the aforementioned interfering antibodies, most commercially available solutions employ dilution methods or pretreatment with blocking agents to eliminate or minimize the presence of interfering antibodies in the samples.

[0048] The dilution method involves serially diluting the sample to reduce the concentration of endogenous interfering antibodies. While this can reduce interference to some extent, its blocking effect is not ideal. Blocking agents, such as heterophile antibody blockers, are typically animal serum or animal-derived immunoglobulins. When co-incubated with the sample, they bind to the heterophile antibodies in the sample, inhibiting their binding to the analyte or label through steric hindrance, thus blocking the interference of heterophile antibodies. The blocking mechanism of anti-animal antibody blockers and immunoglobulin blockers is consistent with that of heterophile antibody blockers.

[0049] Therefore, by adding an inhibitor to the sample to be tested, the specific binding between the inhibitor and the interfering antibody can reduce the amount of interfering antibody. However, the effectiveness of eliminating interference is usually determined by the type or subtype of the interfering antibody, the type or subtype of the inhibitor, and also by factors such as the concentration of the interfering antibody and the concentration of the inhibitor. Generally speaking, an inhibitor targets only a single interfering antibody. Due to individual differences, clinical samples vary greatly, and the types and concentrations of interfering antibodies differ. Currently, common inhibitors cannot achieve ideal blocking effects.

[0050] In some implementations... The blocking agent includes a single blocking agent or a combination blocking agent; Preferably, the single blocking agent includes a biological blocking agent or a chemical blocking agent.

[0051] Unlike existing common interfering antibody blocking agents, the blocking reagent described in this application weakens or eliminates interference by destroying the structure of interfering substances in the test sample. It is universally applicable, targeting almost all interfering antibodies. Furthermore, depending on the specific type of interfering antibody, different biological blocking agents, chemical blocking agents, or a combination of both can be selected, and the concentration of the blocking reagent can be controlled to minimize its impact on the antigen or specific antibody and destroy the interfering antibody structure as much as possible, achieving a relatively thorough elimination of interference and resulting in good blocking efficacy.

[0052] In some embodiments, the blocking agent comprises a chemical blocking agent selected from compounds that can disrupt antibody structures, such as protein denaturants, strong acids or bases, or reducing agents.

[0053] The blocking reagent described in this application may independently contain one protein denaturant or reducing agent, or may simultaneously contain one protein denaturant and one reducing agent. The protein denaturant described in this application may also contain two or more protein denaturants; it may contain two or more denaturants; it may contain two or more protein denaturants and one reducing agent, or two or more protein denaturants and two or more reducing agents, or one protein denaturant and two or more denaturants; this application does not impose any limitations herein.

[0054] In this embodiment, the protein denaturing agent can disrupt the secondary or tertiary structure of the interfering antibody by breaking its hydrophobic bonds, hydrogen bonds, van der Waals forces, etc., thereby weakening or eliminating the interference of the interfering antibody. Similarly, the reducing agent can disrupt the structure of the interfering antibody by breaking its disulfide bonds, thus weakening or eliminating its interference.

[0055] In some specific embodiments, chemical blocking agents may include, but are not limited to, strong acids (e.g., hydrochloric acid), strong bases (e.g., sodium hydroxide), readily soluble salts (e.g., calcium chloride, potassium thiocyanate), SDS (Sodium dodecyl sulfate), heavy metal salts (e.g., copper sulfate), urea, guanidine hydrochloride, guanidine isothiocyanate, and some organic solvents (e.g., methanol, ethanol, acetone), cysteine, reduced glutathione, β-mercaptoethanol, DTT (dithiothreitol), and TCEP (tris-(2-carboxyethyl)phosphine), etc.

[0056] In some embodiments, the blocking agent comprises a biological blocking agent selected from proteolytic enzymes. The blocking agent described in this application may independently comprise one proteolytic enzyme, or it may comprise two or more proteolytic enzymes.

[0057] In this embodiment, the proteolytic enzyme breaks down the peptide bonds of the interfering antibody, thereby destroying the structure of the interfering antibody and weakening or eliminating its interference.

[0058] In some specific embodiments, the proteolytic enzyme may be selected from cysteine ​​proteases, serine proteases, aspartic proteases, or metalloproteinases.

[0059] Cysteine ​​proteases, also known as thiol proteases, contain a thiol group (cysteine) in their active site. This thiol group catalyzes the hydrolysis of antibody peptide bonds. Cysteine ​​proteases include, but are not limited to, papain, bromelain, and fig protease. For example, papain can hydrolyze lysine, arginine, and the carboxyl terminus of proteins. Its optimal pH for application is 5–7, and it can be used to weaken or eliminate interference from various types of interfering antibodies.

[0060] Serine proteases are endopeptidases, also known as alkaline proteases. Their active site contains a serine residue, which acts as a catalytic group to cleave the peptide bonds of interfering antibodies, thereby disrupting their structure. Serine proteases include, but are not limited to, subtilisin, trypsin, chymotrypsin, and elastase. For example, trypsin can hydrolyze the carboxyl terminus of lysine and arginine, and its optimal pH for application is 7–9.

[0061] Aspartic proteases are endopeptidases whose active site contains an aspartic acid residue. This aspartic acid residue acts as a catalytic group, interfering with the cleavage of peptide bonds in antibodies, thereby disrupting the antibody's structure. Aspartic proteases include, but are not limited to, pepsin and cathepsin D. Taking pepsin as an example, it tends to cleave peptide bonds of amino acids or amino acids with carboxyl terms such as phenylalanine, tryptophan, glutamic acid, tyrosine, or leucine. The optimal pH for its application is 1–4.

[0062] In some embodiments, the proteolytic enzyme may also include specific endonucleases corresponding to the type of interfering antibody, such as IdeS, Lys-C, Lys-N, etc.

[0063] Among them, the endonuclease IdeS (Immunoglubulin G-degrading enzyme of Streptococcus pyogenes) comes from Streptococcus pyogenes and is a common thiol protease from type A streptococci. It has intrapeptidase activity that hydrolyzes IgG and can recognize the hinge region of interfering antibodies, namely the space between the CH1 and CH2 domains, and specifically degrade IgG, thereby destroying the structure of interfering antibodies.

[0064] The endonuclease Lys-C is a lysine protease that specifically hydrolyzes the carboxyl terminus of lysine residues, thereby disrupting the structure of interfering antibodies.

[0065] The endonuclease Lys-N is also a lysine protease that can specifically hydrolyze the amino terminus of lysine, thereby achieving the effect of destroying and interfering with the antibody structure.

[0066] The blocking reagent described in the embodiments of this application may independently contain one specific endonuclease, or may contain two or more specific endonucleases; the specific endonuclease may also be used in combination with other proteolytic enzymes (such as thiol proteases, serine proteases or aspartic proteases).

[0067] In some preferred embodiments, the proteolytic enzyme is selected from at least one of papain, pepsin, trypsin, subtilisin, bromelain, endonuclease IdeS, endonuclease Lys-C, and endonuclease Lys-N.

[0068] When the blocking agent described in this application includes two proteolytic enzymes, such as a mixture of alkaline protease and papain, the interfering antibody is enzymatically hydrolyzed under alkaline conditions. The alkaline protease has cleavage sites at the amide bonds at the carboxyl ends of all hydrophobic and aromatic amino acids on its carboxyl side chain, catalyzing the hydrolysis of peptide bonds within the interfering antibody molecule. The peptide bonds catalyzed by papain and alkaline protease are complementary, which can maximally hydrolyze the interfering antibody, thereby improving the effect of weakening / eliminating the interference of the interfering antibody.

[0069] In some embodiments, the composite blocking agent comprises a protease and an enzyme-catalyzing promoter. The enzyme-catalyzing promoter provides optimal reaction conditions for the protease, and / or acts as an activator of the protease. The enzyme-catalyzing promoter includes at least one of an acid, a base, a metal salt, or an oligopeptide.

[0070] In some embodiments, the content of the proteolytic enzyme in the blocking agent is 0.5-5 mg / mL, and the content of the enzyme-catalyzing reaction promoter in the composite blocking agent is 0.5-500 mM.

[0071] In some specific embodiments, the blocking reagent may contain pepsin and hydrochloric acid. In this case, hydrochloric acid provides suitable reaction conditions for pepsin, and at the same time, hydrochloric acid acts as an activator for pepsin to enzymatically decompose the structure of interfering antibodies. The hydrochloric acid environment enables pepsin to maintain high activity, thereby improving the efficiency of pepsin in hydrolyzing the peptide bonds of interfering antibodies and achieving the effect of destroying as much of the interfering antibody structure as possible, greatly reducing / eliminating the interference of interfering antibodies in the immunoassay system. Meanwhile, hydrochloric acid itself also has the effect of breaking the hydrogen bonds of interfering antibodies. Therefore, when hydrochloric acid and pepsin are used in combination, the effect of destroying the structure of interfering antibodies is better, which can further enhance the effect of reducing / eliminating the interference of interfering antibodies.

[0072] The blocking reagent contains pepsin at a concentration of 0.1–1 mg / mL and hydrochloric acid at a concentration of 1–500 mM. For example, the pepsin concentration can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.8 mg / mL, or 1 mg / mL, and the hydrochloric acid concentration can be 1 mM, 10 mM, 30 mM, 50 mM, 70 mM, 90 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, or 500 mM.

[0073] Preferably, the pepsin content is 0.1~0.2 mg / mL and the hydrochloric acid content is 10~100 mM; more preferably, the pepsin content is 0.1 mg / mL and the hydrochloric acid content is 50 mM.

[0074] In this embodiment, by leveraging the synergistic effect between hydrochloric acid and pepsin, specific enzyme-catalyzing promoters in the blocking reagent, and by controlling the concentrations of hydrochloric acid and pepsin in the blocking reagent, not only can the destructive effect of the blocking reagent on the structure of interfering antibodies be enhanced, but the influence of the blocking reagent on the immune response can also be reduced. In other words, by controlling the content of pepsin and hydrochloric acid within the aforementioned range, the influence of the blocking reagent on the specific antigen-antibody response can be minimized, thereby reducing the impact of the blocking reagent on the immunoassay results and ensuring the accuracy of the test results.

[0075] In some specific embodiments, the blocking agent may contain papain and reduced glutathione. In this case, both reduced glutathione and papain have sulfhydryl groups. Reduced glutathione acts as an activator for papain to enzymatically degrade the structure of interfering antibodies. The two work synergistically to enhance the effect of papain, making the blocking agent more effective in destroying the structure of interfering antibodies and effectively enhancing the effect of weakening / eliminating the interference of interfering antibodies.

[0076] The blocking reagent contains papain at a concentration of 0.1–5 mg / mL and reduced glutathione at a concentration of 1–50 mM. For example, the papain concentration can be 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL; and the reduced glutathione concentration can be 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

[0077] Preferably, the papain content is 1-3 mg / mL and the reduced glutathione content is 5-20 mM; more preferably, the papain content is 2 mg / mL and the reduced glutathione content is 10 mM.

[0078] In this embodiment, by leveraging the synergistic effect between the specific enzymatic reaction promoters reduced glutathione and papain in the blocking reagent, and by controlling the concentrations of reduced glutathione and papain in the blocking reagent, not only can the destructive effect of the blocking reagent on the structure of interfering antibodies be enhanced, but the influence of the blocking reagent on the immune response can also be reduced. Specifically, controlling the content of reduced glutathione and papain within the aforementioned range minimizes the impact of the blocking reagent on the specific antigen-antibody response, reduces the influence of the blocking reagent on the immunoassay results, and ensures the accuracy of the test results.

[0079] In some specific embodiments, the blocking agent may comprise trypsin, calcium chloride, and sodium hydroxide, wherein the Ca... 2+ Sodium hydroxide acts as a trypsin activator, enhancing the effectiveness of trypsin. When trypsin and calcium chloride are used together, their synergistic effect results in a better destructive effect on interfering antibodies. Sodium hydroxide regulates the pH of the blocking agent, allowing trypsin to function under suitable acidic or alkaline conditions. Furthermore, when sodium hydroxide is used in combination with calcium chloride, it simultaneously activates trypsin, further enhancing the destructive effect on interfering antibodies. Additionally, sodium hydroxide itself has a certain effect on breaking the hydrogen bonds of interfering antibodies; therefore, the combined use of trypsin, calcium chloride, and sodium hydroxide can further enhance the elimination of interfering antibody interference.

[0080] The blocking reagent contains trypsin at a concentration of 0.05–5 mg / mL, calcium chloride at a concentration of 1–500 mM, and sodium hydroxide at a concentration of 0.005 mM–0.05 mM. For example, the trypsin concentration can be 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, etc.; the calcium chloride concentration can be 1 mM, 10 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, etc.; and the sodium hydroxide concentration can be 0.005 mM, 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, etc.

[0081] Preferably, the trypsin content is 0.1~2 mg / mL, the calcium chloride content is 50~200 mM, and the sodium hydroxide content is 0.01~0.02 mM; more preferably, the trypsin content is 0.5 mg / mL, the calcium chloride content is 100 mM, and the sodium hydroxide content is 0.01 mM.

[0082] In this embodiment, by leveraging the synergistic effect between the specific enzymatic reaction promoters calcium chloride and sodium hydroxide in the blocking reagent and trypsin, and by controlling the concentrations of trypsin, calcium chloride, and sodium hydroxide in the blocking reagent, not only can the destructive effect of the blocking reagent on the structure of interfering antibodies be enhanced, but the influence of the blocking reagent on the immune response can also be reduced, thus ensuring the accuracy of the detection results.

[0083] In some embodiments, the blocking agent further includes a solvent; preferably, the solvent is selected from physiological saline.

[0084] The solvent described in this application allows the concentration of the blocking reagent to be controlled within a suitable range, enabling the blocking reagent to effectively eliminate interference from interfering antibodies while minimizing its impact on the specific antigen-antibody reaction, thus ensuring the accuracy of the immunoassay results. When physiological saline is chosen as the solvent, it can adjust the osmotic pressure, and its pH of 7 will not affect the pH of the components in the blocking reagent, thereby avoiding any impact on the blocking effect.

[0085] In some implementations, a blocking reagent may be added to the sample to be tested, followed by incubation and the addition of a detection reagent.

[0086] In some implementations, a blocking reagent and a detection reagent may be added sequentially to the sample to be tested, without incubation between the addition of the blocking reagent and the detection reagent.

[0087] In this embodiment, the blocking reagent is incubated with the sample to be tested before the detection reagent is added, and the detection reagent is added directly without allowing incubation time after adding the blocking reagent. Both of these blocking methods can achieve good clearance of interfering antibodies in the sample to be tested, which can reduce the possibility of false positives or false negatives in the test results.

[0088] When the blocking reagent is added to the test sample, it can immediately destroy the structure of the interfering antibody in the test sample, and the destruction effect is good. Therefore, it not only has a good effect of weakening or even eliminating the signal (false positive) or shielding the signal (false negative) brought by the interfering antibody, but also has a fast effect of weakening or eliminating the interference of the interfering antibody, resulting in a good blocking effect.

[0089] In some implementations, the sample to be tested does not require additional diluent treatment.

[0090] In this embodiment, there is no need for an additional step of diluting the test sample, which reduces the cumbersome dilution steps when performing immunoassay on the test sample and improves the detection efficiency.

[0091] In some embodiments, the blocking agent is stored separately.

[0092] In this embodiment, the prepared blocking reagent is stored separately and not together with the detection reagent. This avoids the blocking reagent from reacting with the detection reagent during storage, which could affect the blocking effect of the blocking reagent and the detection results of the detection reagent, thereby improving the accuracy of the immunoassay results.

[0093] A second aspect of this application provides a diagnostic product or diagnostic reagent comprising the blocking reagent described in the first aspect of this application; wherein the diagnostic product includes a reagent kit, a chip, or a test strip. Therefore, the blocking reagent can be configured as a diagnostic reagent for use in an immunoassay process; for example, the blocking reagent can be configured as a diagnostic reagent as a component of a reagent kit.

[0094] In some embodiments, diagnostic products or diagnostic reagents containing the blocking agent are stored separately. For example, when the blocking agent is configured as a diagnostic reagent and is part of a kit, the blocking agent is stored separately from other test reagents in the kit and is not exposed to contact before use to avoid reaction between the blocking agent and the test reagent components.

[0095] In this embodiment, the kit consisting of a detection reagent and a blocking reagent can be used in a chemiluminescence detection system, such as in an immunoassay analysis platform for photo-induced chemiluminescence detection and magnetic particle chemiluminescence detection.

[0096] Specifically, the detection reagents used in the photo-induced chemiluminescence platform include: The R1 reagent includes luminescent microparticles and antigens / antibodies bound to them. The luminescent microparticles are doped with dimethylthiophene and lanthanide metal complexes, which can react with singlet oxygen to generate a detectable signal. The antigens / antibodies bound to the luminescent microparticles can specifically bind to the analyte. R2 reagent includes a marker (e.g., biotin) and an antigen / antibody bound thereto, wherein the antigen / antibody bound to the first marker can specifically bind to the analyte; The R3 reagent includes photosensitive microparticles and a bound marker (e.g., avidin), wherein the photosensitive microparticles are doped with a photosensitizer and can generate singlet oxygen upon photoexcitation. The markers in both the R3 and R2 reagents can be selected from members of a specific binding pair, and the markers in the R3 and R2 reagents can bind specifically.

[0097] The above-mentioned reagents R1, R2, R3, and blocking reagents should be stored separately.

[0098] The kit composed of the above reagents can be used for indirect immunoassay analysis on a photo-induced chemiluminescence detection platform. When the analyte is present, it forms an immune complex with the antigen / antibody in reagents R1 and R2 through a double-antibody sandwich mechanism. Photosensitive microparticles, stimulated by excitation light, generate singlet oxygen. The luminescent microparticles react with this singlet oxygen to generate a detectable light signal, thus enabling immunoassay of the analyte based on photo-induced chemiluminescence detection.

[0099] In some specific embodiments, reagents R1, R2, and R3 further include buffer solutions, which may contain protein stabilizers and preservatives; preferably, the buffer solutions may be selected from HEPES buffer, Tris-HCl buffer, or PBS buffer.

[0100] In some specific embodiments, the lanthanide metal complex is selected from one or more of europium, terbium, and samarium.

[0101] This application also provides a method for using a photo-induced chemiluminescence detection kit, comprising: adding a blocking reagent to the sample to be tested, wherein the blocking reagent destroys the structure of the interfering antibody in the sample to be tested, and then adding the detection reagent.

[0102] In some embodiments, the detection reagent used in the magnetic particle chemiluminescence detection platform includes: R1 reagent includes magnetic microparticles and antigens / antibodies bound thereto, wherein the antigens / antibodies bound to the magnetic microparticles can specifically bind to the analyte. R2 reagents include chemiluminescent labels (e.g., acridinium esters) and antigens / antibodies bound thereto, wherein the antigens / antibodies bound to the chemiluminescent labels can specifically bind to the analyte; R3 reagents include luminescent substrates that can chemically react with chemiluminescent labels.

[0103] The above-mentioned reagents R1, R2, R3, and blocking reagents should be stored separately.

[0104] The kit composed of the above reagents can be used for double-antibody sandwich immunoassay analysis on a magnetic microparticle chemiluminescence detection platform. When the analyte is present, it forms an immune complex with the antigen / antibody in reagents R1 and R2 through a double-antibody sandwich reaction. An external magnetic field causes the immune complex to aggregate with the magnetic microparticles. A light signal is generated through the reaction between the luminescent substrate and the chemiluminescent label, thus enabling immunoassay of the analyte based on magnetic microparticle chemiluminescence detection.

[0105] In some specific embodiments, reagents R1, R2, and R3 further include buffer solutions, which may contain protein stabilizers and preservatives; preferably, the buffer solutions may be selected from HEPES buffer, Tris-HCl buffer, or PBS buffer.

[0106] This application also provides a method for using a magnetic microparticle chemiluminescence detection kit, comprising: adding a blocking reagent to the sample to be tested, wherein the blocking reagent destroys the structure of the interfering antibody in the sample to be tested, and then adding the detection reagent.

[0107] In other embodiments, the kit may include: Solid-phase carriers coated with antigens / antibodies; Enzyme-labeled antigens / antibodies (e.g., horseradish peroxidase HRP); Enzyme substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine) chromogenic solution).

[0108] The above-mentioned testing reagents and blocking reagents should be stored separately.

[0109] This application also provides a method for using an enzyme-linked immunosorbent assay (ELISA) kit, comprising: adding a blocking reagent to the sample to be tested, wherein the blocking reagent destroys the structure of the interfering antibody in the sample to be tested, and then adding the above-mentioned detection reagent.

[0110] The kit composed of the above reagents can be used for competitive immunoassay analysis on an enzyme-linked immunosorbent assay (ELISA) platform. When the analyte is present, it specifically binds to the antigen / antibody on the solid-phase carrier to form an antigen-antibody complex. The enzyme-labeled antigen / antibody also binds to the solid-phase carrier. The enzyme substrate reacts with the enzyme to generate a detectable signal, thus enabling the immunoassay of the analyte based on ELISA.

[0111] In other embodiments, the test strip comprises a sample pad prepared by treatment with the blocking reagent improved in the first aspect of this application. Preferably, the blocking reagent comprises papain and reduced glutathione.

[0112] This application also provides a method of using a test strip, comprising: dropping the sample to be tested onto a sample pad treated with a blocking reagent, wherein the blocking reagent destroys the structure of the interfering antibody in the sample to be tested.

[0113] This application also provides the application of blocking reagents as described in the first aspect of this application or diagnostic products or reagents as described in the second aspect of this application in various immunoassay analysis platforms, such as chemiluminescence detection systems, enzyme-linked immunosorbent assay (ELISA) systems, or immunochromatographic detection systems.

[0114] This application also provides the application of blocking reagents as described in the first aspect of this application or diagnostic products or reagents as described in the second aspect of this application in various immunoassay modalities, such as indirect methods, double-antibody sandwich methods, or competitive methods.

[0115] This application also provides the use of blocking reagents as described in the first aspect of this application or diagnostic products or reagents as described in the second aspect of this application in the qualitative detection of antigens or antibodies, such as HBc-IgM, Rubella IgM, HBs Ag, HBc Ab, cytomegalovirus IgM, etc.

[0116] III. Specific Implementation Examples To make the present invention easier to understand, the present application will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not limited to the scope of application of the present application. Unless otherwise specified, the raw materials or components used in the present application can be obtained commercially or by conventional methods.

[0117] Example 1: Application of the blocking method in the detection of HBc-IgM by photo-induced chemiluminescence 1. Experimental Procedure (1) Kit preparation Reagent 1: HBc Ag was coated with aldehyde-based luminescent microparticles at a mass ratio of 10:0.1 and diluted to 25 μg / mL with 50 mM HEPES buffer containing protein stabilizers and preservatives to prepare Reagent 1.

[0118] Reagent 2: Mouse anti-human IgM was labeled with N-hydroxysuccinimide biotin (BNHS) at a molar ratio of 20:1 and diluted to 3 μg / mL with 100 mM Tris-HCl buffer containing protein stabilizer and preservative to prepare Reagent 2.

[0119] Reagent 3: Aldehyde-based photosensitive microparticles are coated with streptavidin at a mass ratio of 10:0.5 and diluted to 50 μg / mL with 50 mM HEPES buffer containing protein stabilizers and preservatives to prepare Reagent 3.

[0120] Reagent 4 (Blocking Reagent): Reagent 4 is prepared by combining different chemical and / or biological reagents. The content of each component is shown in the table below.

[0121] Table 1. Preparation of Reagent 4

[0122] (2) Detection of HBc-IgM using an indirect method 10 μL of the sample to be tested, 25 μL of reagent 4, 25 μL of reagent 1 and 25 μL of reagent 2 were added to the reaction wells in sequence. After reacting for 15 min, reagent 3 was added and reacted for 10 min. The signal value was read at a wavelength of 620 nm.

[0123] If the sample to be tested contains specific HBc-IgM, it can form a luminescent microparticle-HBc Ag-HBc-IgM-mouse anti-human IgM-biotin-avidin-photosensitive microsphere bridge to generate a signal; otherwise, it cannot.

[0124] 2. Experimental Results Table 2. Signal values ​​of four samples tested with different reagents.

[0125] Table 3. Ratios of different reagents 4 to the control

[0126] 3. Experimental Data Analysis As shown in the table above, the addition of chemical, biological, or combined blocking agents all had a certain blocking effect on negative samples 2-6 with excessively high detection signal values. However, the blocking effect of chemical blocking agents (②-⑥) was not as good as that of biological blocking agent (⑦), and it had a greater impact on positive samples. Biological blocking agent (⑦) had a significant blocking effect on false positive samples and had little impact on positive samples. The best effect was achieved by combined blocking agents (⑧, ⑨, ⑩), because the enzyme reaction promoter is an activator of proteolytic enzymes (Ca). 2+ When combined with trypsin, reduced glutathione, and papain, or under optimal conditions for proteolytic enzymes (hydrochloric acid and pepsin), the effects of proteolytic enzymes can be further enhanced.

[0127] The blocking method, which involves adding blocking reagents to disrupt the structure of interfering substances, is applicable to photo-induced chemiluminescence platforms and can significantly suppress false positive sample signals without affecting positive samples.

[0128] Example 2: Optimal Concentration Optimization of Blocking Agent in Blocking Methods 1. Experimental Procedure The optimal concentration of the blocking methods for the two effective combination of compound blocking agents (⑨, ⑩) used in Example 1 was optimized. Based on the components of the two blocking reagents (⑨, ⑩) in Example 1, the concentration of each component was adjusted to prepare Reagent 4 of the HBc-IgM photochemiluminescence reagent kit. The optimal concentration conditions were then screened by testing samples. The content of each component in Reagent 4 is shown in the table below.

[0129] Table 4. Preparation of Reagent 4

[0130] 2. Experimental Results (1) The results of testing the sample using reagent 4 of combination one.

[0131] Table 5. Results of Concentration Optimization (Signal Value) Test

[0132] (2) The results of testing the sample using reagent 4 of combination two.

[0133] Table 6. Results of Concentration Optimization (Signal Value) Test

[0134] 3. Experimental Data Analysis As shown in Table 5, for combination one: When the papain concentration was 2 mg / mL, its efficiency in destroying interfering antibodies reached saturation, and further increasing the papain concentration had no significant difference. High concentrations of reduced glutathione showed high inhibitory effects on both false-positive and true-positive samples. Therefore, the optimal concentration of combination one is papain at no less than 2 mg / mL and reduced glutathione at approximately 10 mM.

[0135] As shown in Table 6, for combination two: Higher pepsin concentrations resulted in greater inhibition of both false and true positive samples. While a hydrochloric acid concentration of 100 mM showed the best inhibitory effect on false positive samples, it also affected the normal antigen-antibody response. Therefore, the optimal concentrations for combination two were pepsin at no less than 0.1 mg / mL and hydrochloric acid at approximately 50 mM.

[0136] Example 3: Application of the blocking method in the detection of Rubella IgM by photo-induced chemiluminescence 1. Experimental Procedure The preparation and detection process of the reagent kit were carried out in accordance with Example 1. The difference between the reagent kit and Example 1 is that: Reagent 1 contains RV Ab coated with aldehyde-based luminescent microspheres at a ratio of 10:0.5 and a concentration of 25 μg / mL; Reagent 2 contains biotin-labeled mouse anti-human IgG monoclonal antibody (RV Ag) at a ratio of 30:1 and a concentration of 4 μg / mL.

[0137] Reagent 4 was prepared using the blocking method of the composite blocking agent (⑩) with better effect in Example 1, and the samples were tested. The test results are shown in the table below.

[0138] 2. Experimental Results Table 7. Detection results of Rubella IgM samples

[0139] 3. Experimental Data Analysis As shown in the table above, the combination of pepsin and hydrochloric acid has a significant effect on eliminating interference. As the concentration of pepsin increases, although the reactivity of positive samples decreases slightly, the reactivity of negative samples with excessively high detection signal values ​​is significantly weakened. The combination of 0.1 mg / mL pepsin and 50 mM hydrochloric acid has the best effect on destroying the interference of interfering antibodies.

[0140] Example 4: Application of the blocking method in the detection of HBsAg by magnetic microparticle chemiluminescence 1. Experimental Procedure (1) Kit preparation Magnetic microparticle suspension: Carboxylated magnetic microparticles were conjugated with Anti-HBs Ag monoclonal antibody at a mass ratio of 10:0.5 and diluted to 0.2 mg / mL with 100 mM Tris-HCl buffer containing protein stabilizers and preservatives to prepare a magnetic microparticle suspension.

[0141] Acridinium ester conjugate: Anti-HBs Ag monoclonal antibody was conjugated with acridinium ester at a mass ratio of 2:1 and diluted to 5 μg / mL with 20 mM PBS buffer containing protein stabilizer and preservative to prepare acridinium ester conjugate.

[0142] Blocking reagent: Dilute pepsin to 0.1 mg / mL with 0.05 mol / L hydrochloric acid to prepare the blocking reagent.

[0143] Luminescent substrates: Luminescent substrate A was prepared using 0.05% H2O2 and 0.5% nitric acid, and luminescent substrate B was prepared using 0.05 mol / L NaOH and 0.1% Triton X-100.

[0144] (2) Detection of HBsAg using the double-antibody sandwich method Take 20 μL of serum sample, add 50 μL of blocking reagent, react for 5 min, then add 50 μL of magnetic microparticle suspension and 100 μL of acridinium ester conjugate sequentially, react for 20 min, and apply an external magnetic field to aggregate the magnetic microparticles and their conjugates; remove the supernatant, wash with 0.1% Tween to remove unbound substances, remove the supernatant by magnetic separation, add 100 μL each of luminescent substrates A and B, and detect the light signal.

[0145] If the sample to be tested contains specific HBsAg, a magnetic microparticle-Anti-HBsAg monoclonal antibody-HBsAg-Anti-HBsAg monoclonal antibody-acridoid ester bridging can be formed to generate a signal; otherwise, it cannot.

[0146] 2. Experimental Results Table 8 Sample Detection (Signal Values)

[0147] 3. Experimental Data Analysis As shown in the table above, the blocking reagent of this application has a significant inhibitory effect on the signal of negative samples 2-5 that were positive in the control group, and a significant enhancement effect on the signal of positive sample 3 that was negative in the control group.

[0148] This blocking method is applicable to magnetic microparticle chemiluminescence platforms and can significantly suppress false positive sample signals and enhance false negative sample signals.

[0149] Example 5: Application of blocking methods in ELISA detection of HBcAb 1. Experimental Procedure (1) Kit preparation Antigen-coated microplates: Dilute HBc Ag 1:2000 with CB solution and add 100 μL / well to the microplate. Incubate at 37°C for 2 h. Discard the liquid from the wells, wash once with PBST, blot dry, add 150 μL / well of 5% skim milk powder, and incubate at 37°C for 1 h. Discard the liquid from the wells, wash once with PBST, blot dry, and store sealed at 4°C for later use.

[0150] HBc Ab enzyme conjugate: HRP and HBc antibody were coupled at a mass ratio of 1:1 using the sodium periodate method, and diluted to 1:2000 with 20 mM PBS buffer containing protein stabilizer and preservative to prepare HBc Ab enzyme conjugate.

[0151] Blocking reagents: Papain and reduced glutathione were diluted to 2 mg / ml and 10 mM respectively using 0.02 mol / L PBS to prepare blocking reagents.

[0152] Colorimetric solutions: Dilute hydrogen peroxide to 0.02% using 0.1M citrate-0.2M disodium hydrogen phosphate buffer to prepare colorimetric solution A; dissolve TMB in ethanol and then dilute it to 0.5 mg / mL with pure water to prepare colorimetric solution B.

[0153] Other reagents can be prepared using existing technologies.

[0154] (2) Sample testing The HBc Ab was detected using a competitive method.

[0155] Add 50 μL of blocking reagent, 50 μL of sample, and 50 μL of enzyme-labeled HBc Ab to the microplate coated with antigen, and react for 30 min. Wash the plate 5 times with PBST and blot dry. Add 50 μL each of chromogenic solutions A and B, and react for 15 min. Add 50 μL of stop solution, and read the OD value at 450 nm within 5 min.

[0156] If the sample to be tested does not contain HBc Ab, an antigen-labeled antibody-HRP bridge will be formed on the microplate, resulting in a high OD value. If the sample to be tested contains HBc Ab, the HBc Ab in the sample will compete with the enzyme-labeled antibody for the coating antigen on the plate, and the OD value will decrease as the concentration of HBc Ab in the sample increases.

[0157] 2. Experimental Results Table 9 Sample Detection (Signal Values)

[0158] 3. Experimental Data Analysis As shown in the table above, the blocking reagent significantly inhibited the signals of negative samples 3 and 4, which were positive in the control group, without affecting the detection of normal negative and positive samples.

[0159] This blocking method is applicable to the ELISA platform and can significantly suppress false positive sample signals, reducing false positives.

[0160] Example 6: Application of the blocking method in cytomegalovirus IgM colloidal gold immunochromatographic test strips 1. Experimental Procedure (1) Preparation of test strips Reference (Zuo Jingjing. Development of Enterovirus EV71-IgM Colloidal Gold Detection Kit [D]. Xiamen University) Prepare CMV IgM colloidal gold immunochromatographic test strips.

[0161] Nitrocellulose (NC) membrane: Cytomegalovirus antigen and goat anti-mouse IgG antibody were diluted with 20 mM PBS buffer. 1 mg / mL cytomegalovirus antigen was coated on the T line of the NC membrane, and 0.5 mg / mL goat anti-mouse IgG antibody was coated on the C line. The membrane was dried overnight at 37°C. Colloidal gold: Colloidal gold solution was prepared using the trisodium citrate reduction method. 100 mL of 0.04% chloroauric acid was added to a clean conical flask and placed on a magnetic stirrer at 300°C and 450 rpm. After boiling, 2 mL of 2% trisodium citrate was added dropwise at a uniform rate. The solution was boiled for 10 minutes, changing from pale yellow to wine red. It was then allowed to cool naturally to room temperature.

[0162] Gold-labeled pads: Mouse anti-human IgM was labeled with colloidal gold at a ratio of 5 μg / mL. Excess sites were blocked with 10% BSA. After the reaction, the supernatant was removed by centrifugation, and the mixture was reconstituted with 20 mM PBS buffer (containing 1% BSA, 0.5% PEG20000, and 0.05% Proclin300). The conjugate pads were pretreated with 20 mM PBS containing 0.1% Tween and dried. The gold-labeled antibody was sprayed onto the conjugate pads and dried at 37°C for 4 hours.

[0163] Sample pads: The sample pads were treated with 20mM PBS containing 2 mg / mL papain, 10 mM reduced glutathione, 3 mM EDTA, 30.1% Triton X-405, 0.1% antipyrine, and 1% valine, and dried overnight at 37°C.

[0164] Assemble the test strip: Attach the NC membrane, absorbent paper, gold label pad, and sample pad to the PVC base plate in sequence to assemble the CMV IgM colloidal gold test strip, cut it into strips, and seal it for storage.

[0165] (2) Sample testing Dilute the serum sample 20-fold with physiological saline. Take 100 μL of the diluted sample and add it to the sample pad. Observe the color development results after 10 minutes. If the T line appears when the C line is visible, it indicates that the cytomegalovirus IgM is positive; if the T line does not appear, it indicates that the cytomegalovirus IgM is negative.

[0166] 2. Experimental Results Table 10

[0167] 3. Experimental Data Analysis As can be seen from the table above, treatment of the sample pad with papain and reduced glutathione can eliminate false positives and restore false negative samples to positive results, without affecting the testing of normal negative and positive samples.

[0168] This blocking method is applicable to immunochromatographic platforms, and can significantly eliminate interference and improve the accuracy of reagent detection.

[0169] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications and revisions can be made to this application within the scope of the claims as prescribed, and without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. A method for blocking interfering substances in an immunoassay, characterized in that, The reaction system for detecting the sample includes a blocking reagent, which can destroy the structure of interfering substances in the sample.

2. The blocking method according to claim 1, wherein the blocking agent comprises a single blocking agent or a combination blocking agent; Preferably, the single blocking agent includes a biological blocking agent or a chemical blocking agent.

3. The blocking method according to claim 2, characterized in that, The chemical blocking agent is selected from chemical reagents that can destroy the antibody structure; preferably selected from at least one of strong acids, strong bases, or reducing agents; more preferably, the reducing agent includes at least one of SDS, cysteine, reduced glutathione, DTT, TCEP, and β-mercaptoethanol.

4. The blocking method according to claim 2, characterized in that, The biological blocking agent is selected from proteolytic enzymes; preferably from cysteine ​​proteases, serine proteases, aspartic proteases, or metalloproteinases; more preferably from at least one of papain, pepsin, trypsin, subtilisin, bromelain, endonuclease IdeS, endonuclease Lys-C, and endonuclease Lys-N.

5. The blocking method according to claim 2, characterized in that, The composite blocker comprises a proteolytic enzyme and an enzyme-catalyzing reaction promoter; the enzyme-catalyzing reaction promoter provides optimal reaction conditions for the proteolytic enzyme, and / or the enzyme-catalyzing reaction promoter is an activator of the proteolytic enzyme; preferably, the enzyme-catalyzing reaction promoter comprises at least one of an acid, a base, a metal salt, and an oligopeptide.

6. The blocking method according to claim 5, characterized in that, The content of the proteolytic enzyme in the blocking reagent is 0.05~5 mg / mL, and the content of the enzyme-catalyzing reaction promoter in the composite blocking agent is 0.5~500 mM; Preferably, the blocking agent comprises pepsin and hydrochloric acid; more preferably, the pepsin content in the blocking agent is 0.1~1 mg / mL and the hydrochloric acid content is 1~500 mM; even more preferably, the pepsin content is 0.1~0.2 mg / mL and the hydrochloric acid content is 10~100 mM. Preferably, the blocking agent comprises papain and reduced glutathione; more preferably, the papain content in the blocking agent is 0.1~5 mg / mL and the reduced glutathione content is 1~50 mM; even more preferably, the papain content is 1~3 mg / mL and the reduced glutathione content is 5~20 mM. Preferably, the blocking agent comprises trypsin, calcium chloride, and sodium hydroxide; more preferably, the trypsin content in the blocking agent is 0.05~5 mg / mL, and the calcium chloride content is 1~500 mM; even more preferably, the trypsin content is 0.1~2 mg / mL, the calcium chloride content is 50~200 mM, and the sodium hydroxide content is 0.005~0.05 mM.

7. The blocking method according to any one of claims 1 to 6, characterized in that, The test sample does not require additional diluent treatment.

8. A diagnostic product or diagnostic reagent, characterized in that, The diagnostic product or diagnostic reagent comprises the blocking reagent according to any one of claims 1 to 7; the diagnostic product includes a kit, chip, or test strip.

9. The use of the blocking agent as described in any one of claims 1 to 7 or the diagnostic product or diagnostic reagent as described in claim 8 in the indirect method, sandwich method or competitive method.

10. The use of the blocking reagent as described in any one of claims 1 to 7 or the diagnostic product or reagent as described in claim 8 in a chemiluminescence detection system, an enzyme-linked immunosorbent assay (ELISA) system, or an immunochromatographic detection system.

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