Use of proteolytic enzymes to reduce or eliminate interfering antibodies in immunoassays

Abstract

The present application provides the use of proteolytic enzymes to reduce or eliminate interfering antibodies in immunoassays for antigens and / or antibodies. The present disclosure utilizes biological methods or biological methods in combination with chemical methods to disrupt the structure of interfering antibodies, thereby attenuating or even eliminating the signal (false positive) or masked signal (false negative) from interfering antibodies, which can significantly improve the accuracy of reagent detection.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics, specifically relating to the application of proteolytic enzymes in immunoassay, and methods and applications for blocking interfering antibodies in immunoassay. Background Technology

[0002] In immunoassays, antibodies such as heterophile antibodies, anti-animal antibodies, and rheumatoid factor may weakly bind to the analyte or marker, interfering with the normal immune response and leading to false positives or false negatives. The common method for removing these irrelevant antibodies is to add active or passive blocking agents. Blocking agents eliminate or reduce interference by binding to interfering antibodies. Passive blocking agents are used at high concentrations and target only a single interfering antibody; active blocking agents do not require excessively high concentrations but cannot block all types of interfering antibodies. Due to individual differences, clinical samples vary greatly, and the types and concentrations of interfering antibodies differ, sometimes adding blocking agents does not achieve the desired blocking effect. Summary of the Invention

[0003] In order to solve one of the aforementioned technical problems in the prior art, this disclosure provides a new blocking method, namely, using biological methods or combined chemical methods to destroy the structure of interfering antibodies (such as heterophilic antibodies, anti-animal antibodies, rheumatoid factor, etc.), thereby weakening or even eliminating the signal (false positive) or the signal (false negative) brought by the interfering antibodies.

[0004] The first aspect of this disclosure provides the use of proteolytic enzymes in the immunoassay of antigens and / or antibodies to reduce or eliminate interfering antibodies. The assay is a non-disease diagnostic assay.

[0005] In some embodiments, the proteolytic enzyme is used in combination with an enzymatic reaction promoter to reduce or eliminate interfering antibodies.

[0006] In some embodiments, the proteolytic enzyme is any proteolytic enzyme capable of disrupting antibody structure. In some embodiments, the proteolytic enzyme includes at least one of endopeptidases, telopeptidases, or dipeptidases. Endopeptidases can cleave large polypeptide chains in the middle to form smaller prion and peptone molecules. Telopeptidases include carboxylpeptidases and aminopeptidases, which hydrolyze the polypeptide chain one by one from the free carboxyl terminus or free amino terminus, respectively, to generate amino acids. Dipeptidases are exopeptidases that act on dipeptide peptide bonds.

[0007] In some embodiments, the proteolytic enzyme is derived from animal viscera, plant stems and leaves, fruits, or prepared by fermentation using microorganisms such as molds, bacteria, yeasts, and actinomycetes.

[0008] In some embodiments, the proteolytic enzymes include, but are not limited to: papain, pepsin, trypsin, subtilisin, bromelin, neutral protease, chymotrypsin, carboxypeptidase, aminopeptidase, endonuclease IdeS (immunoglobulin G degrading enzyme), endonuclease Lys-C (lysine endonuclease, which cleaves the carboxyl terminus of lysine), endonuclease Lys-N (lysine endonuclease, which cleaves the amino terminus of lysine), and all other proteolytic enzymes that can disrupt or interfere with antibody structure.

[0009] In some embodiments, the proteolytic enzymes are classified into four main categories based on their active sites: aspartic proteases, serine proteases, cysteine ​​proteases, and metalloproteinases. Different proteolytic enzymes have different cleavage sites. For example, papain belongs to the cysteine ​​protease class, with a suitable pH range of 5–7, and its specificity for cleavage is the carboxyl group of Lys / Arg / Phe; pepsin belongs to the aspartic protease class, with a suitable pH range of 1–4, and its specificity for cleavage is the carboxyl groups of aromatic carboxyl and amino groups, as well as the carboxyl groups of Leμ / Asp / Glμ; trypsin belongs to the serine / metalloproteinase class, with a suitable pH range of 7–9, and its specificity for cleavage is the carboxyl group of Lys / Arg.

[0010] In some embodiments, the proteolytic enzyme is present in the blocking reagent at a concentration of 0.5-5 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, or any value between them.

[0011] In some embodiments, the enzyme-catalyzing reaction promoter includes chemical reagents capable of promoting and / or activating the enzyme-catalyzing reaction. In some specific embodiments, the enzyme-catalyzing reaction promoter includes, but is not limited to: acids (e.g., hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), perchloric acid (HClO4), hydrobromic acid (HBr), hydroiodic acid (HI), etc.), bases (e.g., sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), barium hydroxide (Ba(OH)2), etc.), metal salts (e.g., calcium chloride, magnesium chloride, potassium chloride, sodium chloride, calcium nitrate, magnesium nitrate, sodium nitrate, potassium nitrate, etc.), oligopeptides (e.g., reduced glutathione, etc.).

[0012] The inventors of this application have discovered that enzyme reaction promoters are activators of proteolytic enzymes. When proteolytic enzymes are used in conjunction with enzyme reaction promoters (such as Ca), [the following applies].2+ Combining proteases with other enzymes (such as trypsin, oligopeptides, or papain) or using them under optimal conditions (such as acid combined with pepsin) can further enhance their effectiveness. Although different enzymes or proteolytic enzymes have different destructive effects and may disrupt antigenic epitopes or affect antigen-antibody reactions, their impact on antigens and specific antibodies can be minimized by selecting different proteases or controlling their concentration and loading order, while destroying as many interfering antibodies as possible.

[0013] In some embodiments, the enzyme-catalyzing reaction promoter is present in the blocking reagent at a concentration of 0.5-500 mM, for example, 0.5 mM, 5 mM, 10 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM or any value between them.

[0014] In some embodiments, papain is used in combination with oligopeptides in the immunoassay. In some preferred embodiments, papain is used in combination with reduced glutathione in the immunoassay. Preferably, 1-3 mg / mL papain is used in combination with 5-20 mM reduced glutathione. More preferably, 1.5-2.5 mg / mL papain and 8-12 mM reduced glutathione are used in combination. Even more preferably, 2 mg / mL papain is used in combination with 10 mM reduced glutathione.

[0015] In some embodiments, pepsin is used in combination with acid in the immunoassay. In some preferred embodiments, pepsin is used in combination with hydrochloric acid in the immunoassay. Preferably, 0.05-0.2 mg / mL pepsin is used in combination with 10-100 mM hydrochloric acid. More preferably, 0.08-0.12 mg / mL pepsin is used in combination with 40-60 mM hydrochloric acid. Even more preferably, 0.1 mg / mL pepsin is used in combination with 50 mM hydrochloric acid.

[0016] In some embodiments, the immunoassay involves the use of trypsin in combination with a calcium salt and a base. In some preferred embodiments, the immunoassay involves the use of trypsin in combination with calcium chloride and sodium hydroxide. Preferably, 0.1-1 mg / mL trypsin is used in combination with 50-150 mM calcium chloride and 0.005-0.05 mM sodium hydroxide. More preferably, 0.2-0.8 mg / mL trypsin, 80-120 mM calcium chloride, and 0.005-0.02 mM sodium hydroxide are used in combination. Even more preferably, 0.5 mg / mL trypsin, 100 mM calcium chloride, and 0.01 mM sodium hydroxide are used in combination.

[0017] In some embodiments, the antibody includes at least one of IgM, IgD, IgG, IgA, IgE, Fab, Fab', F(ab')2, Fv, scFv, or VH.

[0018] In some embodiments, the interfering antibody includes one or more of an interfering antibody and cytokines.

[0019] In some embodiments, the interfering antibody includes one or more of heterophilic antibodies, anti-animal antibodies, or rheumatoid factor.

[0020] In some embodiments, the immunoassay includes immunochromatographic immunoassay, enzyme-linked immunosorbent assay (ELISA), or chemiluminescent immunoassay.

[0021] In some specific embodiments, the immunoassay includes photoluminescence detection, magnetic microparticle chemiluminescence detection, colloidal gold immunochromatography, latex immunochromatography, radioimmunoassay (RIA), or time-resolved fluorescence immunoassay (TRFIA). The immunoassay is also applicable to various immunoassay modalities, such as double-antibody sandwich assays, competitive assays, and indirect assays.

[0022] A second aspect of this disclosure provides a detection kit for antigen and / or antibody immunoassay, the kit comprising a blocking reagent comprising a proteolytic enzyme.

[0023] In some embodiments, the proteolytic enzyme is any proteolytic enzyme capable of disrupting antibody structure. In some embodiments, the proteolytic enzyme includes at least one of endopeptidases, telopeptidases, or dipeptidases. Endopeptidases can cleave large polypeptide chains in the middle to form smaller prion and peptone molecules. Telopeptidases include carboxylpeptidases and aminopeptidases, which hydrolyze the polypeptide chain one by one from the free carboxyl terminus or free amino terminus, respectively, to generate amino acids. Dipeptidases are exopeptidases that act on dipeptide peptide bonds.

[0024] In some embodiments, the proteolytic enzyme is derived from animal viscera, plant stems and leaves, fruits, or prepared by fermentation using microorganisms such as molds, bacteria, yeasts, and actinomycetes.

[0025] In some embodiments, the proteolytic enzymes include, but are not limited to: papain, pepsin, trypsin, subtilisin, bromelin, neutral protease, chymotrypsin, carboxypeptidase, aminopeptidase, endonuclease IdeS (immunoglobulin G degrading enzyme), endonuclease Lys-C (lysine endonuclease, which cleaves the carboxyl terminus of lysine), endonuclease Lys-N (lysine endonuclease, which cleaves the amino terminus of lysine), and all other proteolytic enzymes that can disrupt or interfere with antibody structure.

[0026] In some embodiments, the proteolytic enzymes are classified into four main categories based on their active sites: aspartic proteases, serine proteases, cysteine ​​proteases, and metalloproteinases. Different proteolytic enzymes have different cleavage sites. For example, papain belongs to the cysteine ​​protease class, with a suitable pH range of 5–7, and its specificity for cleavage is the carboxyl group of Lys / Arg / Phe; pepsin belongs to the aspartic protease class, with a suitable pH range of 1–4, and its specificity for cleavage is the carboxyl groups of aromatic carboxyl and amino groups, as well as the carboxyl groups of Leμ / Asp / Glμ; trypsin belongs to the serine / metalloproteinase class, with a suitable pH range of 7–9, and its specificity for cleavage is the carboxyl group of Lys / Arg.

[0027] In some embodiments, the proteolytic enzyme is present in the blocking reagent at a concentration of 0.5-5 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, or any value between them.

[0028] In some embodiments, the blocking agent further includes an enzyme reaction promoter.

[0029] In some embodiments, the enzyme-catalyzing reaction promoter is selected from chemical reagents capable of promoting and / or activating enzyme-catalyzing reactions. In some specific embodiments, the enzyme-catalyzing reaction promoter includes, but is not limited to: acids (e.g., hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), perchloric acid (HClO4), hydrobromic acid (HBr), hydroiodic acid (HI), etc.), bases (e.g., sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), barium hydroxide (Ba(OH)2), etc.), metal salts (e.g., calcium chloride, magnesium chloride, potassium chloride, sodium chloride, calcium nitrate, magnesium nitrate, sodium nitrate, potassium nitrate, etc.), oligopeptides (e.g., reduced glutathione), etc.

[0030] In some embodiments, the enzyme-catalyzing reaction promoter is present in the blocking reagent at a concentration of 0.5-500 mM, for example, 0.5 mM, 5 mM, 10 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM or any value between them.

[0031] In some embodiments, the blocking agent comprises papain and oligopeptides. In some preferred embodiments, the blocking agent comprises papain and reduced glutathione. Preferably, the blocking agent comprises 1-3 mg / mL papain and 5-20 mM reduced glutathione. More preferably, the blocking agent comprises 1.5-2.5 mg / mL papain and 8-12 mM reduced glutathione. Even more preferably, the blocking agent comprises 2 mg / mL papain and 10 mM reduced glutathione.

[0032] In some embodiments, the blocking agent comprises pepsin and acid. In some preferred embodiments, the blocking agent comprises pepsin and hydrochloric acid. Preferably, the blocking agent comprises 0.05-0.2 mg / mL pepsin and 10-100 mM hydrochloric acid. More preferably, the blocking agent comprises 0.08-0.12 mg / mL pepsin and 40-60 mM hydrochloric acid. Even more preferably, the blocking agent comprises 0.1 mg / mL pepsin and 50 mM hydrochloric acid.

[0033] In some embodiments, the blocking agent comprises trypsin, a calcium salt, and a base. In some preferred embodiments, the blocking agent comprises trypsin, calcium chloride, and sodium hydroxide. Preferably, the blocking agent comprises 0.1-1 mg / mL trypsin, 50-150 mM calcium chloride, and 0.005-0.05 mM sodium hydroxide. More preferably, the blocking agent comprises 0.2-0.8 mg / mL trypsin, 80-120 mM calcium chloride, and 0.005-0.02 mM sodium hydroxide. Even more preferably, the blocking agent comprises 0.5 mg / mL trypsin, 100 mM calcium chloride, and 0.01 mM sodium hydroxide.

[0034] A third aspect of this disclosure provides a method for blocking interfering antibodies in the immunoassay of antigens and / or antibodies, comprising the step of mixing the above-described test kit with the sample to be tested.

[0035] In some embodiments, the interfering antibody includes one or more of an interfering antibody and cytokines.

[0036] In some embodiments, the interfering antibody includes heterophile antibodies, anti-animal antibodies, or rheumatoid factor.

[0037] The fourth aspect of this disclosure provides the application of the above-described detection kit or the above-described method for blocking interfering antibodies in the immunoassay of antigens and / or antibodies.

[0038] This invention provides a novel blocking method that utilizes proteolytic enzymes or a combination of proteolytic enzymes and enzymatic reaction promoters to destroy interfering antibodies, replacing traditional blocking agents and significantly improving the accuracy of reagent detection. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0040] definition

[0041] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0042] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0043] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0044] The term "antigen" as used in this article refers to a substance that can stimulate the body to produce an immune response and can bind to immune response products, antibodies and sensitized lymphocytes, in vivo and in vitro to produce an immune effect.

[0045] The terms “antibody” and “immunoglobulin” as used herein are used in the broadest sense, including any isotype of antibody or immunoglobulin, 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 that contain the antigen-binding portion of an antibody and non-antibody proteins.

[0046] As used herein, the term "antibody" includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding moieties," "antigen-binding polypeptides," or "immunobinding agents"), or their single chains. An "antibody" is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds, or its antigen-binding moieties.

[0047] The term "immunoglobulin" as used in this article refers to five main categories: IgA, IgD, IgE, IgG, and IgM. These main categories can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant heavy chain domains corresponding to different categories of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. Different types (κ or λ) of immunoglobulins have essentially the same CL length, but different classes of immunoglobulins have different CH lengths. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The hinge region, located between CH1 and CH2, is rich in proline and easily stretches and bends, thereby altering the distance between antigen-binding sites and facilitating antibody binding to antigenic epitopes located at different positions.

[0048] The term "interfering antibody" as used in this article refers to antibodies such as heterophile antibodies, anti-animal antibodies, and rheumatoid factor that may weakly bind to the analyte or marker in immunoassays, interfering with the normal immune response and leading to false positives or false negatives. These irrelevant antibodies are the interfering antibodies described in this article.

[0049] As used in this article, the term "blocking reagent" refers to a substance or reagent that can reduce or eliminate interfering antibodies in immunoassays. In this article, blocking reagents include proteolytic enzymes and, optionally, enzyme-catalyzing agents.

[0050] 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 or interfere with antibody structure to promote and / or activate enzymatic reactions, such as acids, bases, metal salts, oligopeptides, etc.

[0051] In this disclosure, proteolytic enzymes are used via biological methods, while enzymatic reaction promoters are used via chemical methods. Chemical and biological methods can be used alone or in combination to achieve better results. Although different enzymatic reaction promoters or proteolytic enzymes have different destructive effects and may affect antigen-antibody reactions, their impact on specific antigen-antibody reactions can be minimized and interfering antibodies destroyed as much as possible by controlling the concentration and sample addition order.

[0052] The blocking method disclosed herein is applicable to various immunoassay platforms, such as gold immunochromatography, ELISA, and chemiluminescence; the blocking method of this invention is also applicable to various immunoassay modalities, such as double-antibody sandwich assays, competitive assays, and indirect assays. In this invention, chemical reagents or proteases can be formulated as reagents and included as part of the kit components.

[0053] The following embodiments are provided to aid in understanding the present invention. However, it should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0054] Example 1

[0055] (1) Kit preparation

[0056] 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.

[0057] Reagent 2: Mouse anti-human IgM was labeled with N-hydroxysuccinimide biotin 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.

[0058] Reagent 3: Papain was prepared into Reagent 3, and the content of each component is shown in Table 1 below.

[0059] Table 1: Preparation of Reagent 3

[0060]

[0061] Universal solution: Use aldehyde-based photosensitive microparticles coated with streptavidin at a mass ratio of 10:0.5, and dilute to 50 μg / mL with 50 mM HEPES buffer containing protein stabilizers and preservatives to prepare the universal solution.

[0062] (2) Operation steps

[0063] HBc-IgM was detected using an indirect method. The serum sample to be tested, identified by clinical standard testing methods, was diluted 20-fold with physiological saline. 10 μL of the diluted serum sample, 25 μL of reagent 3, 25 μL of reagent 1, and 25 μL of reagent 2 were added sequentially to the reaction wells. After reacting for 15 min, universal solution was added, and the reaction was allowed to proceed for another 10 min. The signal value was read at a wavelength of 620 nm. If the sample contains specific HBc-IgM, a bridging reaction can be formed between luminescent microparticles, HBc Ag (reagent 1), HBc-IgM (sample), mouse anti-human IgM, biotin (reagent 2), avidin, and photosensitive microspheres (universal solution), generating a signal; otherwise, no bridging reaction will occur.

[0064] (3) Test Results

[0065] Different test samples were tested using the above kit and operating procedures, and the results are shown in Table 2 below.

[0066] Table 2: Signal values ​​and blocking effects of proteolytic enzymes as blocking agents in test samples

[0067] Reagent 3 control group Experimental group 1 Ratio of experimental group to control group Negative sample 1 1841 2523 1.37 Negative sample 2 41410 1363 0.03 negative sample 3 100854 5338 0.05 negative sample 4 159963 4114 0.03 5 negative samples 193151 2928 0.02 Negative sample 6 133820 4787 0.04 Positive sample 1 34999 80299 2.29 Positive sample 2 93571 69698 0.74

[0068] As shown in Table 2 above, the addition of proteolytic enzymes has a certain blocking effect on negative samples 2 to 6 with excessively high detection signal values, but the reduction in positive samples is significantly smaller than that of false positive samples.

[0069] Example 2

[0070] The experimental methods, raw materials, and procedures were the same as in Example 1, except that the components of reagent 3 were different, as shown in Table 3 below. The test results are shown in Table 4.

[0071] Table 3: Preparation of Reagent 3

[0072]

[0073] Table 4: Signal values ​​and blocking effects of different reagents on samples 3

[0074]

[0075]

[0076] As shown in Table 4 above, compared with the addition of proteolytic enzyme alone (experimental group 1), the combination of proteolytic enzyme and enzyme reaction promoter (experimental group 2) has a better blocking effect on negative samples 2-6 with excessively high detection signal values, and does not affect positive samples.

[0077] Example 3

[0078] The experimental methods, raw materials, and procedures were the same as in Example 1, except that the components of reagent 3 were different, as shown in Table 5 below. The test results are shown in Tables 6 and 7.

[0079] Table 5: Preparation of Reagent 3

[0080]

[0081] Table 6: Signal values ​​of samples detected by different reagents

[0082] Reagent 3 control group Experimental group 2 Experimental group 3 Experimental group 4 Negative sample 1 1841 764 1803 535 Negative sample 2 41410 847 4157 1123 negative sample 3 100854 3257 11354 957 negative sample 4 159963 2207 8433 2413 5 negative samples 193151 2166 5597 1552 Negative sample 6 133820 2733 6752 1701 Positive sample 1 34999 62715 44585 25634 Positive sample 2 93571 61732 36004 10663

[0083] Table 7: Blocking Effect

[0084] ratio Experimental group 2 / Control group Experimental group 3 / Control group Experimental group 4 / Control group Negative sample 1 0.41 0.98 0.29 Negative sample 2 0.02 0.10 0.03 negative sample 3 0.03 0.11 0.01 negative sample 4 0.01 0.05 0.02 5 negative samples 0.01 0.03 0.01 Negative sample 6 0.02 0.05 0.01 Positive sample 1 1.79 1.27 0.73 Positive sample 2 0.66 0.38 0.11

[0085] As shown in Tables 6 and 7 above, different proteolytic enzymes combined with different enzyme-catalyzing agents can significantly inhibit the generation of false positive signals in negative samples without affecting positive samples.

[0086] Example 4

[0087] The optimal concentrations of the two effective enzyme reaction promoters and proteolytic enzyme combinations (experimental groups 2 and 4) used in Example 3 were optimized. The optimal concentration conditions were screened by preparing reagent 3 of the HBc-IgM photocatalytic chemiluminescence kit and testing the samples. The experimental methods, materials, and procedures were the same as in Example 1. The content of each component of reagent 3 is shown in Table 8 below. The detection results are shown in Table 9 below.

[0088] Table 8: Preparation of Reagent 3

[0089]

[0090] Table 9: Concentration Optimization (Signal Value)

[0091]

[0092]

[0093] As shown in Table 9 above, for combination 1: papain and reduced glutathione, the efficiency of papain in destroying interfering antibodies reached saturation at a concentration of 2 mg / mL, and further increasing the concentration of papain 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 for combination 1 is papain at least 2 mg / mL and reduced glutathione at approximately 10 mM. For combination 2: pepsin and hydrochloric acid, the higher the concentration of pepsin, the greater the inhibitory effect on both false-positive and true-positive samples. Although a hydrochloric acid concentration of 100 mM showed the best inhibitory effect on false-positive samples, it affected the normal antigen-antibody reaction. Therefore, the optimal concentration for combination 2 is pepsin at least 0.1 mg / mL and hydrochloric acid at approximately 50 mM.

[0094] Example 5

[0095] 1. Reagent kit preparation

[0096] 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.

[0097] 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.

[0098] Blocking reagent: Dilute pepsin to 0.1 mg / mL with 0.05 M hydrochloric acid to prepare the blocking reagent.

[0099] Luminescent substrates: Luminescent substrate A was prepared using 0.05% H2O2 and 0.5% nitric acid, and luminescent substrate B was prepared using 0.05M NaOH and 0.1% Triton X-100.

[0100] 2. Operating Procedures

[0101] HBsAg was detected using a double-antibody sandwich assay. 20 μL of serum sample was taken, and 50 μL of blocking reagent was added. After reacting for 5 min, 50 μL of magnetic microparticle suspension and 100 μL of acridinium ester conjugate were added sequentially. After reacting for 20 min, an external magnetic field was applied to aggregate the magnetic microparticles and their conjugates. The supernatant was removed, and the sample was washed with 0.1% Tween to remove unbound substances. After magnetic separation and removal of the supernatant, 100 μL each of luminescent substrates A and B were added, and the optical signal was detected. If the sample contained specific HBsAg, a bridge could be formed between the magnetic microparticles, anti-HBsAg monoclonal antibody, HBsAg (analyte), and acridinium ester, generating a signal; otherwise, no bridge could be formed.

[0102] 3. Test Results

[0103] The above-mentioned kit was used to test different clinically standardized test samples, and the results are shown in Table 10 below.

[0104] Table 10: Sample Detection (Signal Values)

[0105]

[0106] As shown in Table 10 above, the blocking reagent significantly inhibited the signals of negative samples 2-5 (which tested positive in the control group) and significantly enhanced the signal of positive sample 3 (which tested negative in the control group).

[0107] Example 6

[0108] 1. Reagent kit preparation

[0109] Antigen-coated microplates: Dilute HBc Ag 1:2000 with CBS buffer 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.

[0110] HBc Ab enzyme conjugate: HRP and HBc antibody were conjugated at a mass ratio of 1:1 using the sodium periodate method, and diluted to 1:2000 with 20mM PBS buffer containing protein stabilizer and preservative to prepare HBc Ab enzyme conjugate.

[0111] Blocking reagents: Papain and glutathione were diluted to 2 mg / ml and 10 mM respectively using 0.02 M PBS to prepare blocking reagents.

[0112] Colorimetric solutions: Hydrogen peroxide was diluted to 0.02% using 0.1M citrate-0.2M disodium hydrogen phosphate buffer to prepare colorimetric solution A; TMB was dissolved in ethanol and then diluted to 0.5 mg / mL with pure water to prepare colorimetric solution B.

[0113] 2. Operating Procedures

[0114] HBc Ab was detected using a competitive assay. In a microplate coated with the antigen, 50 μL of blocking reagent, 50 μL of sample, and 50 μL of enzyme-labeled HBc Ab were added sequentially, and the reaction was allowed to proceed for 30 min. The plate was washed 5 times with PBST and blotted dry. 50 μL each of chromogenic solutions A and B were added, and the reaction was allowed to proceed for 15 min. 50 μL of stop solution was added, and the OD value was read at 450 nm within 5 min. If the sample contained no HBc Ab, an antigen-labeled antibody-HRP bridge would form on the microplate, resulting in a higher OD value. If the sample contained HBc Ab, the HBc Ab in the sample would compete with the enzyme-labeled antibody for the coating antigen on the plate, and the OD value would decrease as the concentration of HBc Ab in the sample increased.

[0115] 3. Test Results

[0116] Different test samples were tested using the kit described above, and the results are shown in Table 11 below.

[0117] Table 11: Sample Detection (Signal Values)

[0118]

[0119] As shown in Table 11 above, the use of 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.

[0120] Example 7

[0121] 1. Preparation of test strips

[0122] Reference (Zuo Jingjing. Development of colloidal gold detection kit for enterovirus EV71-IgM [D]. Xiamen University, 2019. DOI:10.27424 / d.cnki.gxmdu.2019.000285.) Toxo CMV colloidal gold immunochromatographic test strips were prepared.

[0123] 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.

[0124] 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 set to 300°C and 450 rpm. After heating to boiling, 2 ml of 2% trisodium citrate was added dropwise at a uniform rate. The mixture was boiled for 10 minutes, after which the solution changed from pale yellow to wine red. The solution was then allowed to cool naturally to room temperature.

[0125] 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.

[0126] Sample pads: Treat sample pads with 20mM PBS containing 2mg / ml papain, 10mM reduced glutathione, 3mM EDTA, 30.1% Triton X-405, 0.1% antipyrine, and 1% valine, and dry overnight at 37°C.

[0127] 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.

[0128] 2. Operating Procedures

[0129] 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.

[0130] 3. Test Results

[0131] Different test samples were tested using the kit described above, and the results are shown in Table 12 below.

[0132] Table 12

[0133] Serial Number CMV IgM sample control group experimental group 1 Negative sample 1 Negative Negative 2 Negative sample 2 (CMV IgG positive) Positive Negative 3 Negative sample 3 (CMV IgG positive) Positive Negative 4 Negative sample 4 (RF positive) Positive Negative 5 Negative sample 5 (RF positive) Positive Negative 6 Negative sample 6 Negative Negative 7 Positive sample 1 (CMV IgG positive) Negative Positive 8 Positive sample 2 Positive Positive

[0134] As can be seen from Table 12 above, treatment of the sample pad with papain and glutathione can eliminate false positives and restore false negative samples to positive results, without affecting the testing of normal negative and positive samples.

[0135] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Proteolytic enzymes reduce or eliminate interference with the use of antibodies in the immunoassay of antigens and / or antibodies.

2. The application according to claim 1, characterized in that, The proteolytic enzyme is used in combination with an enzyme-catalyzing reaction promoter to reduce or eliminate interfering antibodies.

3. The application according to claim 1 or 2, characterized in that, The proteolytic enzyme includes at least one of endopeptidase, telopeptidase, or dipeptidase. Preferably, the proteolytic enzyme includes at least one of papain, pepsin, trypsin, subtilisin, bromelain, neutral protease, chymotrypsin, carboxypeptidase, aminopeptidase, endonuclease IdeS, endonuclease Lys-C, or endonuclease Lys-N.

4. The application according to claim 2 or 3, characterized in that, The enzyme reaction promoter includes chemical reagents capable of promoting and / or activating enzyme reactions; Preferably, the enzyme reaction promoter includes at least one of an acid, a base, a metal salt, and an oligopeptide; More preferably, the acid includes at least one selected from hydrochloric acid, sulfuric acid, nitric acid, perchloric acid, hydrobromic acid, and hydroiodic acid. More preferably, the alkali includes at least one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide. More preferably, the metal salt includes at least one of calcium chloride, magnesium chloride, potassium chloride, sodium chloride, calcium nitrate, magnesium nitrate, sodium nitrate, and potassium nitrate. More preferably, the oligopeptide includes reduced glutathione.

5. The application according to any one of claims 1-4, characterized in that, In the aforementioned immunoassay, papain and oligopeptides are used in combination; Preferably, papain is used in combination with reduced glutathione; More preferably, 1-3 mg / mL papain is used in combination with 5-20 mM reduced glutathione; or In the aforementioned immunoassay, pepsin is used in combination with acid; Preferably, pepsin is used in combination with hydrochloric acid; More preferably, 0.05-0.2 mg / mL pepsin is used in combination with 10-100 mM hydrochloric acid; or In the aforementioned immunoassay, trypsin is used in combination with calcium salts and alkali; Preferably, trypsin is used in combination with calcium chloride and sodium hydroxide; More preferably, 0.1-1 mg / mL trypsin, 50-150 mM calcium chloride, and 0.005-0.05 mM sodium hydroxide are used in combination.

6. A kit for the immunoassay of antigens and / or antibodies, the kit comprising a blocking reagent comprising a proteolytic enzyme.

7. The detection kit according to claim 6, characterized in that, The proteolytic enzyme includes at least one of endopeptidase, telopeptidase, or dipeptidase. Preferably, the proteolytic enzyme comprises at least one selected from papain, pepsin, trypsin, subtilisin, bromelain, neutral protease, chymotrypsin, carboxypeptidase, aminopeptidase, endonuclease IdeS, endonuclease Lys-C, or endonuclease Lys-N. More preferably, the content of the proteolytic enzyme in the blocking reagent is 0.5-5 mg / mL.

8. The detection kit according to claim 6 or 7, characterized in that, The blocking agent also includes an enzyme reaction promoter. Preferably, the enzyme reaction promoter is selected from chemical reagents capable of promoting and / or activating enzyme reactions. Preferably, the enzyme reaction promoter includes at least one of an acid, a base, a metal salt, and an oligopeptide. More preferably, the acid includes at least one selected from hydrochloric acid, sulfuric acid, nitric acid, perchloric acid, hydrobromic acid, and hydroiodic acid. More preferably, the alkali includes at least one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide. More preferably, the metal salt includes at least one selected from calcium chloride, magnesium chloride, potassium chloride, sodium chloride, calcium nitrate, magnesium nitrate, sodium nitrate, and potassium nitrate. More preferably, the oligopeptide includes reduced glutathione. More preferably, the content of the enzyme reaction promoter in the blocking reagent is 0.5-500 mM.

9. The detection kit according to any one of claims 6-8, characterized in that, The blocking agent comprises papain and oligopeptides; preferably, the blocking agent comprises papain and reduced glutathione; more preferably, the blocking agent comprises 1-3 mg / mL papain and 5-20 mM reduced glutathione; or The blocking agent comprises pepsin and acid; preferably, the blocking agent comprises pepsin and hydrochloric acid; more preferably, the blocking agent comprises 0.05-0.2 mg / mL pepsin and 10-100 mM hydrochloric acid; or The blocking agent includes trypsin, calcium salt and base. Preferably, the blocking agent includes trypsin, calcium chloride and sodium hydroxide. More preferably, the blocking agent includes 0.1-1 mg / mL trypsin, 50-150 mM calcium chloride and 0.005-0.05 mM sodium hydroxide.

10. A method for blocking interfering antibodies in the immunoassay of antigens and / or antibodies, comprising the step of mixing the reagents in the test kit of any one of claims 6-8 with the sample to be tested.

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