Epstein-barr virus magnetic microparticle chemiluminescence detection reagent kit

Abstract

This invention relates to the field of virus detection, specifically to an Epstein-Barr virus (EBV) magnetic particle chemiluminescence immunoassay kit. Through antigen screening, this invention obtains an EBV magnetic particle chemiluminescence immunoassay kit using the natural antigen gp125 protein as the antigen, and optimizes the reagents therein, reducing detection background while increasing antibody stability. Experiments show that the EBV magnetic particle chemiluminescence immunoassay kit of this invention has good stability, high specificity, and high sensitivity. Furthermore, the kit has a simple structure, low time cost for EBV detection, and good stability. It can be combined with a fully automated chemiluminescence analyzer to achieve fully automated high-throughput detection, and has strong clinical application significance.

Description

Technical Field

[0001] This invention relates to the field of virus detection, specifically to an EB virus magnetic particle chemiluminescence detection kit. Background Technology

[0002] Epstein-Barr virus (EBV) belongs to the γ subfamily of the Herpesviridae family and primarily infects B lymphocytes. EBV infection causes abnormal cell proliferation and transformation. Studies have found that EBV is associated with susceptibility and is closely linked to various human malignancies, such as nasopharyngeal carcinoma, infectious mononucleosis (IM), and Burkitt lymphoma. In the later stages of EBV replication, a large amount of VCA structural protein is synthesized, which combines with viral DNA to form the nucleocapsid, and further assembles into a complete virion. The titer of EBV capsid antigen IgM antibody can be used to assess the clinical infection cycle in IM patients.

[0003] Existing methods for detecting Epstein-Barr virus (EBV) all have certain drawbacks. For example, virus isolation methods are time-consuming and require demanding culture conditions, making them unsuitable for routine clinical applications. Immunoenzyme immunoassays suffer from poor stability, are cumbersome to operate, and have high technical requirements, making them inconvenient for grassroots applications. Enzyme-linked immunosorbent assays (ELISA) have long reaction times and low automation. Colloidal gold chromatography is expensive and has low accuracy. In contrast, magnetic particle chemiluminescence immunoassay offers good stability, simple operation, rapid response, high specificity, and high sensitivity, enabling fully automated high-throughput sample detection. However, current methods for magnetic particle chemiluminescence immunoassay suffer from problems such as poor antibody stability and susceptibility to background interference. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an EB virus magnetic particle chemiluminescence detection kit.

[0005] This invention provides a reagent combination comprising an enzyme conjugate, a sample diluent, and a magnetic microparticle suspension;

[0006] The enzyme conjugate is anti-human IgM;

[0007] The sample diluent includes anti-human IgG;

[0008] The magnetic microparticles in the magnetic microparticle suspension are coated with EB virus capsid antigen; the molecular weight of the antigen is 125±10kD.

[0009] In this invention, the method for preparing the EB virus capsid antigen is as follows: infecting cells with an EB virus strain, lysing the infected cells, and separating and purifying them to obtain the EB virus capsid antigen.

[0010] Furthermore, in the reagent combination described in this invention,

[0011] The enzyme conjugate is horseradish peroxidase-labeled anti-human IgM;

[0012] The sample diluent is a PBS buffer and contains 1% to 3% (v / v) anti-human IgG.

[0013] The buffer solution for the magnetic microparticle suspension is Tris buffer containing 20%–50% fetal bovine serum by volume. The working concentration of EB virus capsid antigen in the magnetic microparticle suspension is 10 μg / mL–250 μg / mL. The diameter of the magnetic microparticles is 0.2 μm–4 μm, and the type of magnetic microparticles is carboxyl magnetic microparticles.

[0014] Furthermore, in the reagent combination described in this invention, the sample diluent further includes 1%–5% (v / v) anti-human IgA, 20%–50% (v / v) fetal bovine serum, 1 mg / mL–5 mg / mL protein stabilizer, and 0.5%–3% (v / v) preservative; the protein stabilizer is a polyhydroxy sugar, including but not limited to sucrose; the preservative includes P300.

[0015] In some specific embodiments of the present invention

[0016] The sample diluent consisted of 2% anti-human IgG, 2% anti-human IgA, 30% fetal bovine serum, 2 mg / mL sucrose, and 1% P300.

[0017] The buffer solution for the magnetic microparticle suspension is a Tris buffer containing 30% fetal bovine serum by volume, and the working concentration of EB virus capsid antigen in the magnetic microparticle suspension is 50 μg / mL.

[0018] In this invention, the anti-human IgG and anti-human IgA in the sample diluent have the same source as the anti-human IgM in the enzyme conjugate. When the anti-human IgM originates from sheep, the anti-human IgG and anti-human IgA also originate from sheep; when the anti-human IgM originates from rabbit, the anti-human IgG and anti-human IgA also originate from rabbit. The source of the anti-human IgM includes sheep, humans, mice, chickens, rabbits, and pigs. In some specific embodiments of this invention, the anti-human IgM is sheep anti-human IgM, the anti-human IgG is sheep anti-human IgG, and the anti-human IgA is sheep anti-human IgA.

[0019] In this invention, adding anti-human IgG from the same source as the antibody to the sample diluent reduces detection background, minimizes IgG interference with IgM, and increases reaction specificity. Furthermore, adding anti-human IgA to the sample diluent further enhances detection specificity and sensitivity.

[0020] In this invention, the magnetic microparticles in the magnetic microparticle suspension are coated with EB virus capsid antigen, which is a natural gp125 antigen. This antigen is purified by lysing cells infected with EB virus strain P3H3, and has a molecular weight of 125±10 kD and a purity ≥30%. Experimental results show that the EB virus magnetic microparticle chemiluminescence detection kit prepared using the natural gp125 protein described in this invention as the antigen exhibits the highest luminescence value and the largest positive-negative contrast, demonstrating good antigenicity and specificity compared to other antibodies.

[0021] In the reagent combination of the present invention, the method for preparing the magnetic microparticle suspension includes: coating EB virus capsid antigen onto magnetic microparticles activated in EDC solution and NHS solution, and then sequentially treating them with a stop solution and a blocking solution to obtain the magnetic microparticle suspension.

[0022] In the method for preparing magnetic microparticle suspension according to the present invention, the activation time of EDC solution and NHS solution is 0.5 to 2 hours; before activation, a step of washing with PBS buffer is included, and the number of washings is 3 to 8; after activation, a step of washing with MES buffer is included, and the number of washings is 2 to 5.

[0023] In the method for preparing magnetic microparticle suspension described in this invention,

[0024] The conditions for coating with natural gp125 antigen are: incubation with shaking at 18–28°C for 1–3 hours.

[0025] The stop solution was a PBS buffer containing 1%–5% PEG 8000 by volume; the stop solution treatment conditions were: shaking at 25°C for 2 hours.

[0026] The blocking solution is a Tris buffer containing a protein stabilizer at a concentration of 1 mg / mL to 5 mg / mL; the protein protectant is a polyhydroxy sugar; most polyhydroxy sugars include, but are not limited to, sucrose; the blocking solution treatment conditions are: blocking 3 times at 25°C, 20 min each time.

[0027] In some specific embodiments of the present invention,

[0028] The termination solution is a PBS buffer containing 1.5% PEG 8000 by volume; the blocking solution is a Tris buffer containing 2 mg / mL sucrose.

[0029] This invention provides a method for detecting EB virus, which involves using the reagent combination described in this invention to detect EB virus in a sample.

[0030] Furthermore, the detection method includes the following steps: mixing the sample, sample diluent, and magnetic microparticle suspension; incubating at a first temperature; adding an enzyme conjugate; incubating at a second temperature; washing; and adding a luminescent reagent to detect luminescence.

[0031] Furthermore, in the aforementioned detection method,

[0032] The volume ratio of sample, sample diluent, magnetic microparticle suspension, and enzyme conjugate was 10:1:2:10;

[0033] The first incubation was at 37°C for 15 minutes;

[0034] The second incubation was carried out at 37°C for 17 minutes;

[0035] The washing reagent is PBS buffer with a pH of 7.5–8.5;

[0036] The luminescent reagent includes solution A and solution B; solution A includes hydrogen peroxide; solution B includes luminol.

[0037] In some specific embodiments, the washing reagent described in this invention is a PBS buffer with a pH of 8.0.

[0038] The detection methods described in this invention include both diagnostic and non-diagnostic methods, which are not limited thereto. The non-diagnostic methods include detecting whether EB virus is present in environmental samples, food, drinking water, or in vitro samples of organisms; the diagnostic methods include detecting whether EB virus is present in organisms.

[0039] This invention provides an EB virus detection kit, which includes calibrator 1, calibrator 0, and the reagent combination described in this invention;

[0040] The calibrator 1 comprises inactivated EB virus capsid antigen IgM antibody-positive human serum and sodium aspartate.

[0041] The calibrator 0 is human serum that is negative for EB virus capsid antigen IgM antibody.

[0042] Furthermore, in the aforementioned EB virus detection kit,

[0043] The concentration of sodium aspartate in calibrator 1 is 1 mg / mL to 5 mg / mL; the S / CO value of positive human serum is 1.75 to 3.25.

[0044] In the calibrator 0, the S / CO value of negative human serum is <0.1.

[0045] The IgM antibody described in Calibrator 1 of this invention is a natural human IgM antibody, which is a tetramer and is easily affected by pH and buffer ion concentration, making it unstable. Therefore, aspartic acid hydrochloride is added to the buffer of Calibrator 1. Experimental results show that after adding aspartic acid hydrochloride to the buffer of Calibrator 1 for 12 months, the decrease in IgM antibody is less than 10%.

[0046] When used in conjunction with instruments such as the applicant's fully automated chemiluminescence analyzer AutoLumo A2000, the reagent kit of this invention can achieve random automated detection of samples, and has the advantages of simple operation, speed and automation.

[0047] This invention, through antigen screening, yielded an Epstein-Barr virus (EBV) magnetic particle chemiluminescence immunoassay kit using the natural antigen gp125 protein as the antigen. The reagents were optimized to reduce background noise and increase the stability of the detection results. Experiments show that the EBV magnetic particle chemiluminescence immunoassay kit of this invention exhibits good stability, high specificity, and high sensitivity. Furthermore, the kit has a simple structure, low time cost for EBV detection, and good stability. It can be combined with a fully automated chemiluminescence analyzer to achieve fully automated high-throughput detection, demonstrating significant clinical application value. Detailed Implementation

[0048] This invention provides an EB virus magnetic particle chemiluminescence 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.

[0049] The detection principle of this application is as follows: First, the natural antigen of the Epstein-Barr virus (EBV) capsid antigen is coated onto magnetic microparticles. During detection, the serum or plasma to be tested is incubated in a reaction vessel. If specific EBV capsid antigen antibodies are present, they will bind to the natural EBV capsid antigen immobilized on the magnetic microparticles. Unbound components are removed by washing. Then, enzyme-labeled anti-human IgM is added, and the mixture is incubated in the reaction vessel. The IgM component in the specific antigen-antibody complex immobilized on the magnetic microparticles binds to the enzyme-labeled antibody. Unbound components are removed by washing again. Substrate hydrogen peroxide and luminol are added. Horseradish peroxidase in the complex immobilized on the magnetic microparticles reacts chemically with the substrate and emits light. The operation can be fully automated using a fully automated chemiluminescence analyzer. The chemiluminescence value is measured to qualitatively determine the presence or absence of EBV capsid antigen IgM antibodies in the sample, thereby determining whether EBV infection has occurred.

[0050] This application also includes a control group. Blank control, negative control, and quality control serum are measured together with the blood sample to be tested to verify the validity of the test. If ① the calibrator 0S / CO value is <0.1, ② 1.75 < the calibrator 1S / CO value is <3.25, the test is valid, further improving the accuracy of the detection.

[0051] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0052] The present invention will be further illustrated below with reference to the embodiments:

[0053] Example 1: Antigen Screening, Preparation, and Reagent Optimization

[0054] 1. Antigen screening

[0055] Five antigens, including the natural gp125, were validated. Suspensions of the five coated antigens were prepared using the same coating process. These suspensions were then combined with enzyme-labeled antibodies diluted 1:3000 (commonly used in our laboratory). Six positive samples (P1–P6) and ten negative samples (N1–N10) were tested. The luminescence intensity and the contrast between positive and negative samples (the average luminescence value of positive samples compared to the average luminescence value of negative samples) were compared. The ratio of the average luminescence value of positive samples to the average luminescence value of negative samples should be greater than 10, and a higher contrast is better. The test results are as follows:

[0056] Table 1. Screening of coating antigens

[0057] Coating antigen P23 P18 gp125 P143 P150 P1 116,702 4,328,530 3,360,821 1,531,412 4,859,942 P2 9,918,536 28,635,155 25,692,034 7,454,062 1,089,217 P3 1,298,665 248,650 3,919,219 199,460 2,148,110 P4 323,360 523,568 2,079,388 189,427 112,995 P5 1,576,040 755,704 2,992,919 1,342,282 2,197,986 P6 621,699 907,277 10,622,815 1,362,911 3,270,188 N1 171,802 246,816 193,076 198,726 345,542 N2 466,610 589,427 300,699 335,771 825,198 N3 144,825 190,762 111,229 96,305 167,067 N4 26,982 237,900 31,042 85,160 153,060 N5 128,278 159,271 108,754 613,708 322,979 N6 32,441 27,356 17,319 120,942 138,298 N7 59,793 122,495 56,978 248,998 271,493 N8 46,097 76,909 58,596 130,764 67,673 N9 127,490 150,584 90,605 90,234 90,818 N10 124,214 126,723 104,587 60,689 77,412 P / N 17.4 30.6 75.6 10.2 9.3

[0058] As shown in the table above, the gp125-coated antigen positive samples had the highest average luminescence value and the greatest contrast between positive and negative results. Therefore, the natural antigen gp125 was selected.

[0059] 2. Preparation of natural antigen gp125

[0060] Cells cultured using the P3H3 virus strain as a standard strain were infected. The cultured cells were then disrupted using physical methods such as repeated freeze-thaw cycles and ultrasonic disruption. The viral lysate was then purified using ultrafiltration, followed by immunoaffinity chromatography to obtain the gp125 protein, ultimately yielding the natural EB virus capsid antigen. The naturally purified antigen (mainly protein) obtained from the organism retains the characteristics of the antigen's original structure (self-modification, correct conformation). The coating antigen of this application has the advantages of high sensitivity and good specificity. The molecular weight of the natural EB virus capsid antigen is 125 ± 10 kDa, and its purity is ≥30%.

[0061] The natural gp125 antigen was coated onto hydroxyl magnetic microparticles with a diameter of 0.2–4 μm for EB virus magnetic microparticle chemiluminescence detection. The working concentration of the natural antigen on the magnetic microparticles was 10–250 μg / mL.

[0062] 3. Optimization of sample dilution solution

[0063] Because the detection is susceptible to background interference, we considered reducing background interference by optimizing the reagents. We observed the effect of adding anti-human IgG antibody and anti-human IgA to the sample diluent on reducing background interference. Six samples (P1–P6) were selected from the sample diluent that were positive for EBV capsid antigen IgM, IgG, and IgA, and ten samples (N1–N10) were negative for EBV capsid antigen IgM but positive for IgG and rheumatoid factor (RF). These samples were divided into four parts for testing: one part served as a control (without anti-human IgG antibody), and the other three parts had a 2% (v / v) mixture of anti-human IgA and anti-human IgG antibodies, anti-human IgG, and anti-human IgA antibodies added to the sample diluent. Positive and negative samples were tested, and the contrast between positive and negative results was calculated. The results are as follows:

[0064] Table 2. Summary of the selection results of anti-human IgG antibodies and anti-human IgA antibodies (adsorbents)

[0065]

[0066] The results showed that after adding anti-human IgG and anti-human IgA antibodies, the luminescence values ​​of EB virus capsid antigen IgM positive, IgG positive, and IgA positive samples increased significantly, and the contrast between positive and negative values ​​increased. Therefore, anti-human IgG and anti-human IgA antibodies were subsequently added to the sample diluent to reduce background interference.

[0067] Example 2 Reagent Kit Preparation Scheme

[0068] I. Reagent Kit Preparation Method 1

[0069] An EB virus capsid antigen IgM antibody magnetic microparticle chemiluminescence detection kit includes an enzyme conjugate, a sample diluent, calibrator 0, calibrator 1, and magnetic microparticles. The magnetic microparticles are coated with natural EB virus capsid antigen. The enzyme conjugate is horseradish peroxidase-labeled anti-human IgM prepared at a working concentration of 1:1000 to 1:5000.

[0070] The method for preparing the EB virus capsid antigen natural antigen coated on the magnetic microparticles is as follows:

[0071] Magnetic microparticle activation: Wash carboxyl magnetic microparticles three times with PBS buffer and discard the supernatant. Activate the magnetic microparticles for 1 hour with EDC and NHS solution diluted with acetic acid. After activation, wash twice with MES buffer and discard the supernatant.

[0072] Coating: The natural EB virus capsid antigen was diluted with MES buffer and coated onto the surface of magnetic microparticles at a rate of 50 μL / mL. The mixture was shaken at 20°C and incubated for 1 hour for adsorption.

[0073] Termination: Discard the coating solution, use PEG 8000 termination solution at 200 μL / test and shake at 20°C for 1 hour to terminate (the volume fraction of PEG 8000 in the termination solution is 1.5%).

[0074] Blocking: Discard the stop solution, and use Tris blocking buffer containing protein protectant at 200 μL / test, shake at 20°C, and block twice for 10 min each time (the protein protectant is sucrose at a concentration of 2 mg / ml).

[0075] Volume adjustment and storage: Adjust the volume to 1 mL using Tris blocking buffer containing protein protectant, and seal for storage.

[0076] The enzyme conjugate is horseradish peroxidase-labeled anti-human IgM (the protein protectant is sucrose at a concentration of 2 mg / ml);

[0077] The sample diluent consists of: 2% (v / v) anti-human IgG, 2% (v / v) anti-human IgA, 30% (v / v) fetal bovine serum, 2 mg / mL sucrose, and 1% (v / v) P300. The anti-human IgG and anti-human IgA secondary antibodies in the sample diluent can effectively bind to IgG and IgA antibodies in the sample, reducing the impact of high concentrations of IgG and IgA antibodies on IgM detection and improving the specificity of the test.

[0078] The calibrator 0 is human serum with an S / CO value < 0.1 that is negative for EB virus capsid antigen IgM antibody;

[0079] The calibrator 1 is an inactivated human serum positive for EB virus capsid antigen IgM antibody with an S / CO value ranging from 1.75 to 3.25. Aspartic acid hydrochloride is added to the diluent of calibrator 1 to increase the ionic strength of the diluent, improve the dispersibility of the IgM antibody, and make the antibody more stable.

[0080] II. Reagent Kit Preparation Method 2:

[0081] An EB virus capsid antigen IgM antibody magnetic microparticle chemiluminescence detection kit includes an enzyme conjugate, a sample diluent, calibrator 0, calibrator 1, and magnetic microparticles. The magnetic microparticles are coated with natural EB virus capsid antigen. The enzyme conjugate is horseradish peroxidase-labeled anti-human IgM prepared at a working concentration of 1:1000 to 1:5000.

[0082] The method for preparing the EB virus capsid antigen natural antigen coated on the magnetic microparticles is as follows:

[0083] Magnetic microparticle activation: Wash carboxyl magnetic microparticles four times with PBS buffer and discard the supernatant. Activate the magnetic microparticles with acetic acid-diluted EDC and NHS solution for 1.5 hours. After activation, wash three times with MES buffer and discard the supernatant.

[0084] Coating: The natural EB virus capsid antigen was diluted with MES buffer and coated onto the surface of magnetic microparticles at a rate of 100 μL / mL. The mixture was shaken at 25°C and incubated for 2 hours for adsorption.

[0085] Termination: Discard the coating solution, use PEG 8000 termination solution at 250 μL / test, shake at 25°C, and terminate for 2 hours;

[0086] Blocking: Discard the stop solution, and use Tris blocking buffer containing protein protectant at 250 μL / test, shaking at 25°C for 3 times, 20 min each time;

[0087] Volume adjustment and storage: Adjust the volume to 2 mL using Tris blocking buffer containing protein protectant, and seal for storage;

[0088] The enzyme conjugate is horseradish peroxidase-labeled anti-human IgM;

[0089] The sample diluent is a PBS buffer containing fetal bovine serum, protein stabilizers, and preservatives. The sample diluent also contains anti-human IgG secondary antibody, which can effectively bind to IgG antibodies in the sample, reduce the impact of high concentrations of IgG antibodies on IgM detection, and improve the specificity of the test.

[0090] The calibrator 0 is human serum with an S / CO value < 0.1 that is negative for EB virus capsid antigen IgM antibody;

[0091] The calibrator 1 is an inactivated human serum positive for EB virus capsid antigen IgM antibody with an S / CO value ranging from 1.75 to 3.25. Aspartic acid hydrochloride is added to the diluent of calibrator 1 to increase the ionic strength of the diluent, improve the dispersibility of the IgM antibody, and make the antibody more stable.

[0092] III. Reagent Kit Preparation Method 3:

[0093] An EB virus capsid antigen IgM antibody magnetic microparticle chemiluminescence detection kit includes an enzyme conjugate, a sample diluent, calibrator 0, calibrator 1, and magnetic microparticles. The magnetic microparticles are coated with natural EB virus capsid antigen. The enzyme conjugate is horseradish peroxidase-labeled anti-human IgM prepared at a working concentration of 1:1000 to 1:5000.

[0094] The method for preparing the EB virus capsid antigen natural antigen coated on the magnetic microparticles is as follows:

[0095] Magnetic microparticle activation: Wash carboxyl magnetic microparticles 5 times with PBS buffer and discard the supernatant. Activate the magnetic microparticles with acetic acid-diluted EDC and NHS solution for 2 hours. After activation, wash 3 times with MES buffer and discard the supernatant.

[0096] Coating: The natural EB virus capsid antigen was diluted with MES buffer and coated onto the surface of magnetic microparticles at a rate of 200 μL / mL. The mixture was shaken at 25°C and incubated for 3 hours for adsorption.

[0097] Termination: Discard the coating solution, use PEG 8000 termination solution at 300 μL / test, shake at 25°C, and terminate for 3 hours;

[0098] Blocking: Discard the stop solution, and use Tris blocking buffer containing protein protectant at 300 μL / test, shake at 25°C, block 3 times, 30 min each time;

[0099] Volume adjustment and storage: Adjust the volume to 3 mL using Tris blocking buffer containing protein protectant, and seal for storage;

[0100] The enzyme conjugate is horseradish peroxidase-labeled anti-human IgM;

[0101] The sample diluent is a PBS buffer containing fetal bovine serum, protein stabilizers, and preservatives. The sample diluent also contains anti-human IgG secondary antibody, which can effectively bind to IgG antibodies in the sample, reduce the impact of high concentrations of IgG antibodies on IgM detection, and improve the specificity of the test.

[0102] The calibrator 0 is human serum with an S / CO value < 0.1 that is negative for EB virus capsid antigen IgM antibody;

[0103] The calibrator 1 is an inactivated human serum positive for EB virus capsid antigen IgM antibody with an S / CO value ranging from 1.75 to 3.25. Aspartic acid hydrochloride is added to the diluent of calibrator 1 to increase the ionic strength of the diluent, improve the dispersibility of the IgM antibody, and make the antibody more stable.

[0104] IV. Screening of Coating Processes for Three Reagent Kits

[0105] The gp125 antigen was prepared into suspensions using three different coating processes at the same concentration. These suspensions were then combined with enzyme-labeled antibodies diluted 1:3000 (commonly used in our laboratory). Six positive samples (P1–P6) and ten negative samples (N1–N10) were tested. The luminescence intensity and the contrast between positive and negative samples (the average luminescence value of positive samples compared to the average luminescence value of negative samples) were compared. The ratio of the average luminescence value of positive samples to the average luminescence value of negative samples should be greater than 10, and a higher contrast is better. The test results are as follows:

[0106] Table 3. Screening of coating processes

[0107] Wrapping process Example 1 Example 2 Example 3 P1 3,350,106 2,985,590 3,082,463 P2 19,755,608 20,905,465 17,076,102 P3 3,895,995 2,745,950 2,757,657 P4 2,970,080 2,170,704 2,238,164 P5 4,728,120 3,267,112 2,978,757 P6 1,865,097 2,721,831 3,868,445 N1 15,406 40,448 79,228 N2 399,830 368,281 282,097 N3 34,475 72,286 33,687 N4 30,946 13,700 93,126 N5 84,834 77,813 26,262 N6 37,323 82,068 51,957 N7 79,379 67,485 70,934 N8 38,291 30,727 75,788 N9 82,470 51,752 71,815 N10 72,642 80,169 13,761 P / N 69.60 65.55 66.78

[0108] As shown in the table above, the average luminescence values ​​of positive samples were highest for all three coating processes, with relatively small differences between positive and negative samples (69.60, 65.55, and 66.78, respectively). Therefore, all three coating processes can be used in this project.

[0109] Example 3: Reagent Kit Performance Evaluation

[0110] The detection method steps are as follows:

[0111] S1. Sample Loading: Take the magnetic microparticle suspension coated with EB virus natural capsid antigen, enzyme conjugate, and sample diluent, and place them into the reagent tray of the fully automated chemiluminescence analyzer via the reagent rack. Send the calibrator or test sample to the aspiration position and load the reaction vessel to the sample loading position.

[0112] First, execute the calibration procedure for the fully automated chemiluminescence analyzer, dispensing 100 μL of sample diluent, 10 μL of calibrator, and 20 μL of magnetic microparticle suspension. Then, when executing the sample detection procedure, dispensing 100 μL of sample diluent, 10 μL of serum or plasma sample, and 20 μL of magnetic microparticle suspension. Mix the reaction solution thoroughly and incubate at 37°C for 15 minutes.

[0113] S2. Add enzyme conjugate: Wash with system washing solution, add 100 μL of enzyme conjugate, mix well, and incubate at 37°C for 17 minutes.

[0114] S3, luminescence: Wash with system washing solution, add 50 μL each of substrate A solution (containing hydrogen peroxide) and substrate B solution (containing luminol), and mix well;

[0115] S4. Result Analysis and Judgment: After 1-5 minutes, the instrument automatically calculates the S / CO value. The validity of the test is verified by measuring the results of the calibrator. If the following conditions are met: ① Calibrator 0 S / CO value < 0.1, ② 1.75 < Calibrator 1 S / CO value < 3.25, the test is valid; when the luminescence value / critical value (S / CO) of the test sample is < 1.00, it is judged as negative; when S / CO ≥ 1.00, it is judged as positive.

[0116] I. Interference assessment for different sample types in the kit:

[0117] Clinical laboratory tests typically use serum or plasma as sample types. Anticoagulants used for plasma samples include sodium citrate, EDTA-2Na, or sodium heparin. Using ordinary serum, procoagulant serum, separated gel serum, EDTA-2Na plasma, sodium citrate plasma, and sodium heparin plasma as research materials, 10 negative samples and 10 positive samples were selected for each sample type. The EB virus capsid antigen IgM antibody magnetic particle chemiluminescence detection kit described above was used for detection to evaluate the interference of different sample types on the kit's detection. The results are shown in Table 4.

[0118] Table 4. Detection of different types of samples by the kit

[0119]

[0120]

[0121] The test results are determined by the cut-off value (CO value). The cut-off value is calculated as the average luminescence value of the quality control serum × 0.4. If the luminescence value of the test sample / the cut-off value (S / CO) < 1.00, it is considered negative. If S / CO ≥ 1.00, it is considered positive.

[0122] As shown in Table 4, the test results of the kit showed little difference when negative and positive samples were tested in the form of ordinary serum, procoagulant serum, separated gel serum, or plasma using EDTA-2Na, sodium citrate, or sodium heparin as anticoagulants. Therefore, the kit has strong anti-interference ability and is suitable for a variety of sample types, including serum and plasma, where the anticoagulant for plasma can be selected from EDTA-2Na, sodium citrate, or sodium heparin.

[0123] II. Evaluation of the stability of the reagent kit:

[0124] Positive samples with three different concentrations of Epstein-Barr virus (EBV) capsid antigen IgM antibody were used as research materials. The EBV capsid antigen IgM antibody magnetic particle chemiluminescence detection kit described above was used for detection. For 12 consecutive months, each sample was measured four times per month to evaluate the detection stability of the kit. The results are shown in Table 5.

[0125] Table 5. S / CO values ​​of the kit for samples with three different antibody concentrations.

[0126]

[0127]

[0128] As shown in Table 5, the coefficient of variation for low-positive samples was 8.86% (the coefficient of variation is calculated using the formula: standard deviation). ÷ average value ×The coefficient of variation (COP) for medium-positive samples was 8.37%, and for high-positive samples it was 4.86%, both less than the industry requirement of 10%, demonstrating the excellent detection stability of the kit. Therefore, the kit exhibits good repeatability and can be used to test low, medium, and high-positive samples.

[0129] III. Evaluation of the specificity of the reagent kit

[0130] Twelve positive samples containing different concentrations of rheumatoid factor (RF) antibody and twelve positive samples containing different concentrations of antinuclear antibody (ANA) were used as research materials. The EB virus capsid antigen IgM antibody magnetic particle chemiluminescence detection kit described above was used for detection to evaluate the kit's detection specificity. The results are shown in Table 6.

[0131] Table 6. S / CO values ​​of the kit for detecting positive samples containing RF antibodies or ANA.

[0132]

[0133]

[0134] As shown in Table 6, when the content of rheumatoid factor RF antibody positive samples reached 120.5 IU / mL and the content of antinuclear antibody ANA positive samples reached 103.748 IU / mL, the S / CO values ​​detected by the kit were all much less than 1.0, proving that rheumatoid factor RF antibody and antinuclear antibody ANA do not interfere with the kit, and the kit has good detection specificity.

[0135] IV. Assessment of interference from endogenous substances:

[0136] Five positive samples and five negative samples were supplemented with 1.0 g / L hemoglobin, 0.4 g / L bilirubin, and 30 g / L triglycerides, respectively. The EB virus capsid antigen IgM antibody magnetic microparticle chemiluminescence detection kit described above was used for detection to evaluate the interference of endogenous substances hemoglobin, bilirubin, and triglycerides on the kit's detection. The results are shown in Tables 7-9:

[0137] Table 7. Detection luminescence values ​​of the kit for samples containing bilirubin.

[0138]

[0139] Table 8. Detection luminescence values ​​of the kit for samples containing hemoglobin

[0140]

[0141]

[0142] Table 9. Detection luminescence values ​​of the kit for samples containing triglycerides

[0143]

[0144] The interference percentage of each sample's luminescence value is calculated by using the average luminescence values ​​of the positive sample and the control. The formula is: Interference percentage = (Average luminescence value of sample - Average luminescence value of control) / Average luminescence value of control × 100%. If the interference percentage of the positive sample is within ±15% and there is no significant change in the background of the negative sample, it will not affect the result determination, and the interference of the kit on endogenous substances in the sample is considered acceptable.

[0145] As shown in Tables 7-9, the interference percentage of positive samples was <15% in the presence of endogenous substances hemoglobin, bilirubin, and triglycerides, demonstrating that the kit can effectively resist interference from endogenous substances. Since the physiological and pathological concentrations of endogenous interfering substances in clinical samples were lower than the test concentrations, 0.4 g / L bilirubin, 30 g / L triglycerides, and 1 g / L hemoglobin can be considered as the tolerance limits for endogenous interference of the kit.

[0146] 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 magnetic particle chemiluminescence detection kit, characterized in that, Includes enzyme conjugate, sample diluent, magnetic microparticle suspension, calibrator 1 and calibrator 0; The enzyme conjugate is a horseradish peroxidase-labeled anti-human IgM antibody; The sample diluent is a PBS buffer containing 2% (v / v) anti-human IgG antibody, 2% (v / v) anti-human IgA antibody, 30% (v / v) fetal serum, 2 mg / mL sucrose, and 1% (v / v) P300. The magnetic microparticles in the magnetic microparticle suspension are coated with EB virus capsid antigen. The method for preparing the EB virus capsid antigen in the magnetic microparticle suspension is as follows: EB virus strain P3H3 is used to infect cells, the infected cells are lysed and isolated and purified to obtain the EB virus capsid antigen; the EB virus capsid antigen is the natural gp125 antigen. The buffer solution for the magnetic microparticle suspension is Tris buffer containing 30% fetal bovine serum by volume, and the working concentration of EB virus capsid antigen in the magnetic microparticle suspension is 50 μg / mL; the diameter of the magnetic microparticles is 0.2 μm to 4 μm, and the type of magnetic microparticles is carboxyl magnetic microparticles. The calibrator 1 comprises 1 mg / mL to 5 mg / mL sodium aspartate and positive human serum containing inactivated EB virus capsid antigen IgM antibody; The calibrator 0 is a negative human serum containing EB virus capsid antigen IgM antibody; The S / CO value of the positive human serum was 1.75~3.25; In the calibrator 0, the S / CO value of negative human serum is <0.

1.

2. The EB virus magnetic particle chemiluminescence detection kit according to claim 1, characterized in that, The magnetic microparticle suspension is prepared by coating EB virus capsid antigen onto magnetic microparticles activated in EDC solution and NHS solution, and then sequentially treating them with a stop solution and a blocking solution to obtain the magnetic microparticle suspension. The activation time for the EDC solution and NHS solution is 0.5 to 2 hours; The activation process also includes a washing step using PBS buffer, with the number of washing cycles being 3 to 8. The activation process also includes a washing step with MES buffer, with the number of washing cycles being 2 to 5.

3. The EB virus magnetic particle chemiluminescence detection kit according to claim 2, characterized in that, In the preparation method of the magnetic microparticle suspension, The conditions for coating with EB virus capsid antigen are: incubation with shaking at 18~28℃ for 1~3 hours; The stop solution was a PBS buffer containing 1%–5% PEG 8000 by volume; the stop solution treatment conditions were: shaking at 25°C for 2 hours. The blocking solution is a Tris buffer containing a protein protectant; the protein protectant is a polyhydroxy sugar; the blocking solution treatment conditions are: blocking 3 times at 25°C, 20 min each time.

Images