Epstein-barr virus early antigen ig a antibody detection kit

Abstract

The present application relates to the field of immune detection technology, in particular to an EB virus early antigen IgA antibody detection kit, which comprises a solid phase carrier suspension, an enzyme conjugate solution and a sample diluent, wherein the surface of the solid phase carrier in the solid phase carrier suspension is coated with EB virus early antigen, the sample diluent comprises a surfactant, and the EB virus early antigen is activated by a non-ionic surfactant. Compared with the existing EB virus detection kit, the detection kit provided by the present application has good stability, high sensitivity, weak background signal and strong specificity, and is suitable for popularization and use.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to an EB virus early antigen IgA antibody detection kit. Background Technology

[0002] Epstein-Barr virus (EBV), also known as human herpesvirus, can only replicate in B lymphocytes, and its infection is associated with a variety of diseases. Based on whether infectious viral progeny are produced after infection, it can be divided into proliferative and non-proliferative infections: antigens associated with proliferative infection include early antigens, membrane antigens, and capsid antigens; identified antigens for non-proliferative infection include nuclear antigens and latent infection proteins. Research on EBV antigens and antibodies is of great significance for revealing the relationship between EBV and nasopharyngeal carcinoma and for the early diagnosis of nasopharyngeal carcinoma.

[0003] Currently, methods for detecting Epstein-Barr virus (EBV) infection include viral culture, immunological detection, and EBV DNA load detection. Because EBV requires host cells to survive, in vitro viral culture has limitations. For example, it typically requires saliva or lymphocytes from patients in the acute phase, the culture time is long, and the positive rate is low, so it is rarely used in clinical testing. EBV exists in the form of circular DNA in the cytoplasm of lymphocytes. 90% of infections are latent; only active EBV can destroy lymphocytes and enter the bloodstream. The vast majority of cell-free DNA fragments in plasma are 82–181 kb in size, so PCR can be used to detect the content of cell-free DNA in plasma. However, it has low sensitivity and poor specificity in early diagnosis.

[0004] Currently, EBV detection mainly employs immunological methods, typically using enzyme immunoassay or immunofluorescence techniques to detect antibodies produced at different stages of infection. However, current EBV testing suffers from several problems that remain to be addressed, including non-specific adsorption of solid-phase antigens into interfering factors during sample testing, leading to cluttered background signals, high false-positive rates, and low sensitivity in immunodiagnostic results. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an EB virus early antigen IgA antibody detection kit. The present invention provides an EB virus early antigen IgA antibody detection kit with good stability, high sensitivity and strong specificity.

[0006] This invention provides a reagent or kit for detecting EB virus early antigen IgA antibody, comprising:

[0007] Solid-phase carrier suspension, enzyme conjugate solution, and sample diluent;

[0008] In the solid-phase carrier suspension, the surface of the solid-phase carrier is coated with early EB virus antigen.

[0009] In the enzyme conjugate solution, the enzyme conjugate is an enzyme-labeled anti-human IgA antibody;

[0010] The sample diluents include calf serum, protein stabilizers, P300, and surfactants.

[0011] The sample diluent of this invention incorporates a surfactant to improve the sensitivity, stability, and specificity of the detection. Studies have shown that the choice of surfactant affects the detection effect of the reagent. In some embodiments, the concentration of the surfactant in the sample diluent is 0.08 vol% to 5 vol%, and the surfactant is selected from at least one of Trixton-100, polyethyleneimine, hexadecyltrimethylammonium bromide, and polydiallyldimethylammonium chloride. Further studies have shown that, compared with other surfactants and protein stabilizers, the addition of Trixton-100 to the sample diluent is more beneficial for cooperating with other reagents and obtaining better detection results.

[0012] In the sample diluent described in this invention, the protein stabilizer, P300, and fetal bovine serum assist the surfactant, allowing for better exposure of the target binding sites in the sample, resulting in more accurate and sensitive detection. In some embodiments, the protein stabilizer is a polyhydroxy sugar, selected from at least one of sucrose or trehalose, specifically sucrose.

[0013] In some specific embodiments, the sample diluent includes water, Trixton-100, calf serum, and P300. This sample diluent has higher compatibility with EB virus early antigen and anti-human IgA antibody, and stronger anti-interference ability, thereby obtaining more accurate and sensitive technical results.

[0014] In some specific embodiments, the sample diluent consists of water, 20 vol%–50 vol% fetal bovine serum, 1–5 mg / mL sucrose, 0.5 vol%–3 vol% P300, and 0.1 vol%–5 vol% Trixton-100. Experiments show that at this composition and concentration, the sample diluent exhibits the strongest anti-interference ability and the highest compatibility with antibodies.

[0015] The solid-phase carrier suspension of the present invention comprises a solid-phase carrier coated with early EB virus antigen and a buffer solution. The preparation of the solid-phase carrier coated with early EB virus antigen includes: activating the early EB virus antigen with a treatment agent containing 0.05 vol% to 0.1 vol% surfactant, mixing the activated solid-phase carrier with the activated antigen, and then incubating and blocking to obtain the solid-phase carrier coated with early EB virus antigen. The surfactant in the treatment agent is a nonionic surfactant.

[0016] Nonionic surfactants have nonpolar hydrophilic groups at their ends, resulting in a milder effect. They can disrupt protein-lipid and lipid-lipid bonds, but have no effect on protein-protein bonds. Therefore, such surfactants preserve the native conformation of proteins, preventing denaturation and ensuring normal function and interaction between antigens and antibodies. In this invention, the antigen is pretreated for activation before coating, and a surfactant is added to the activator to increase its hydrophilicity and solubility in aqueous solution, thereby improving its coating efficiency. Compared with other antigen activators, this invention selects nonionic surfactants for addition, thereby better opening disulfide bonds in proteins, exposing more reaction sites, and improving detection sensitivity. In the embodiments of this invention, the nonionic surfactants used in the antigen activation step include at least one of L7600, Tween 20, alkyl glucoside, n-octylglutamic acid, vitamin E polyethylene glycol succinate, or Trixton-100. In some embodiments, Tween 20 is used as the surfactant for activating the antigen. In some specific embodiments, the treatment agent for early EB virus antigen includes water and 0.1 vol% Tween 20. The activation conditions for the early antigen of the EB virus include a reaction at 15-25°C for 1 hour;

[0017] Before the antibody is coated onto the solid-phase support, the solid-phase support is also activated. In this invention, the matrix of the solid-phase support includes polystyrene-phenylene crosslinked resin, derived plastics, magnetic microspheres, nitrocellulose membranes, glass cellulose membranes, or nylon membranes. In some specific embodiments, magnetic microspheres are preferred as the matrix of the solid-phase support. The activating agents for the solid-phase support of this invention include water, acetic acid, EDC, and NHS. The concentration of acetic acid is 0.1 vol% to 5 vol%, the concentration of EDC is 0.5 to 1 mg / ml, and the concentration of NHS is 0.5 to 1 mg / ml. The activation conditions for the solid-phase support include a reaction at 20–28°C for 1 hour.

[0018] The activated antigen was mixed with a solid-phase carrier and incubated to coat the solid-phase carrier with the antigen. The incubation conditions were 20°C with shaking for 1 hour; during the coating process, the concentration of the EB virus early antigen was 0.1 vol% to 5 vol%.

[0019] After coating, the coated product also needs to be blocked. The blocking reagent described in this invention is PEG8000.

[0020] The blocked solid-phase carrier was resuspended in buffer solution to prepare a solid-phase carrier suspension. In the solid-phase carrier suspension of this invention, the concentration of the solid-phase carrier coated with EB virus early antigen is 10 μL / mL, and the buffer solution is a Tris blocking solution containing a protein protectant. The protein protectant is sucrose, with a concentration of 0.1 vol% to 5 vol% in the buffer solution, and the concentration of Tris in the buffer solution is 0.5 mol / L.

[0021] In this invention, a solid-phase carrier coated with early EB virus antigen is responsible for capturing antibodies in the analyte, while an enzyme conjugate is used to detect the captured antibodies. In this invention, the enzyme conjugate solution comprises an enzyme conjugate and a buffer solution. The enzyme conjugate is a horseradish peroxidase-labeled anti-human IgA antibody. The concentration of the horseradish peroxidase-labeled anti-human IgA antibody in the enzyme conjugate solution is 1:(1000–5000), and the buffer solution is PBS solution.

[0022] Preferably, it also includes a calibrator solution, wherein the calibrator includes calibrator 1 and calibrator 2;

[0023] The calibrator 1 solution includes negative human serum and PBS buffer, wherein the negative human serum is serum from a person who has not been infected with EB virus, and the S / CO value of the negative human serum is <0.1;

[0024] The calibrator 2 solution includes positive human serum and PBS buffer. The positive human serum is an inactivated anti-human IgA antibody, and the S / CO value of the positive human serum is 1.75 to 3.25.

[0025] This invention also provides a method for detecting early EB virus antigen, comprising detecting a sample using a reagent or kit for detecting EB virus early antigen IgA antibody according to this invention. The sample includes serum / plasma.

[0026] This invention provides an Epstein-Barr virus (EBV) early antigen IgA antibody detection kit, comprising a solid-phase carrier suspension, an enzyme conjugate solution, and a sample diluent. The solid-phase carrier suspension contains EBV early antigen coated on its surface, which is activated using a nonionic surfactant. Compared to existing EBV detection kits, the kit provided by this invention exhibits better stability, higher sensitivity, weaker background signal, and stronger specificity. Experimental verification shows that after 14 days of storage at 37°C, the luminescence value variation of positive samples is within ±20%. The kit also demonstrates strong anti-interference capabilities; hemoglobin, triglycerides, and bilirubin at concentrations of 50–3000 mg / dL do not significantly interfere with the detection of false positives and weakly positive samples. Detailed Implementation

[0027] This invention provides an EB virus early antigen IgA antibody detection kit. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0028] The kit provided by this invention pre-treats the antigen before coating, increasing its hydrophilicity and solubility in aqueous solution, thereby improving its coating efficiency. It also provides a pretreatment agent for activating solid-phase antigens in immunodiagnostic reagents, including at least one nonionic surfactant that can break disulfide bonds in proteins, exposing more reaction sites and improving detection sensitivity. The nonionic surfactant has a nonpolar hydrophilic terminal group, resulting in a milder effect; it can disrupt protein-lipid and lipid-lipid bonds, but has no effect on protein-protein binding. Therefore, this surfactant preserves the native conformation of the protein, preventing denaturation and ensuring normal function and interaction between the antigen and antibody.

[0029] This invention provides a sample diluent for use in the dilution of clinical serum / plasma samples, comprising at least one surfactant, which can reduce non-specific adsorption caused by the binding of solid-phase antigens with interfering factors in the sample during sample detection, reduce background signals and false positive detection rates of immunodiagnostic results, and improve the specificity of the detection method.

[0030] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0031] Example 1

[0032] Preparation of EB virus early antigen IgA antibody detection kit (magnetic microsphere chemiluminescence indirect method):

[0033] The formulation includes an enzyme conjugate solution, a sample diluent, calibrator 1, calibrator 2, and a solid-phase carrier suspension. In this embodiment, the solid-phase carrier is a magnetic microsphere, which is coated with recombinant EB virus early antigen.

[0034] I. Preparation of solid-phase carrier suspension:

[0035] 1. Antigen activation: The recombinant antigen of early EB virus was activated using an activation stage treatment agent containing 0.1 vol% alkyl glucoside (APG) activator. The activation conditions included a reaction at 15–25°C for 1 hour.

[0036] 2. Activation of Magnetic Microspheres: Magnetic microspheres contain carboxyl active groups, with approximately 30 μg of carboxyl groups per gram (g) of magnetic beads (dry weight). The microspheres are uniform in size, with a diameter of 1.5-3 μm. Take 10 μL of the stock solution of magnetic microspheres (30 mg / mL), wash the carboxyl-containing magnetic microspheres three times with PBS buffer, and discard the supernatant. Add acetic acid diluted with EDC and NHS solution to activate the magnetic microspheres for 1 hour at 20-28℃. After activation, wash twice with MES buffer and discard the supernatant. The activation reagents include water, acetic acid, EDC, and NHS, where the concentration of acetic acid is 0.1 vol%-5 vol%, the concentration of EDC is 0.5-1 mg / mL, and the concentration of NHS is 0.5-1 mg / mL.

[0037] 3. Coating: Dilute the recombinant EB virus early antigen with MES buffer and coat it onto the surface of magnetic microspheres at a rate of 50 μL / mL. Shake at 20°C and incubate for 1 hour for adsorption.

[0038] 4. Termination: Discard the coating solution, use PEG 8000 termination solution at 200 μL / test, shake at 20℃, and terminate for 1 hour;

[0039] 5. Blocking: Discard the stop solution, and use Tris blocking buffer containing protein protectant at a rate of 200 μL / test. Shake at 20°C for 20 min each time.

[0040] 6. Volume adjustment and storage: Adjust the volume to 1 mL using Tris blocking buffer containing protein protectant, and seal for storage;

[0041] II. Preparation of Sample Diluent

[0042] Sample dilution: The sample diluent is PBS buffer. Samples are diluted using a sample diluent containing 0.12 vol% cetyltrimethylammonium bromide (CTAB) surfactant. The sample diluent also includes 20%–50% fetal bovine serum (FBS), 1 mg / mL–5 mg / mL protein stabilizer, and 0.5%–3% preservative. The protein stabilizer is a polyhydroxy sugar selected from at least one of sucrose or trehalose; sucrose is used in this embodiment. The preservative includes P300.

[0043] III. Preparation of enzyme conjugates:

[0044] Horseradish peroxidase-labeled anti-human IgA was dissolved in a buffer solution, which was PBS buffer.

[0045] IV. Preparation of Standards:

[0046] Calibrator 1: The buffer is PBS buffer, and the S / CO value of negative human serum should be <0.1.

[0047] Calibrator 2: The buffer is PBS buffer, which includes inactivated EB virus early antigen IgA antibody-positive human serum with an S / CO value of 1.75 to 3.25.

[0048] Example 2

[0049] The nonionic surfactant contained in the activation stage treatment agent is L7600, with a concentration of 0.05 vol%.

[0050] The surfactant contained in the sample dilution agent was PEI (0.08 vol%).

[0051] The solid support is a polystyrene-phenylene crosslinked resin.

[0052] The other steps are the same as in Example 1.

[0053] Example 3

[0054] The nonionic surfactant contained in the activation stage treatment agent is alkyl glucoside (APG) at a concentration of 0.08 vol%.

[0055] The sample diluent contained cetyltrimethylammonium bromide (CTAB) as a surfactant at a concentration of 0.1 vol%.

[0056] The solid support is a type of magnetic microsphere.

[0057] The other steps are the same as in Example 1.

[0058] Example 4

[0059] The nonionic surfactant contained in the activation stage treatment agent is vitamin E polyethylene glycol succinate (TPGS) at a concentration of 0.1 vol%.

[0060] The surfactant contained in the sample diluent was polydiallyl dimethyl ammonium chloride (PDDA) at a concentration of 0.15 vol%.

[0061] The solid support is a nitrocellulose membrane (Nc).

[0062] The other steps are the same as in Example 1.

[0063] Example 5

[0064] Evaluation of different nonionic surfactants in the activation phase treatment: Using serum / plasma as samples, automated analysis was performed using the fully automated chemiluminescence analyzer AutoLumo A2000, AutoLumo A2000Plus, or AutoLumo A2000 Plus B manufactured by Zhengzhou Antu Bioengineering Co., Ltd. The detection steps included: mixing the test sample, the magnetic bead suspension coated with antigen, and the sample diluent; reacting at 37°C for 10–20 min; then washing with PBS buffer; adding the enzyme-labeled solution; reacting at 37°C for 10–20 min; then washing with PBS buffer; adding luminescent substrate solution A and luminescent substrate solution B; reacting at 37°C for 5 min; and detecting the luminescence value. The luminescence value was used to determine whether the sample was positive. The volume ratio of sample, sample diluent, magnetic microparticle suspension, and enzyme conjugate was 10:1:2:10.

[0065] The other steps were the same as in Example 1. During the activation stage, five nonionic surfactants—L7600, Tween20 (T-20), alkyl glucoside (APG), n-octylglutamate (OGP), and vitamin E polyethylene glycol succinate (TPGS)—were added to the treatment agent. The test group without added surfactants and anionic surfactants (SDS) served as a control. Ten EBV early antigen IgA positive samples (P1-P10) and ten EBV early antigen IgA negative samples (N1-N10) were detected using the developed EBV early antigen IgA antibody detection kit (magnetic microsphere chemiluminescence indirect method). The results are shown in Table 1.

[0066] Table 1. Evaluation of different nonionic surfactants in the activation stage treatment agent

[0067]

[0068]

[0069] As shown in Table 1, without adding surfactants or anionic surfactants, good contrast between positive and negative results cannot be achieved when other parameters remain unchanged. After activating the antigen using the T-20 nonionic surfactant, the kit's detection results exhibit a large positive / negative contrast ratio (P / N), which is an important indicator for evaluating kit performance. Therefore, T-20 is considered to have good antigen activation ability.

[0070] Example 6

[0071] In addition to diluting the samples, the sample diluent should create a buffer environment conducive to antigen-antibody binding, providing optimal reaction conditions for immunodiagnosis. To investigate the effects of different active agents and suitable concentrations in the sample diluent on clinical testing, we studied the effects of Trixton-100, polyethyleneimine (PEI), hexadecyltrimethylammonium bromide (CTAB), and polydiallyldimethylammonium chloride (PDDA) in the sample diluent, and investigated the addition of different concentrations of the active agent Trixton-100. The prepared EBV early antigen IgA antibody detection kit (magnetic microsphere chemiluminescence indirect method) was used to test 10 EBV early antigen IgA positive samples (P1-P10) and 10 EBV early antigen IgA negative samples (N1-N10). The results are shown in Tables 2-1 and 2-2:

[0072] Table 2-1. Effects of different sample dilutions on clinical testing

[0073]

[0074]

[0075] Table 2-2. Effects of different concentrations of surfactant in sample dilution agents on clinical testing.

[0076]

[0077] As shown in Tables 2-1 and 2-2, the average ratio of positive to negative samples increased when 1 vol% Trixton-100 was added, but the average ratio of both positive and negative samples decreased as the Trixton-100 concentration increased to 1 vol%. This indicates that the appropriate surfactant concentration has a significant impact on the performance of the kit.

[0078] Example 1

[0079] The antigen activator was prepared using T-20 activator, and the concentration of Trixton-100 added to the sample dilution agent was 0.1%. The EB virus early antigen IgA antibody detection kit (magnetic microsphere chemiluminescence indirect method) was prepared, and the rest was the same as in Example 1.

[0080] 1. Stability assessment of the reagent kit:

[0081] The above-mentioned kit was used to test 10 EB early antigen IgA positive samples (P1-P10) and 10 EB early antigen negative samples (N1-N10) to assess the stability of the kit after being placed at 37℃ for 7, 10, and 14 days. The results are expressed as S / CO, as shown in Table 3. The results in Table 3 were processed to obtain the amplitude of signal value changes, as shown in Table 4.

[0082] Table 3. Results of stability testing of the reagent kit

[0083]

[0084] Table 4. Stability assessment of the kit

[0085]

[0086]

[0087] The stability assessment of the kit generally requires that the luminescence value variation of positive samples be within ±20%, and that negative samples should not produce positive results. As shown in Tables 3 and 4, after storage at 37℃ for 7, 10, and 14 days, the luminescence value variation of positive samples remained within ±20%, and no positive results were observed in negative samples. This indicates that antigen activation and the addition of an active agent during sample dilution do not adversely affect the stability of the kit.

[0088] 2. Specificity evaluation of the kit:

[0089] Adding surfactants may interfere with the specificity of the kit. Therefore, two samples, negative and weakly positive, were used, and different concentrations of hemoglobin, triglycerides, and bilirubin were added to examine the specificity and anti-interference ability of the kit. The results are shown in Table 5-7:

[0090] Table 5. Assessment of Hemoglobin Interference

[0091]

[0092]

[0093] Table 6. Triglyceride interference assessment

[0094]

[0095] Table 7. Bilirubin Interference Assessment

[0096]

[0097]

[0098] As shown in Table 5, when the hemoglobin concentration reaches 1000 mg / dL, the results for negative samples remain negative, without producing false positives. Furthermore, 1000 mg / dL of hemoglobin does not significantly interfere with the detection of weakly positive samples in the kit. According to the results in Table 6, when the triglyceride concentration reaches 3000 mg / dL, the results for negative samples remain negative, without producing false positives. Furthermore, 3000 mg / dL of triglycerides does not significantly interfere with the detection of weakly positive samples in the kit. Table 7 shows that when the bilirubin concentration reaches 50 mg / dL, the results for negative samples remain negative, without producing false positives, and 50 mg / dL of bilirubin does not significantly interfere with the detection of weakly positive samples in the kit.

[0099] The anti-interference assessment of the kit requires that the deviation of the detection results of interfering samples from those of samples with only solvent added be within ±10%. The assessments of all three interfering factors met this requirement. This indicates that activating the antigen and adding the active agent during sample dilution does not adversely affect the specificity of the kit. Therefore, the antigen activator and sample dilution agent provided by this invention can be applied to immunodiagnostic kits for viral infection biomarkers.

[0100] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An EB virus early antigen IgA antibody detection kit, characterized in that, include: Solid-phase carrier suspension, enzyme conjugate solution, and sample diluent; In the solid-phase carrier suspension, the surface of the solid-phase carrier is coated with early EB virus antigen. In the enzyme conjugate solution, the enzyme conjugate is an enzyme-labeled anti-human IgA antibody; The sample diluent includes fetal bovine serum, a protein stabilizer, P300, and a surfactant. The protein stabilizer is a polyhydroxy sugar selected from at least one of sucrose or trehalose. The surfactant has a concentration of 0.08 vol% to 5 vol%. The surfactant is selected from at least one of Trixton-100, polyethyleneimine, hexadecyltrimethylammonium bromide, and polydiallyldimethylammonium chloride. The preparation of a solid-phase carrier coated with early EB virus antigen includes: activating the early EB virus antigen with a treatment agent containing 0.05 vol% to 0.1 vol% surfactant, mixing it with the activated solid-phase carrier, and then incubating and blocking to obtain a solid-phase carrier coated with early EB virus antigen. The surfactant in the treatment agent is a nonionic surfactant.

2. The reagent kit according to claim 1, characterized in that, The surfactant in the sample diluent is Trixton-100, and the protein stabilizer is sucrose.

3. The EB virus early antigen IgA antibody detection kit according to claim 1 or 2, characterized in that, The sample diluent consists of 20 vol% to 50 vol% fetal bovine serum, 1 to 5 mg / mL sucrose, 0.5 vol% to 3 vol% P300, and 0.1 vol% to 5 vol% Trixton-100.

4. The reagent kit according to claim 1, characterized in that, The nonionic surfactant includes at least one of L7600, Tween 20, alkyl glucoside, n-octylglutamic acid, vitamin E polyethylene glycol succinate, or Trixton-100.

5. The reagent kit according to claim 4, characterized in that, The treatment agents for early EB virus antigens include water and 0.1 vol% Tween 20; The activating agents of the solid support include water, 0.1 vol% to 5 vol% acetic acid, 0.5 to 1 mg / mL EDC, and 0.5 to 1 mg / mL NHS.

6. The kit according to claim 4 or 5, characterized in that, The activation conditions for the EB virus early antigen include a reaction at 15-25°C for 1 hour; The activation conditions for the solid support include reacting at 20~28℃ for 1 hour; The incubation conditions are 20°C and shaking incubation for 1 hour; The blocking reagent is PEG 8000.

7. The kit according to claim 1, characterized in that, The matrix of the solid support includes polystyrene-phenylene crosslinked resin, derived plastics, magnetic microspheres, nitrocellulose membranes, glass cellulose membranes, or nylon membranes.

8. The reagent kit according to claim 1, characterized in that, In the solid-phase carrier suspension, the concentration of the solid-phase carrier coated with the early antigen of EB virus is 10 μL / mL, and the buffer is a Tris blocking solution containing sucrose, wherein the sucrose concentration is 0.1 vol%~5 vol% and the Tris concentration is 0.5 mol / L. In the enzyme conjugate solution, the concentration of horseradish peroxidase-labeled anti-human IgA antibody is 1:(1000~5000), and the buffer is PBS buffer.

9. The reagent kit according to claim 1, characterized in that, It also includes a calibration solution, wherein the calibrators include calibrator 1 and calibrator 2; The calibrator 1 solution includes negative human serum and PBS buffer, wherein the negative human serum is serum from a person who has not been infected with EB virus, and the S / CO value of the negative human serum is <0.1; The calibrator 2 solution includes positive human serum and PBS buffer. The positive human serum is an inactivated anti-human IgA antibody with an S / CO value of 1.75 to 3.25.

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