Hepatitis b virus core antibody igm type photochemical chemiluminescence detection kit and detection method

Abstract

The present disclosure provides a photo-activated chemiluminescence detection kit and method for detecting hepatitis B virus core IgM type antibody. The detection kit comprises: reagent 1, luminescent microspheres coated with hepatitis B core antigen (HBcAg), which can react with singlet oxygen and generate a detectable light signal; reagent 2, anti-IgM antibody labeled with a first partner; and reagent 3, blocking reagent, which can reduce or eliminate interfering antibodies. The detection kit of the present disclosure destroys the structure of interfering antibodies by chemical and / or biological methods, thereby weakening or even eliminating the signal or shielded signal caused by interfering antibodies, which can significantly improve the accuracy of hepatitis B virus core IgM type antibody detection.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics, specifically relating to a photo-induced chemiluminescence detection kit and a photo-induced chemiluminescence detection method for HBc-IgM. Background Technology

[0002] Hepatitis B virus (HBV) is a virus that causes life-threatening liver infections. Most people are asymptomatic upon initial infection. However, some develop acute symptoms that can last for weeks, including jaundice (yellowing of the skin and eyes), dark urine, extreme fatigue, nausea, vomiting, and abdominal pain. Acute hepatitis can lead to acute liver failure and death. Among the long-term complications of HBV infection, some individuals develop advanced liver diseases such as cirrhosis and hepatocellular carcinoma, resulting in high morbidity and mortality rates.

[0003] In hepatitis B virus (HBV) immunoassay, 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

[0004] To address one of the aforementioned technical problems in the existing technology, this disclosure provides a novel photo-induced chemiluminescence detection method for detecting hepatitis B virus core antibody IgM (HBc-IgM). This method uses chemical and / or biological 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) caused by the interfering antibodies.

[0005] The first aspect of this disclosure provides a photo-induced chemiluminescence detection kit for hepatitis B virus core antibody IgM, the detection kit comprising:

[0006] Reagent 1 consists of luminescent microspheres coated with hepatitis B virus core antigen, wherein the luminescent microspheres react with singlet oxygen to generate a detectable light signal.

[0007] Reagent 2, anti-IgM antibody labeled with the first pair,

[0008] Reagent 3, blocking reagent, which can reduce or eliminate interfering antibodies.

[0009] In some implementations...

[0010] The blocking agent includes a single blocking agent or a combination blocking agent;

[0011] Preferably, the single blocking agent includes a biological blocking agent or a chemical blocking agent.

[0012] In some embodiments, the biological blocking agent includes a proteolytic enzyme.

[0013] 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. Exopeptidases 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] In some embodiments, the chemical blocking agent includes a chemical reagent that can disrupt the antibody structure.

[0019] In some embodiments, the chemical blocking agent includes at least one of a strong acid, a strong base, or a reducing agent.

[0020] In some embodiments, the strong acid includes at least one of hydrochloric acid, sulfuric acid, nitric acid, perchloric acid, hydrobromic acid, and hydroiodic acid.

[0021] In some embodiments, the strong base includes at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide.

[0022] In some embodiments, the reducing agent includes cysteine, reduced glutathione, SDS, DTT, TCEP, β-mercaptoethanol, etc.

[0023] In some embodiments, the composite blocker includes a proteolytic enzyme and an enzyme-catalyzing reaction promoter, wherein the enzyme-catalyzing reaction promoter is capable of promoting and / or activating the enzyme-catalyzing reaction.

[0024] In some embodiments, the enzyme reaction promoter includes at least one of an acid, a base, a metal salt, and an oligopeptide.

[0025] In some specific embodiments, the enzyme-catalyzed reaction promoters include, but are 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.).

[0026] 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.

[0027] In some embodiments, the enzyme-catalyzing reaction promoter is present in the composite inhibitor 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In some embodiments, the detection kit further includes reagent 4, which is coated with photosensitive microspheres containing a second pairing agent that can specifically bind to the first pairing agent, and the photosensitive microspheres can release singlet oxygen upon photoexcitation.

[0032] The second aspect of this disclosure provides a photo-induced chemiluminescence detection method for hepatitis B virus core antibody IgM, the detection method comprising using the detection kit described in the first aspect to detect whether hepatitis B virus core antibody IgM is present in the sample to be tested.

[0033] In some embodiments, the blocking reagent in the test kit can block interfering antibodies in the sample to be tested.

[0034] In some embodiments, the interfering antibody includes heterophile antibodies, anti-animal antibodies, or rheumatoid factor.

[0035] In some embodiments, the detection method includes the following steps:

[0036] (1) The sample to be tested is mixed with reagent 3, reagent 1 and reagent 2 in sequence to carry out the first reaction and obtain the first reactant;

[0037] (2) The first reactant is reacted with singlet oxygen in a second reaction, and the light signal is detected and analyzed.

[0038] The third aspect of this disclosure provides the application of the detection kit described in the first aspect or the detection method described in the second aspect in the indirect detection of hepatitis B virus core antibody IgM.

[0039] This invention provides a novel detection kit and method for hepatitis B virus core antibody IgM. It utilizes proteolytic enzymes or a combination of proteolytic enzymes and enzyme reaction promoters to destroy interfering antibodies, replacing traditional blocking agents to block interfering antibodies, which can significantly improve the accuracy of reagent detection. Detailed Implementation

[0040] 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.

[0041] definition

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Hepatitis B virus (HBV) is a DNA virus belonging to the family Hepadnavividae. After infection with HBV, a large amount of surface antigen remains in the blood, resulting in HBsAgemia. HBsAg itself is not the complete HBV virus, but rather its outer shell. It is not infectious but has antigenicity and is only one of the markers of HBV infection. "HBV antigen" refers to any HBV antigen or protein, including core proteins such as HBcAg or HBcAG and HBeAg or HBeAG, and envelope proteins such as HBsAg or HBsAG.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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 refers to 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 refers to 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.

[0052] 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.

[0053] As used herein, the terms "first pair" and "second pair" refer to specific binding pairs, where the first pair is one member of a specific binding pair, and the second pair is the other. The term "specific binding pair" as used herein refers to a pair of molecules capable of specifically binding 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, widely found in plant and animal tissues, has two ring structures: an imidazoline ring and a thiophene ring, with the imidazoline ring being the primary binding site for avidin. Activated biotin can couple to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents; while avidin is a protein secreted by Streptomyces with a molecular weight of 65 kDa. The avidin molecule consists of four identical peptide chains, each capable of binding one biotin. In this article, avidin can be selected from egg avidin, streptavidin, yolk avidin, neutral avidin, and avidin-like substances.

[0054] 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.

[0055] Example 1

[0056] (1) Kit preparation

[0057] 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.

[0058] 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.

[0059] Reagent 3: Prepared as shown in Table 1 below.

[0060] Table 1: Preparation of Reagent 3

[0061]

[0062] 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.

[0063] (2) Operation steps

[0064] 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.

[0065] (3) Sample testing

[0066] Different test samples were tested using the above kit and operating procedures. The results are shown in Tables 2 and 3 below.

[0067] Table 2: Signal values ​​of samples detected by different reagents

[0068] Reagent 3 Comparison 2 3 4 5 6 7 Negative sample 1 1841 1670 376 508 980 1440 2523 Negative sample 2 41410 400 489 896 1822 2608 1363 negative sample 3 100854 500 619 907 914 3343 5338 negative sample 4 159963 2269 1643 1121 3643 3159 4114 5 negative samples 193151 741 2454 956 2353 4144 2928 Negative sample 6 133820 2610 1029 884 2536 12654 4787 Positive sample 1 34999 3365 4449 8368 8935 4210 80299 Positive sample 2 93571 9165 6106 13101 6703 5328 69698

[0069] Table 3: Blocking Effect

[0070] Ratio of Reagent 3 experimental group to control group 2 / Control 3 / Control 4 / Control 5 / Control 6 / Control 7 / Control Negative sample 1 0.91 0.20 0.28 0.53 0.78 1.37 Negative sample 2 0.01 0.01 0.02 0.04 0.06 0.03 negative sample 3 0.00 0.01 0.01 0.01 0.03 0.05 negative sample 4 0.01 0.01 0.01 0.02 0.02 0.03 5 negative samples 0.00 0.01 0.00 0.01 0.02 0.02 Negative sample 6 0.02 0.01 0.01 0.02 0.09 0.04 Positive sample 1 0.10 0.13 0.24 0.26 0.12 2.29 Positive sample 2 0.10 0.07 0.14 0.07 0.06 0.74

[0071] As shown in Tables 2-3 above, the addition of chemical or biological blocking agents has a certain blocking effect on negative samples 2-6 with excessively high detection signal values. However, after adding biological blocking agents (group 7), the decrease in positive samples was significantly smaller than that of false positive samples. Furthermore, chemical blocking agents (groups 2-6) had a greater impact on positive samples. Therefore, the blocking effect of biological blocking agents (group 7) is better than that of chemical blocking agents (groups 2-6).

[0072] Example 2

[0073] 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 4 below. The test results are shown in Tables 5 and 6.

[0074] Table 4: Preparation of Reagent 3

[0075]

[0076] Table 5: Signal values ​​of samples detected by different reagents

[0077]

[0078]

[0079] Table 6: Blocking Effect

[0080] Ratio of Reagent 3 experimental group to control group 7 / Control 8 / Control Negative sample 1 1.37 0.41 Negative sample 2 0.03 0.02 negative sample 3 0.05 0.03 negative sample 4 0.03 0.01 5 negative samples 0.02 0.01 Negative sample 6 0.04 0.02 Positive sample 1 2.29 1.79 Positive sample 2 0.74 0.66

[0081] As shown in Tables 5 and 6 above, compared with the biological blocking agent containing only proteolytic enzyme (Group 7), the combined blocking agent (Group 8) using proteolytic enzyme and enzyme reaction promoter has a better blocking effect on negative samples 2-6 with excessively high detection signal values, and does not affect positive samples.

[0082] Example 3

[0083] 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 7 below. The test results are shown in Tables 8 and 9.

[0084] Table 7: Preparation of Reagent 3

[0085]

[0086]

[0087] Table 8: Signal values ​​of samples detected by different reagents

[0088] Reagent 3 Comparison 8 9 10 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

[0089] Table 9: Blocking Effect

[0090] Ratio of Reagent 3 experimental group to control group 8 / Control 9 / Control 10 / Control 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

[0091] As shown in Tables 8 and 9 above, the combined blocking agents composed of different proteolytic enzymes and different enzyme reaction promoters can significantly inhibit the generation of false positive signals in negative samples without affecting positive samples.

[0092] Example 4

[0093] The optimal concentrations of the two effective enzyme reaction promoters and proteolytic enzyme combinations (groups 8 and 10) 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 10 below. The detection results are shown in Table 11 below.

[0094] Table 10: Preparation of Reagent 3

[0095]

[0096] Table 11: Concentration Optimization (Signal Value)

[0097]

[0098]

[0099] As shown in the table 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.

[0100] 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. A photocatalytic chemiluminescence detection kit for hepatitis B virus core antibody IgM, characterized in that, The detection kit includes: Reagent 1 consists of luminescent microspheres coated with hepatitis B virus core antigen, wherein the luminescent microspheres react with singlet oxygen to generate a detectable light signal. Reagent 2, anti-IgM antibody labeled with the first pair, Reagent 3, blocking reagent, which can reduce or eliminate interfering antibodies.

2. The detection kit according to claim 1, characterized in that, 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.

3. The detection kit according to claim 2, characterized in that, The biological blocking agent includes proteolytic enzymes; Preferably, the proteolytic enzyme includes at least one of endopeptidase, telopeptidase, or dipeptidase; More 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. Preferably, the content of the proteolytic enzyme in the blocking reagent is 0.5-5 mg / mL.

4. The detection kit according to claim 2, characterized in that, The chemical blocking agent includes chemical reagents that can disrupt the antibody structure; Preferably, the chemical blocking agent includes at least one of a strong acid, a strong base, or a reducing agent; More preferably, the strong acid includes at least one of hydrochloric acid, sulfuric acid, nitric acid, perchloric acid, hydrobromic acid, and hydroiodic acid; More preferably, the strong base includes at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide; More preferably, the reducing agent includes at least one of cysteine, reduced glutathione, SDS, DTT, TCEP, and β-mercaptoethanol.

5. The detection kit according to claim 2, characterized in that, The composite blocker includes a proteolytic enzyme and an enzyme reaction promoter, wherein the enzyme reaction promoter can promote and / or activate the enzyme reaction. Preferably, the enzyme reaction promoter includes at least one of an acid, a base, a metal salt, and an oligopeptide; Preferably, the content of the enzyme-catalyzing reaction promoter in the composite inhibitor is 0.5-500 mM.

6. The detection kit according to any one of claims 1-5, 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.

7. The detection kit according to any one of claims 1-6, characterized in that, The detection kit further includes reagent 4, which is coated with photosensitive microspheres containing a second pairing agent. The second pairing agent can specifically bind to the first pairing agent, and the photosensitive microspheres can release singlet oxygen upon photoexcitation.

8. A photo-induced chemiluminescence detection method for hepatitis B virus core antibody IgM, characterized in that, The detection method includes using the detection kit according to any one of claims 1-7 to detect whether the hepatitis B virus core antibody IgM is present in the sample to be tested.

9. The detection method according to claim 8, characterized in that, The detection method includes the following steps: (1) The sample to be tested is mixed with reagent 3, reagent 1 and reagent 2 in sequence to carry out the first reaction and obtain the first reactant; (2) The first reactant is reacted with singlet oxygen in a second reaction, and the light signal is detected and analyzed.

10. The application of the detection kit according to any one of claims 1-7 or the detection method according to claim 8 or 9 in the indirect detection of hepatitis B virus core antibody IgM.

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