Detection kit and detection method for EB (Epstein-Barr) virus capsid antigen IgG (Immunoglobulin G) antibody

Abstract

The invention relates to a preparation method of an EB (Epstein-Barr) virus capsid antigen-magnetic bead complex, which comprises the following steps: adding 0.5-2M of PB buffer solution and EB virus capsid antigen into magnetic beads, uniformly mixing at 15-30 DEG C, and standing for 2-3 hours; wherein the EB virus capsid antigen is a VCA-P18 antigen and / or a VCA-P23 antigen; adding a confining liquid, and uniformly mixing at 15-37 DEG C for 1-3 hours; removing supernate, and adding a magnetic bead diluent for resuspension; the magnetic bead diluent comprises about 0.1 M of a Tris-HCl buffer solution, Casein of which the mass volume ratio is 0.2%-1.0%, MAK33 of which the mass volume ratio is (10-30) [mu] g / mL, and TW20 of which the mass volume ratio is 0.3%-0.8%.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a detection kit and a detection method for Epstein-Barr virus capsid antigen IgG antibody. Background Art

[0002] Epstein-Barr virus (EBV) belongs to the herpesvirus family and is called human herpesvirus type IV. It mainly infects human epithelial cells and B cells through saliva. It is closely related to the occurrence of many diseases and threatens human health. More than 90% of individuals have been infected with EBV before the age of 20, and most people show asymptomatic infection or self-limited infectious mononucleosis (IM). EBV is also associated with the occurrence of many malignant tumors, including nasopharyngeal carcinoma (NPC), lymphoid malignancies, post-transplant lymphoproliferative disease (PTLD), systemic lupus erythematosus (SLE) and gastric cancer (GC).

[0003] After EBV infection, the body will produce an immune response against multiple EB virus antigens and produce a variety of EB virus antibodies, including EBV capsid antigen antibodies (EBV-VCA-IgG / IgA / IgM), EBV early antigen antibodies (EBV-EA-IgA), EBV nuclear antigen antibodies (EBV-NA-IgG / IgA), Rta protein antibodies (Rta-IgG) and Zta protein antibodies (Zta-IgG), etc.

[0004] Different antibodies appear at different stages of viral infection. VCA-IgG antibodies appear at the beginning of infection along with VCA-IgM antibodies and can persist in the body for many years or for life. EBV nucleoprotein (EBNA) is produced during the recovery period of primary infection, and EBNA-IgG antibody positivity is often considered a specific indicator of previous infection. Therefore, tracking the EBV antibody spectrum helps in the diagnosis of EBV infection and is an indispensable basis for clinical diagnosis.

[0005] At present, there are gold label method and enzyme-linked immunosorbent assay (ELISA) for the determination of IgG antibodies to EB virus capsid antigen. The gold label method has low sensitivity and can only be qualitative but not quantitative; the ELISA method has poor quantitative accuracy, poor anti-interference ability, long operation time, low degree of automation, and the results are greatly affected by human factors. Therefore, a more suitable method for detecting VCA-IgG levels is urgently needed. Summary of the invention

[0006] In view of the technical problems existing in the prior art, the present invention proposes a method for preparing an Epstein-Barr virus capsid antigen-magnetic bead complex, comprising: adding 0.5-2M PB buffer and Epstein-Barr virus capsid antigen to magnetic beads, and mixing and placing at 15-30 degrees for 2-3 hours; wherein the Epstein-Barr virus capsid antigen is VCA-P18 antigen and / or VCA-P23 antigen; adding blocking solution, and mixing at 15-37 degrees for 1-3 hours; removing the supernatant, and adding magnetic bead diluent for resuspending; the magnetic bead diluent comprises about 0.1M Tris-HCl buffer, 0.2%-1.0% Casein by mass volume ratio, (10-30)ug / mL MAK33 and 0.3%-0.8% TW20 by mass volume ratio; preferably, the magnetic bead diluent comprises about 0.1M Tris-HCl buffer, 0.5% Casein by mass volume ratio, 20ug / mL MAK33 and 0.05% TW20 by mass volume ratio.

[0007] The method as described above further comprises: after adding blocking solution to react for 1-3 hours, removing the supernatant, and adding magnetic bead diluent to wash the reaction product once or multiple times.

[0008] In the method described above, the blocking solution includes 0.1 M PBS buffer and 1% BSA in a mass-to-volume ratio.

[0009] The present application relates to an Epstein-Barr virus capsid antigen-magnetic bead complex prepared according to any of the above methods, wherein the Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex or a VCA-P23-magnetic bead complex.

[0010] The present application relates to a detection kit for IgG antibodies to Epstein-Barr virus capsid antigens, comprising: a VCA-P18-magnetic bead complex and / or a VCA-P23-magnetic bead complex; an acridinium ester-anti-human IgG conjugate; and a sample diluent, wherein the sample diluent comprises approximately 0.1M PBS buffer and (10-30)% newborn calf serum in a mass-to-volume ratio; preferably, the sample diluent comprises approximately 0.1M PBS buffer and 20% newborn calf serum in a mass-to-volume ratio.

[0011] In the kit as described above, the VCA-P18-magnetic bead complex and / or VCA-P23-magnetic bead complex is prepared by any of the methods described above, or the VCA-P18-magnetic bead complex and / or VCA-P23-magnetic bead complex is selected from the Epstein-Barr virus capsid antigen-magnetic bead complex described above.

[0012] A method for detecting Epstein-Barr virus capsid antigen IgG antibody in a sample comprises: obtaining a sample; mixing the sample, an Epstein-Barr virus capsid antigen-magnetic bead complex and a sample diluent to obtain an antigen-antibody complex; wherein the Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex and / or a VCA-P23-magnetic bead complex; preferably, the Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex and a VCA-P23-magnetic bead complex; adding an acridinium ester-labeled anti-human IgG antibody to the antigen-antibody complex for binding reaction; adding an excitation solution to the binding reaction product; detecting the luminescence intensity of the excitation light; and obtaining the detection result of the Epstein-Barr virus capsid antigen IgG antibody.

[0013] In the detection method described above, the addition ratio of sample, Epstein-Barr virus capsid antigen-magnetic bead complex, acridinium ester-labeled anti-human IgG antibody and sample diluent is 1:(4-6):(8-12):(3-6), preferably 1:5:10:4.

[0014] In the detection method as described above, the ratio of the added amount of VCA-P18-magnetic bead complex to that of VCA-P23-magnetic bead complex is 1:(0.5-3), preferably 1:(1-2), and more preferably 1:1.

[0015] In the detection method as described above, the excitation solution includes: a mixed solution of hydrogen peroxide and nitric acid and a sodium hydroxide solution.

[0016] The present invention adopts magnetic particles as the solid phase carrier of the antigen, and utilizes chemiluminescent immunoassay technology to realize single-person, random, rapid, and automated detection on a fully automatic chemiluminescent immunoassay instrument. The indirect method adopted in the technical principle selects the two most valuable dominant antigens in the EB virus capsid antigen protein, avoiding the problem of low sensitivity caused by the incomplete sites of conventional chemiluminescent detection kits on the market. This kit is suitable for the quantitative detection of EB virus capsid antigen IgG antibodies in human serum samples, and is used for clinical auxiliary diagnosis of EB virus infection. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] In the following detailed description, reference may be made to the specific embodiments that are part of the present application and are used to illustrate the present application. Each specific embodiment of the present application is described in sufficient detail below so that a person of ordinary skill in the art with relevant knowledge and skills in the art can implement the technical solution of the present application. It should be understood that other embodiments may also be used or the embodiments of the present application may be changed.

[0019] The professional terms used in this application have the following meanings:

[0020] The "capsid" mentioned herein refers to the protein shell of the virus, which is used to encapsulate the viral genetic material (nucleic acid).

[0021] The "EB virus capsid antigen" mentioned herein is an antigenic substance produced after an organism is infected with EB virus, including but not limited to P18, P23, etc. EB virus is a human herpes virus, mainly transmitted through saliva. After infection, it can lurk in human lymphocytes for a long time, and can be activated and cause disease under certain conditions. It is usually related to a variety of diseases, such as infectious mononucleosis, nasopharyngeal carcinoma, Burkitt's lymphoma, etc.

[0022] "P18" and "VCA-P18" as used herein have the same meaning, and "P23" and "VCA-P23" have the same meaning. Both P18 and P23 are Epstein-Barr virus capsid antigens. The P18 antigen is highly immunogenic in humans and contains B cell epitopes presented in the carboxyl region. The P23 antigen can display the VCA antibody spectrum in serological analysis.

[0023] The "EB virus capsid antigen-magnetic bead complex" mentioned herein refers to the EB virus capsid antigen labeled with magnetic beads, which allows the antigen to bind to a specific antibody and also has magnetism and can be adsorbed in a magnetic field. In some embodiments, the EB virus capsid antigen-magnetic bead complex is specifically a VCA-P18-magnetic bead complex and / or a VCA-P23-magnetic bead complex.

[0024] The "Tris-HCl buffer" mentioned in this article refers to a certain amount of hydrochloric acid added to an aqueous solution containing a certain amount of Tris to adjust the pH value of the solution. It has strong alkalinity, and only this buffer system is needed to prepare a buffer with a wide pH range from acidic to alkaline; it has little interference with biochemical processes and does not react with calcium, magnesium and heavy metal ions.

[0025] The "Casein" mentioned in this article refers to sodium caseinate, also known as sodium caseinate, sodium caseinate, sodium caseinate or sodium casein. It is the sodium salt of casein, the main protein in cow's milk. It is a safe and harmless thickener and emulsifier.

[0026] The "MAK33" mentioned in this article refers to the cleaning antibody / specific interference elimination protein, which is available from companies such as Roche and can be used to reduce nonspecific interference in the immunoassay process.

[0027] The "TW20" mentioned in this article refers to polysorbate 20, also known as Tween 20, which is a surfactant, a yellow or amber clear oily liquid used as an emulsifier, dispersant, solubilizer, stabilizer, etc.

[0028] Unless otherwise specified, the percentage of a component in a reagent referred to in this article represents the mass-to-volume ratio of the component in the reagent, for example, the mass of the component added per liter of reagent.

[0029] The present application relates to a detection kit for Epstein-Barr virus capsid antigen IgG antibody, which includes: VCA-P18-magnetic bead complex and / or VCA-P23-magnetic bead complex, acridinium ester-anti-human IgG conjugate and sample diluent.

[0030] The sample diluent comprises about 0.1M PBS buffer and (10-30)% newborn calf serum in a mass-volume ratio; preferably, the sample diluent comprises about 0.1M PBS buffer and 20% newborn calf serum in a mass-volume ratio.

[0031] In some embodiments, the kit of the present application does not limit the preparation method and acquisition method of the EB virus capsid antigen-magnetic bead complex, and it can be purchased directly or made by yourself. In some embodiments, the EB virus capsid antigen-magnetic bead complex is prepared by the following method:

[0032] Add 0.5-2M PB buffer and Epstein-Barr virus capsid antigen to the magnetic beads, mix and place at 15-30 degrees for 2-3 hours; wherein the Epstein-Barr virus capsid antigen is VCA-P18 antigen and / or VCA-P23 antigen; add blocking solution, mix and place at 15-37 degrees for 1-3 hours; remove the supernatant, and add magnetic bead diluent to resuspend.

[0033] In some embodiments, the magnetic bead diluent comprises about 0.1M Tris-HCl buffer, 0.2%-1.0% Casein by mass volume ratio, (10-30)ug / mL MAK33, and 0.3%-0.8% TW20 by mass volume ratio; preferably, the magnetic bead diluent comprises about 0.1M Tris-HCl buffer, 0.5% Casein by mass volume ratio, 20ug / mL MAK33, and 0.05% TW20 by mass volume ratio.

[0034] In some embodiments, after adding blocking solution and reacting for 1-3 hours, the supernatant is removed and magnetic bead diluent is added to wash the reaction product once or multiple times.

[0035] In some embodiments, the blocking solution includes 0.1 M PBS buffer and 1% BSA in a weight-to-volume ratio.

[0036] In some embodiments, the Epstein-Barr virus capsid antigen-magnetic bead complex is specifically a VCA-P18-magnetic bead complex and / or a VCA-P23-magnetic bead complex.

[0037] A method for detecting IgG antibodies of Epstein-Barr virus capsid antigen in a sample comprises: obtaining a sample; mixing the sample, Epstein-Barr virus capsid antigen-magnetic bead complex and sample diluent to obtain an antigen-antibody complex. The Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex and / or a VCA-P23-magnetic bead complex; preferably, the Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex and a VCA-P23-magnetic bead complex; adding an acridinium ester-labeled anti-human IgG antibody to the antigen-antibody complex for binding reaction; adding an excitation solution to the binding reaction product; detecting the luminescence intensity of the excitation light; and obtaining the detection result of IgG antibodies of Epstein-Barr virus capsid antigen.

[0038] In some embodiments, the addition ratio of the sample, Epstein-Barr virus capsid antigen-magnetic bead complex, acridinium ester-labeled anti-human IgG antibody, and sample diluent is 1:(4-6):(8-12):(3-6), preferably 1:5:10:4.

[0039] In some embodiments, the ratio of the added amount of VCA-P18-magnetic bead complex to that of VCA-P23-magnetic bead complex is 1:(0.5-3), preferably 1:(1-2), and more preferably 1:1.

[0040] In some embodiments, the stimulating solution includes: a mixed solution of hydrogen peroxide and nitric acid and a sodium hydroxide solution.

[0041] The present application will illustrate the technical solution of the present application through the following examples. Those skilled in the art should understand that the present application should not be limited to the following examples.

[0042] Example 1 Preparation method of VCA-P18-magnetic bead complex

[0043] 1) Take the magnetic bead concentrate stock solution, remove the supernatant, and mix thoroughly with activation buffer (100mM MES buffer, pH6.0); adsorb the magnetic beads and remove the supernatant; repeat 3 times; in some embodiments, the present application does not limit the type of magnetic beads. In this embodiment, the magnetic beads selected are carboxyl magnetic beads, and this embodiment does not limit the specific type of carboxyl magnetic beads used. In this embodiment, the magnetic bead concentrate stock solution used is purchased directly, and the product is purchased from JSR Life Sciences, and the article number is: MS160-Carboxyl 10%. In some embodiments, the magnetic bead concentrate is only a storage state of the magnetic beads and is not a limitation of the present application. In fact, the applicant can choose any magnetic beads, and the selected magnetic beads can have any initial state. According to the experimental requirements, the magnetic beads can be purchased directly or prepared by the experimenter.

[0044] 2) Add activation buffer, mix thoroughly, add EDC (10 mg / ml), and mix at room temperature for 30 min; unless otherwise specified, the room temperature in this embodiment is 18°C-28°C; preferably, the room temperature is 20°C-25°C, which will not be repeated below.

[0045] 3) adsorption, remove the supernatant, wash twice with PB (pH 8.0) buffer, add PB (pH 8.0) buffer, add VCA-P18 antigen to the magnetic beads, react at room temperature for 2 hours, and remove the supernatant. In this example, VCA-P18 antigen was purchased from Meridian Company, product number R01571.

[0046] 4) Add blocking solution, block at room temperature for 2 hours, adsorb on magnetic stand for about 2-3 minutes, remove supernatant with a pipette, and the remaining component is VCA-18-magnetic bead complex. Further, add magnetic bead diluent to wash the complex several times, for example, wash 4 times, adsorb the complex, remove supernatant, and add magnetic bead diluent to resuspend the complex. In this embodiment, the blocking solution includes: 0.1M PBS buffer, 1% BSA.

[0047] In order to screen a magnetic bead diluent with better stability, in this embodiment, three different magnetic bead diluents were selected for experiment. The components of the three magnetic bead diluents respectively include:

[0048] Magnetic bead diluent 1: 0.1M Tris-HCl buffer, 0.5% Casein by mass volume ratio, and 0.05% TW20 by mass volume ratio;

[0049] Magnetic bead diluent 2: 0.1 M Tris-HCl buffer, 5% BSA by mass volume ratio, and 0.05% TW20 by mass volume ratio;

[0050] Magnetic bead diluent 3: 0.1 M Tris-Hcl buffer, 0.5% Casein in a mass-volume ratio, 20 ug / mL MAK33, and 0.05% TW20 in a mass-volume ratio.

[0051] The above reagent components were purchased directly. This application does not limit the production company and product number of the reagent components. According to the applicant's repeated experimental results, the reagent components produced by different companies or with different product numbers have a negligible effect on the final VCA-IgG detection results.

[0052] Example 2 Preparation of VCA-P23-magnetic bead complex

[0053] The preparation method of the VCA-P23-magnetic bead complex can refer to the preparation method of the VCA-P18-magnetic bead complex in Example 1, except that the VCA-P18 antigen is replaced by VCA-P23, which will not be described in detail here.

[0054] Example 3 Effect of magnetic bead diluent on the stability of antigen-magnetic bead complex (mixed complex of VCA-P18-magnetic bead complex and VCA-P23-magnetic bead complex)

[0055] In this embodiment, the inventors took the mixed complex of VCA-P18-magnetic bead complex and VCA-P23-magnetic bead complex in a 1:1 ratio and mixed it evenly with magnetic bead diluent, and further divided the mixed complex into a 4°C group and a 37°C group, and placed the 4 degree group in an environment of about 4°C and the 37°C group in an environment of about 37°C, and detected the luminescence values ​​of different samples on 3 days, 6 days and 10 days respectively (using the company's self-developed fully automatic chemiluminescence immunoassay, ZETA C21 for detection), and calculated the change trend of the luminescence value of 37 degrees relative to 4 degrees on the same day, that is, the change range, to verify the effect of magnetic bead diluent on the stability of the antigen-magnetic bead mixed complex. The test results are shown in Table 1 below, where:

[0056] The range of change is [(luminescence value at 37 degrees - luminescence value at 4 degrees) / luminescence value at 4 degrees] * 100% detected on the same day. The smaller the decrease, the better the stability of the antigen-magnetic bead complex and the better the performance of the magnetic bead diluent.

[0057] Table 1 Effect of magnetic bead diluent on the stability of antigen-magnetic bead complex

[0058]

[0059] It can be clearly seen from Table 1 that the stability of the antigen-magnetic bead mixed complex is best when the magnetic bead diluent includes 0.1M Tris-HCl buffer, 0.5% Casein, 20ug / mL MAK33 and 0.05% TW20. The TW20 mentioned in this application refers to Tween 20.

[0060] Example 4 Acridinium ester-mouse anti-human IgG complex

[0061] 1) Take mouse anti-human IgG into an EP tube, add 0.1M PB buffer to 0.3ml, take acridinium ester working solution, add it to the above protein solution, mix immediately, wrap a layer of aluminum foil outside the centrifuge tube to avoid light, place it on a rotary mixer, at room temperature, mix and react for a while;

[0062] 2) Take 1% Lys, add it to the above centrifuge tube, keep it away from light, place it on a rotary mixer, and mix it at room temperature for 30 minutes;

[0063] 3) The acridinium ester-mouse anti-human IgG complex was purified, glycerol was added, and stored at -20 degrees for future use.

[0064] Example 5 Detection method

[0065] This application is based on magnetic particle chemiluminescence technology, combined with a fully automatic chemiluminescence instrument to determine anti-EBV-VCA-IgG antibodies in human sample serum. The basic detection process is as follows:

[0066] (1) Mix the obtained human sample serum, the antigen-magnetic bead complex prepared according to the method of Example 1 and / or Example 2, and the sample diluent, and react the anti-EBV-VCA-IgG antibody in the positive sample with the antigen on the antigen-magnetic bead complex to form an antigen-antibody complex. The antigen-magnetic bead complex can be a VCA-P18-magnetic bead complex, a VCA-P23-magnetic bead complex, or a mixed complex of the VCA-P18-magnetic bead complex and the VCA-P23-magnetic bead complex in a certain ratio, and the mixing ratio can be (1-10): (1-10), for example, the ratio is 1:1, 2:1, 1:2, 3:1, 1:3, 2:3, 3:2... This article will not repeat them.

[0067] (2) Under the action of the magnetic field, the antigen-antibody complex is adsorbed to the wall of the reaction tube, and a cleaning solution (including 0.1 M PBS buffer and 0.05% TW20) is added to wash away the unbound substances.

[0068] (3) adding an acridinium ester-mouse anti-human IgG complex to the antigen-antibody complex, so that the antigen-antibody complex binds to the IgG antibody on the acridinium ester-mouse anti-human IgG complex to form an antibody-antigen-antibody conjugate.

[0069] Among them, the addition ratio of sample, total amount of antigen-magnetic bead complex, acridinium ester-IgG complex and sample diluent is approximately 1:5:10:4.

[0070] (4) After adding a cleaning solution (including 0.1M PBS buffer and 0.05% TW20) to wash the precipitated antibody-antigen-antibody conjugate, an excitation solution is added. The excitation solution includes a mixed solution of hydrogen peroxide and nitric acid and a sodium hydroxide solution. Under alkaline conditions, the hydrogen peroxide in the excitation solution reacts with the acridinium ester to form an excited acridinone, which releases photons in the process of returning to the ground state, forming a luminescent reaction, and the luminescence intensity of the reaction can be detected using a luminometer.

[0071] (5) Within the detection range, the luminescence intensity is proportional to the concentration of EBV-VCA-IgG in the sample. Therefore, the EBV-VCA-IgG concentration of the sample to be tested is calculated and read by the software fitting curve.

[0072] Among them, steps 1 to 5 can all be completed automatically by the instrument. This application does not limit the specific model of the instrument used. In this embodiment, the instrument used is the company's self-developed fully automatic chemiluminescence immunoassay analyzer, and the following embodiments are luminescence values ​​measured by the software ZETA C21 in this instrument.

[0073] This example uses the commonly used enzyme-linked immunosorbent assay (ELISA) as a reference reagent for EBV-VCA-IgG detection, and uses the test results as a positive control. For the reference reagent, a test result ≥20RU / mL is considered positive; a test result <20RU / mL is considered negative. According to the test results of this application, the cutoff value (Cutoff) is used as the criterion for determining whether the test result is negative or positive. A result greater than the Cutoff value is considered a positive result, and a result less than the Cutoff value is considered a negative result.

[0074] Example 6 Screening of antigen-magnetic bead complexes

[0075] In this example, VCA-P18-magnetic bead complex (hereinafter referred to as P18 complex), VCA-P23-magnetic bead complex (hereinafter referred to as P23 complex) and mixtures of P18 complex and P23 complex in different proportions were mixed with the sample to be tested, and the VCA-IgG antibody content in the sample was detected by the method described in Example 5, wherein the magnetic bead diluent was the magnetic bead diluent 3 shown in Example 3, and the sample diluent included: 0.1M PBS buffer and 5% BSA. The test results are shown in Table 2:

[0076] Table 2 Screening of antigen-magnetic bead complex

[0077]

[0078]

[0079]

[0080] As shown in Table 2, when the P18 complex and the P23 complex were mixed in a 1:1 ratio, the best positive coincidence rate was 100%, while when the P23 complex was tested alone, the negative coincidence rate was even higher, reaching 95%.

[0081] Example 7 Screening of sample diluents

[0082] In this example, the P18 complex and the P23 complex were mixed in a ratio of 1:1 for further screening of sample diluents with better sensitivity and specificity for VCA-IgG antibody detection. The magnetic bead diluent used in this example was the magnetic bead diluent 3 shown in Example 3.

[0083] In this embodiment, three sample diluents are involved, each comprising:

[0084] Sample diluent 1: 0.1 M PBS buffer and 5% BSA;

[0085] Sample diluent 2: 0.1 M PBS buffer, 5% BSA and 0.05% TW20;

[0086] Sample diluent 3: 0.1 M PBS buffer and 20% newborn calf serum.

[0087] The test results and analysis results of different sample dilutions on the samples are shown in Table 3:

[0088] Table 3

[0089]

[0090]

[0091] As shown in Table 3, when the sample diluent includes 0.1M PBS buffer and 20% newborn calf serum, not only can the 100% positive coincidence rate after the improvement of Example 6 be maintained, but the negative coincidence rate can also be increased to 95%, which can be used for VCA-IgG antibody detection of clinical samples.

[0092] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

1. A method for preparing an Epstein-Barr virus capsid antigen-magnetic bead complex, comprising: Add 0.5-2M PB buffer and Epstein-Barr virus capsid antigen to the magnetic beads, mix and place at 15-30 degrees for 2-3 hours; wherein the Epstein-Barr virus capsid antigen is VCA-P18 antigen and / or VCA-P23 antigen; Add blocking solution and mix at 15-37 degrees for 1-3 hours; The supernatant is removed and the beads are resuspended in a magnetic bead diluent; the magnetic bead diluent comprises about 0.1M Tris-HCl buffer, 0.2%-1.0% Casein by mass volume ratio, (10-30)ug / mL MAK33 and 0.3%-0.8% TW20 by mass volume ratio; preferably, the magnetic bead diluent comprises about 0.1M Tris-HCl buffer, 0.5% Casein by mass volume ratio, 20ug / mL MAK33 and 0.05% TW20 by mass volume ratio.

2. The method according to claim 1, further comprising: After adding blocking solution and reacting for 1-3 hours, remove the supernatant and add magnetic bead diluent to wash the reaction product once or multiple times.

3. The method according to claim 1, wherein the blocking solution comprises 0.1 M PBS buffer and 1% BSA in a mass volume ratio.

4. An Epstein-Barr virus capsid antigen-magnetic bead complex prepared according to any one of claims 1 to 3, wherein the Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex or a VCA-P23-magnetic bead complex.

5. A detection kit for EB virus capsid antigen IgG antibody, comprising: VCA-P18-magnetic bead complex and / or VCA-P23-magnetic bead complex; Acridinium ester-anti-human IgG conjugate; as well as A sample diluent, wherein the sample diluent comprises about 0.1M PBS buffer and (10-30)% newborn calf serum in a mass-volume ratio; preferably, the sample diluent comprises about 0.1M PBS buffer and 20% newborn calf serum in a mass-volume ratio.

6. The kit according to claim 5, wherein the VCA-P18-magnetic bead complex and / or the VCA-P23-magnetic bead complex is composed of Prepared by the method of any one of claims 1 to 3, or the VCA-P18-magnetic bead complex and / or VCA-P23-magnetic bead complex is selected from The Epstein-Barr virus capsid antigen-magnetic bead complex according to claim 4.

7. A method for detecting EB virus capsid antigen IgG antibodies in a sample, comprising: Get samples; The sample, the Epstein-Barr virus capsid antigen-magnetic bead complex and the sample diluent are mixed to obtain an antigen-antibody complex; wherein the Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex and / or a VCA-P23-magnetic bead complex; preferably, the Epstein-Barr virus capsid antigen-magnetic bead complex is a VCA-P18-magnetic bead complex and a VCA-P23-magnetic bead complex; adding an acridinium ester-labeled anti-human IgG antibody to the antigen-antibody complex for binding reaction; adding a stimulating solution to the binding reaction product; detecting the luminous intensity of the excitation light; and Obtain the test results of EB virus capsid antigen IgG antibody.

8. The detection method according to claim 7, wherein the addition ratio of the sample, Epstein-Barr virus capsid antigen-magnetic bead complex, acridinium ester-labeled anti-human IgG antibody and sample diluent is 1:(4-6):(8-12):(3-6), preferably 1:5:10:

4.

9. The detection method according to claim 7, wherein: The ratio of the added amount of the VCA-P18-magnetic bead complex to the VCA-P23-magnetic bead complex is 1:(0.5-3), preferably 1:(1-2), and more preferably 1:

1.

10. The detection method according to claim 7, wherein: The exciting solution includes: a mixed solution of hydrogen peroxide and nitric acid and a sodium hydroxide solution.

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