WO2023109535A1 - 抗马立克氏病毒单克隆抗体、其杂交瘤细胞株及检测试剂盒应用 - Google Patents

抗马立克氏病毒单克隆抗体、其杂交瘤细胞株及检测试剂盒应用 Download PDF

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WO2023109535A1
WO2023109535A1 PCT/CN2022/136408 CN2022136408W WO2023109535A1 WO 2023109535 A1 WO2023109535 A1 WO 2023109535A1 CN 2022136408 W CN2022136408 W CN 2022136408W WO 2023109535 A1 WO2023109535 A1 WO 2023109535A1
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marek
virus
antibody
monoclonal antibody
serum
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French (fr)
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李泽君
石晓娜
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中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus

Definitions

  • the invention relates to the technical field of veterinary diagnostic biological products, in particular to an anti-Marek's virus monoclonal antibody, a hybridoma cell line thereof, and a Marek's virus antibody blocking ELISA detection kit containing the monoclonal antibody.
  • Marek's disease is a malignant lymphoproliferative neoplastic disease of chickens caused by Marek's disease virus (MDV). It can cause tumors in the internal organs and skin of infected chickens, and at the same time invade the central nervous system of infected chickens, resulting in paralysis or death of infected chickens. After inoculation of HVT (Marek's disease turkey herpes virus vaccine) or CVI-988 vaccine (chicken Marek's disease live vaccine), such cases of MDV-induced tumors in chicken flocks are not uncommon, which has caused huge damage to the domestic poultry industry. Economic losses. There is an urgent need to develop kits for specific diagnosis of Marek's disease and effective monitoring of MDV immune antibodies.
  • HVT Marek's disease turkey herpes virus vaccine
  • CVI-988 vaccine chicken Marek's disease live vaccine
  • Marek's disease virus belongs to the family Herpesviridae, subfamily Alpha-Herpesviridae, and belongs to the genus Marekvirus.
  • MDV virions are typical of herpes viruses. There are two forms of virus particles: one is a complete virus particle with an envelope, with a diameter of about 273-400nm, which mainly exists in chicken feather bursa epithelial cells, and its resistance is relatively strong; the other is a virus particle without a capsule.
  • Membrane virions which are approximately 85-100 nm in diameter, are strictly cell-associated viruses and are less resistant.
  • the main source of MDV infection is diseased chickens and infected chickens.
  • Chicks are most susceptible to MDV, and there is a longer incubation period from virus infection to onset.
  • the clinical symptoms of chickens infected with MDV mainly include weight loss, intermittent limb paralysis, and mental depression.
  • the virulence of MDV has been continuously enhanced, and some sick chickens have tumors at the age of 3-4 weeks, and the tumors are already very serious at the age of 8-9 weeks.
  • Marek's virus Although a preliminary diagnosis can be made based on the clinical symptoms and necropsy lesions of the disease, laboratory tests should still be combined to confirm the disease.
  • immunological methods commonly used to identify Marek's virus include immunofluorescence (FA), enzyme-linked immunosorbent assay (ELISA), and virus neutralization assay (VN). MDV against chicken feather sacs can also be identified using the agar diffusion test (AGP).
  • AGP agar diffusion test
  • the method of molecular diagnosis of Marek's virus is the diagnosis of specific pathogenic genes, including polymerase chain reaction (PCR), fluorescent quantitative PCR and so on.
  • PCR polymerase chain reaction
  • PCR fluorescent quantitative PCR
  • the present invention will solve the technical problem of the lack of high-sensitivity Marek's virus antibody detection products on the market at present, and provide an anti-Marek's virus monoclonal antibody, which can be used to detect Marek's virus rapidly and sensitively, and is effective for Marek's virus It provides good methods and kits for the clinical diagnosis of HBV and the evaluation of its vaccine immunity.
  • an anti-Marek's virus monoclonal antibody is provided.
  • the monoclonal antibody specifically binds to the gB protein of Marek's virus.
  • the monoclonal antibody is produced by a hybridoma cell line with a preservation number of CCTCC NO: C2021104.
  • the antibody subclass of the monoclonal antibody is IgG2a, and its light chain is ⁇ chain.
  • a hybridoma cell line secreting and producing the above-mentioned monoclonal antibody is also provided.
  • the preservation number of the hybridoma cell line is CCTCC NO: C2021104.
  • a Marek's virus antibody detection kit comprising the above-mentioned monoclonal antibody.
  • the kit of the present invention also includes ELISA plate coated with Marek's virus antigen, positive control serum, negative control serum, sample diluent, 10 times concentrated washing solution, Marek's virus monoclonal antibody, enzyme-labeled secondary antibody , chromogenic solution, stop solution, sealing film and kit instructions.
  • the enzyme-labeled secondary antibody was goat anti-mouse enzyme-labeled secondary antibody.
  • Said kit adopts blocking ELISA method to detect Marek's virus antibody, comprising the following steps:
  • the monoclonal antibody, enzyme-labeled secondary antibody, chromogenic solution and stop solution according to claim 1 or 2 are sequentially added;
  • blocking rate (%) (negative control well average OD450nm value-sample well average OD450nm value)/negative control well average OD450nm value ⁇ 100%, Calculate the blocking rate of the serum sample to be tested to obtain the detection result.
  • the judgment criteria of the test results are: when the blocking rate is ⁇ 26.4%, it is positive for Marek’s virus antibody; when the blocking rate is ⁇ 19.2%, it is negative for Marek’s virus antibody; Repeated testing is required, and if the blocking rate is still less than 26.4% in repeated testing results, it is judged to be negative for Marek's virus antibody.
  • the application of the above-mentioned monoclonal antibody in the preparation of products for diagnosing or treating Marek's disease is also provided.
  • the application of the above Marek's virus antibody detection kit in the preparation of products for diagnosing Marek's disease is also provided.
  • the invention utilizes the anti-Marek's virus monoclonal antibody to prepare the blocking ELISA kit for detecting Marek's virus antibody, which has strong specificity and high sensitivity, and is useful in the epidemiological investigation of Marek's virus, the evaluation of vaccine immune efficacy and Marek's disease It has a good application prospect in diagnosis.
  • Fig. 1 is the indirect immunofluorescence diagram of the reaction between the monoclonal antibody 1B7 of Example 1 of the present invention and CVI-988;
  • Fig. 2 is the indirect immunofluorescence diagram of the reaction between the monoclonal antibody 1B7 of Example 1 of the present invention and the MDV-gB protein;
  • Fig. 3 is a graph showing the results of antibody subclass identification of the Marek's virus monoclonal antibody 1B7 in Example 1 of the present invention.
  • the hybridoma cell 1B7 strain secreting anti-chicken Marek's disease virus gB protein monoclonal antibody of the present invention has been preserved in the China Center for Type Culture Collection (CCTCC for short, address: China. Wuhan. Wuhan University) on November 30, 2021.
  • the deposit number is CCTCC NO: C2021104.
  • the present invention firstly uses the PCAGGS-MDV-gB plasmid to penetrate into the leg muscles of Balb/C mice through a gene transfer instrument, and utilizes monoclonal antibody preparation technology to screen A hybridoma cell line 1B7 that stably secretes anti-Marek's virus monoclonal antibody was obtained.
  • the secreted monoclonal antibody can specifically bind to CVI-988 and the gB protein of Marek's virus.
  • the subtype of the monoclonal antibody is IgG2a subclass, appeal type. Then use the purified Marek's virus antigen to coat the ELISA plate.
  • a blocking ELISA Blockinge ELISA, B-ELISA
  • the B-ELISA method for detecting Marek's virus antibody of the present invention shows good application prospects in the diagnosis of Marek's disease and its antibody detection because of its higher sensitivity and specificity.
  • CVI-988 vaccine virus mouse myeloma cell line (SP2/0), PCAGGS-MDV-gB eukaryotic expression plasmids were constructed by our laboratory; Marek's virus negative and positive sera were prepared and preserved by our laboratory.
  • 6-week-old and 8-12-week-old female BALB/c mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.
  • Freund's complete adjuvant, Freund's incomplete adjuvant and HRP goat anti-mouse IgG were purchased from Sigma Company; Quick Antibody was purchased from Biodragon Company; PEG1450, HAT, HT were purchased from Sigma Company; DMEM medium, fetal bovine serum GIBCO Company; TMB Chromogenic solution was purchased from Wuhan Boster Company.
  • the optimal concentration of coating antigen and positive in the indirect ELISA method was determined by square array titration.
  • the purified MDV antigen, mouse positive serum and negative serum were diluted twice, and the antigen and serum concentration with OD 450nm value around 1.0 and the largest P/N value were selected as the optimal working concentration.
  • mice peritoneal macrophages as feeder cells according to conventional methods.
  • splenocytes and myeloma cells SP2/0 were fused at a ratio of 10:1.
  • the fused cells were fused under the action of the agent PEG1450, and the fused cells were seeded in a 96-well plate and cultured in a hypoxanthine-aminopterin-thymidine (HAT) selective medium.
  • HAT hypoxanthine-aminopterin-thymidine
  • the hybridoma cells positive for antibody secretion were screened by indirect ELISA, and cloned three times in 96-well cell culture plates according to the limited dilution method.
  • mice Take female BALB/c mice aged 8-12 weeks, inject 0.5ml sterilized liquid paraffin into each mouse, and inject 2-5 ⁇ 104 hybridoma cells into the abdominal cavity after 7-10 days, and the mice can be seen 7-10 days after injection.
  • the abdomen was obviously enlarged, ascites was collected, centrifuged at 12,000 rpm for 10 minutes, the supernatant was collected, and the antibody titer of the ascites was detected by the established ELISA method, and stored at -80°C for future use.
  • SPF chicken fibroblast CEF was prepared and cultured in a 6-well cell culture plate. After the cells grew into a single layer, they were infected with the CVI-988 strain, and a negative control well was set at the same time. After 120 hours of infection, discard the supernatant, fix the cells with 4% paraformaldehyde, wash three times with PBST, add hybridoma cell culture supernatant, incubate at 37°C for 1 hour, wash three times with PBST, add FITC-goat anti-mouse IgG antibody, continue for 37 hours Incubate at °C for 1 h, wash with PBST three times, and finally observe under a microscope under fluorescence. Those with green fluorescence are judged as positive; those without fluorescence are judged as negative.
  • 293T was cultured in a 96-well culture plate. After the cells grew into a single layer, the recombinant eukaryotic expression plasmid pCAGGS-MDV-gB was transfected into the 293T cells, and a negative control well was set at the same time.
  • the supernatant of hybridoma cells was detected by indirect ELISA method, and the fusion pore cells were screened by indirect ELISA method.
  • a strain capable of stably secreting anti-chicken Marek's disease virus gB was obtained
  • the hybridoma cell line of protein monoclonal antibody, the cell line is named 1B7, and this hybridoma cell line has been preserved in the China Center for Type Culture Collection (CCTCC for short, address: China.Wuhan. Wuhan University), the preservation number is CCTCC C2021104. And the viability of the culture has been tested by the collection center (CCTCC) on December 6, 2021, and the result is survival.
  • CTCC China Center for Type Culture Collection
  • the culture supernatant and ascites of the positive hybridoma cells were respectively diluted 10 times, and detected by the established indirect ELISA method, and mouse positive and negative serum controls were established at the same time.
  • the P/N value is ⁇ 2.1
  • the maximum dilution of the antibody is its antibody titer.
  • Marek's virus blocking ELISA antibody detection method 1. Marek's virus blocking ELISA antibody detection method
  • Dilute the qualified purified antigen with coating solution Dilute according to 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 in turn, and add to 96-well ELISA microplate respectively, each dilution has 8 wells, 100 ⁇ L per well . Place at 4°C for 15-18 hours; discard the coating solution, add PBST washing solution, leave for 3 minutes, discard the washing solution, wash 3 times, add blocking solution, 100 ⁇ L per well, place at 20-25°C for 1 hour, discard blocking solution.
  • the positive serum and negative serum were diluted according to 1:200, 1:400, 1:800 and 1:1600, respectively added to the microplate wells coated with different antigen concentrations, 100 ⁇ L per well.
  • After adding serum incubate at 37°C for 1 h, discard the serum diluent, wash with PBST 3 times; each time for 3 min; add 1:2000 diluted goat anti-mouse IgG enzyme-labeled secondary antibody (0.8 mg/ml), 100 ⁇ L per well, 37 Place at °C for 1 hour, wash 3 times as above, discard the washing solution, add TMB substrate solution, 100 ⁇ L per well, place in the dark at room temperature for 10 minutes, add 50 ⁇ L of stop solution per well.
  • the OD450nm value of each well was measured on a microplate reader.
  • the antigen concentration when the ratio of negative serum OD450nm value and positive serum OD450nm value is the largest is determined as the optimal antigen coating concentration.
  • the dilution and action time of the serum are determined in accordance with the above 2
  • the monoclonal antibody is in accordance with the conditions determined in the above 4
  • Carry out the enzyme-labeled antibody according to the conditions determined in the above 5 add the substrate TMB, and develop the color at room temperature in the dark, the color development time is 5, 10, 15 and 20 minutes, and determine the best color development according to the operating procedure time.
  • Critical value average inhibition rate of negative samples + 2 times or 3 times standard deviation.
  • the purified antigen that passed the CVI-988 purified virus test was diluted with the coating solution, and then 2-fold ratio dilution was performed. Dilute the antigen at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 in sequence, a total of 6 dilutions of antigen, positive serum and negative serum at 1:5, 1:10 respectively , 1:20 and 1:40 dilutions for ELISA test, the OD 450nm values of coated virus, positive serum and negative serum at each dilution are shown in Table 2. From the results in Table 2, it can be seen that the optimal antigen dilution is 1:800, and the working concentration of the serum to be tested is 1:10.
  • P stands for positive serum
  • N stands for negative serum
  • P/N stands for the ratio of positive serum to negative serum
  • Coat the enzyme-labeled plate with the optimal concentration of antigen use the determined blocking solution and conditions, the dilution of serum, the dilution of monoclonal antibody and the enzyme-labeled antibody to perform blocking ELISA test, add the substrate TMB, and develop color at room temperature in the dark.
  • the color development time was 5, 10, 15 and 20 minutes
  • the blocking rate was the highest when the color development time was 10 minutes at room temperature, so it was determined that the best color development time was 10 minutes at room temperature.
  • avian Newcastle disease virus H5, H7, H9 subtype avian influenza virus (AIV), avian reovirus (ARV), avian adenovirus type 4 (FADV-4)
  • ILTV chicken infectious laryngotracheitis virus
  • ILTV chicken infectious laryngotracheitis virus
  • Chicken Marek's virus-positive serum was diluted 1:2 to 1:1024 times, and detected by the established blocking ELISA method and indirect immunofluorescence (IFA) test in parallel.
  • the results show that the IFA titer of the serum to be tested is 1:64, while the antibody titer of the blocking ELISA is 1:512 (see Table 7), the blocking ELISA antibody titer is 8 times more sensitive than the neutralization test, and has a good sensitivity.
  • the kit of the invention is used for detecting Marek's virus antibody in chicken serum.
  • the kit contains the components listed in Table 11 below.
  • the 10-fold concentrated wash solution is diluted 10-fold with deionized or distilled water before use.
  • Sample preparation Dilute the serum to be tested by 1:10 with the sample diluent for determination; if the antibody titer of the serum to be tested is to be measured, first dilute the serum with the sample diluent by 1:5, and then perform a 2-fold ratio Dilutions were performed for each dilution.
  • the blocking rate (PI) is calculated with the following formula:
  • PI (1-average OD 450nm value of sample wells/average OD 450nm value of negative control wells) ⁇ 100%
  • the blocking rate (PI) of the sample is between 19.2% and 26.4%, it is judged as suspicious; the test should be repeated once more, and if the PI value is still ⁇ 26.4%, it is judged as chicken Marek's virus antibody negative.
  • the highest dilution factor of the positive sample determined by ELISA is the ELISA antibody titer of the serum sample.
  • Kit components of different batch numbers should not be mixed, and cross-contamination should be prevented when different reagents are used.
  • TMB substrate solution and stop solution are irritating to the skin, and should not be exposed to strong light or oxidizing agents.

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Abstract

本发明公开了一种抗马立克氏病毒单克隆抗体。本发明还公开了马立克氏病毒抗体检测试剂盒，以及上述单克隆抗体在制备诊断或治疗马立克氏病的产品中的应用。本发明利用抗马立克氏病毒的单克隆抗体制备的检测马立克氏病毒抗体的阻断ELISA试剂盒，特异性强、敏感性高，在马立克氏病毒的流行病学调查、疫苗免疫效力评估和马立克氏病的诊断方面具有很好的应用前景。

Description

抗马立克氏病毒单克隆抗体、其杂交瘤细胞株及检测试剂盒应用 技术领域
本发明涉及兽用诊断生物制品技术领域，尤其涉及一种抗马立克氏病毒单克隆抗体、其杂交瘤细胞株、以及包含该单克隆抗体的马立克氏病毒抗体阻断ELISA检测试剂盒。
背景技术
马立克氏病(Marek’disease，MD)是由马立克氏病毒(Marek’s disease virus，MDV)引起的鸡的一种恶性淋巴组织增生性肿瘤病。能够引起感染鸡的内脏和皮肤出现肿瘤，同时侵袭感染鸡的中枢神经系统，导致感染鸡出现瘫痪或死亡的现象。在接种HVT(马立克氏病火鸡疱疹病毒疫苗)或者CVI-988疫苗(鸡马立克氏病活疫苗)后，MDV在鸡群中诱发肿瘤的这种病例屡见不鲜，给国内养禽业造成了巨大的经济损失。急需开发特异性诊断马立克氏病和有效监测MDV免疫抗体的试剂盒。
马立克氏病毒(Marek’s disease virus，MDV)是属于疱疹病毒科，α-疱疹病毒亚科，马立克病毒属。MDV病毒粒子呈典型的疱疹病毒形态。病毒粒子有两种存在形式：一种是完整的带有囊膜的病毒粒子，直径约为273-400nm，主要存在于鸡羽毛囊上皮细胞中，其抵抗力较强；另一种是没有囊膜的病毒粒子，其直径约为85-100nm，是严格的细胞结合性病毒，其抵抗力较弱。MDV的主要传染源是病鸡和带毒鸡，感染鸡的羽毛囊上皮细胞内如果存在病毒，即可通过水平传播的方式进行传播。雏鸡最容易感染MDV，从病毒感染到发病有较长的潜伏期。感染MDV病鸡的临床症状主要包括体重下降、间歇性肢体麻痹、精神沉郁等。近些年来，MDV毒力的不断增强，部分病鸡3-4周龄就出现肿瘤，8-9周龄时肿瘤已经很严重。
虽然根据该病的临床症状与剖检病变可以做出初步诊断，但仍应结合实验室检测以确诊该病。目前鉴定马立克氏病毒通常采用的免疫学方法有免疫荧光技术(FA)、酶联免疫吸附试验(ELISA)、病毒中和试验(VN)。针对鸡羽毛囊的MDV还可以采用琼脂扩散试验(AGP)进行鉴定。分子诊断马立克氏病毒的方法是针对特异性致病基因的诊断，包括聚合酶链式反应(PCR)、荧光定量PCR等。但是，这些鉴定诊断方法的敏感性都不高，需要研制更高敏感性的马立克氏病毒抗体检测试剂盒。
发明内容
本发明要解决目前市场上缺乏高敏感性的马立克氏病毒抗体检测产品的技术问题，提供一种抗马立克氏病毒单克隆抗体，该单克隆抗体可用于快速、灵敏地检测马立克氏病毒，对马立克氏病毒的临床诊断及其疫苗免疫效力评估提供很好的方法和试剂盒。
为了解决上述技术问题，本发明通过如下技术方案实现:
在本发明的一个方面，提供了一种抗马立克氏病毒单克隆抗体。
优选的，所述单克隆抗体与马立克氏病毒的gB蛋白特异性结合。
更优选的，所述单克隆抗体由保藏号为CCTCC NO：C2021104的杂交瘤细胞株产生。
所述单克隆抗体的抗体亚类为IgG2a，其轻链为к链。
在本发明的另一方面，还提供了一种分泌产生上述单克隆抗体的杂交瘤细胞株。
优选的，该杂交瘤细胞株的保藏号为CCTCC NO：C2021104。
在本发明的另一方面，还提供了一种马立克氏病毒抗体检测试剂盒，包含上述的单克隆抗体。
优选的，本发明试剂盒还包含马立克氏病毒抗原包被的ELISA酶标板、阳性对照血清、阴性对照血清、样品稀释液、10倍浓缩洗涤液、马立克氏病毒单克隆抗体、酶标二抗、显色液、终止液、封板膜和试剂盒说明书。其中酶标二抗为羊抗鼠酶标二抗。
所述试剂盒采用阻断ELISA方法检测马立克氏病毒抗体，包括以下步骤：
用纯化的马立克氏病毒抗原包被ELISA酶标板；
将待测血清样品与包被抗原作用后，依次加入权利要求1或2所述的单克隆抗体、酶标二抗、显色液和终止液；
利用酶标仪读取吸光度值OD 450nm,并按公式：阻断率(％)＝(阴性对照孔平均OD 450nm值-样品孔平均OD 450nm值)/阴性对照孔平均OD 450nm值×100％，计算待测血清样品的阻断率，得检测结果。
所述检测结果的判定标准为：阻断率≥26.4％时为马立克氏病毒抗体阳性；阻断率≤19.2％时为马立克氏病毒抗体阴性；19.2％＜阻断率＜26.4％时为可疑，需重复检测，若重复检测结果阻断率仍小于26.4％，则判定为马立克氏病毒抗体阴性。
在本发明的另一方面，还提供了上述单克隆抗体在制备诊断或治疗马立克氏病的产品中的应用。
在本发明的另一方面，还提供了上述马立克氏病毒抗体检测试剂盒在制备诊断马立克氏病的产品中的应用。
本发明利用抗马立克氏病毒单克隆抗体制备的检测马立克氏病毒抗体的阻断ELISA试剂盒，特异性强、敏感性高，在马立克氏病毒的流行病学调查、疫苗免疫效力评估和马立克氏病的诊断方面具有很好的应用前景。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是本发明实施例1的单克隆抗体1B7与CVI-988反应的间接免疫荧光图；
图2是本发明实施例1的单克隆抗体1B7与MDV-gB蛋白反应的间接免疫荧光图；
图3是本发明实施例1的马立克氏病毒单克隆抗体1B7的抗体亚类鉴定结果图。
本发明分泌抗鸡马立克病病毒gB蛋白单克隆抗体的杂交瘤细胞1B7株，已于2021年11月30日保藏于中国典型培养物保藏中心(简称CCTCC，地址：中国.武汉.武汉大学)，保藏号为CCTCC NO：C2021104。
具体实施方式
为了开发更高灵敏度的特异性针对马立克氏病毒的诊断试剂盒，本发明首先采用PCAGGS-MDV-gB质粒经基因导入仪打入Balb/C小鼠腿部肌肉，利用单克隆抗体制备技术，筛选获得了1株稳定分泌抗马立克氏病毒单克隆抗体的杂交瘤细胞株1B7，其分泌的单克隆抗体可以与CVI-988、及马立克氏病毒的gB蛋白特异性结合，该单克隆抗体亚类为IgG2a亚类、к型。然后用纯化的马立克氏病毒抗原包被ELISA板，将待测血清与包被抗原作用后，依次加入1B7单抗、酶标抗鼠二抗、显色液和终止液，利用酶标仪读取吸光度值OD 450nm，通过计算待测血清样品的阻断率，建立了检测马立克氏病毒抗体的阻断ELISA(Blockinge ELISA,B-ELISA)方法。本发明检测马立克氏病毒抗体的B-ELISA方法，以其更高的敏感性和特异性在马立克氏病的诊断及其抗体检测方面表现出很好的应用前景。
实施例1马立克氏病毒单克隆抗体的制备和鉴定
1.材料和方法
1.1病毒、细胞株、质粒及血清
CVI-988疫苗毒、小鼠骨髓瘤细胞株(SP2/0)、PCAGGS-MDV-gB真核表达质粒由本实验室自己构建；马立克氏病毒阴性和阳性血清由本实验室制备、保存。
1.2实验动物
6周龄和8～12周龄雌性BALB/c小鼠，购自上海杰思捷实验动物有限责任公司。
1.3主要试剂
弗氏完全佐剂、弗氏不完全佐剂和HRP羊抗鼠IgG购自Sigma公司；QuickAntibody购自Biodragon公司；PEG1450、HAT、HT购自Sigma公司；DMEM培养基、胎牛血清GIBCO公司；TMB显色液购自武汉博士德公司。
1.4病毒抗原的制备
将CVI-988疫苗毒株从液氮中取出，放入37℃水浴锅中融化后接种SPF鸡胚成纤维细胞，连续观察5-6d，待CPE达80％以上时，收取感染细胞并用少量PBS重悬，并冻融5-6次后， 8000rpm离心10min取上清，将上清分装后于-70℃保存，用作包被抗原。
1.5 MDV单克隆抗体的制备
1.5.1小鼠免疫
用PCAGGS-MDV-gB质粒经基因导入仪打入小鼠腿部肌肉，选用6～8周龄雌性Balb/C小鼠，每只200μg；每隔两周分别进行第二次和第三次免疫，剂量与首免相同，当小鼠的抗体效价达到1:10 4以上的时候，取抗体效价最高的小鼠脾脏用于细胞融合。三免两周后进行加强免疫，三天后开始细胞融合。
1.5.2间接ELISA方法的建立
采用方阵滴定法确定间接ELISA方法中包被抗原和阳性的最佳浓度。将纯化的MDV抗原、鼠阳性血清和阴性血清分别作2倍倍比稀释，选择OD 450nm值在1.0左右，且P/N值最大的抗原和血清浓度作为其最佳工作浓度。
1.5.3阳性杂交瘤细胞株的建立和筛选
按常规方法制备小鼠腹腔巨噬细胞作为饲养层细胞，在加强免疫后第三天，按常规淋巴细胞杂交瘤技术，脾细胞与骨髓瘤细胞(SP2/0)按10:1的比例在融合剂PEG1450作用下融合，将融合的细胞接种在96孔板中，在次黄嘌呤-氨基蝶呤-胸苷(HAT)选择性培养基下培养。经间接ELISA筛选抗体分泌阳性的杂交瘤细胞，按有限稀释法在96孔细胞培养板中进行3次克隆。
1.5.4单克隆抗体腹水的制备和效价测定
取8～12周龄雌性BALB/c小鼠，每只腹腔注射0.5ml灭菌液体石蜡，7～10天后腹腔注入2～5×10 4个杂交瘤细胞，注射后7～10天可见小鼠腹部明显膨大，采取腹水，12000rpm离心10min，收集上清液，并用建立的ELISA方法检测腹水的抗体效价，-80℃保存备用。
1.5.5单克隆抗体的间接免疫荧光鉴定(IFA)
制备SPF鸡成纤维细胞CEF并于6孔细胞培养板中培养，待细胞长成单层后，感染CVI-988毒株，同时设置阴性对照孔。感染120h后，弃上清，细胞用4％多聚甲醛固定，经PBST洗涤三次，加入杂交瘤细胞培养上清，37℃孵育1h，PBST洗涤三次，加入FITC-羊抗鼠IgG抗体，继续37℃孵育1h，PBST洗涤三次，最后在荧光下显微镜下观察，凡有绿色荧光者判为阳性；无荧光者判为阴性。
1.5.6抗MDV-gB蛋白单克隆抗体的鉴定
将293T培养于96孔培养板，待细胞长成单层后，将重组真核表达质粒pCAGGS-MDV-gB转染293T细胞，同时设阴性对照孔。转染24h后，弃上清，细胞用4％的多聚甲醛固定，经PBST洗涤一次，加入杂交瘤细胞培养上清，37℃孵育1h，PBST洗涤三次，加入FITC-羊抗 鼠IgG抗体，继续37℃孵育1h，PBST洗涤三次，最后在荧光显微镜下观察，凡有特异的绿色荧光者判为阳性；无荧光者判为阴性。
2.结果
2.1细胞融合和杂交瘤细胞的建立
利用淋巴细胞杂交瘤技术，通过间接ELISA检测方法检测杂交瘤细胞上清，通过间接ELISA方法对融合孔细胞进行筛选，经过鉴定和亚克隆纯化，共获得1株能够稳定分泌抗鸡马立克病病毒gB蛋白单克隆抗体的杂交瘤细胞株，将该细胞株命名为1B7，且此杂交瘤细胞株已于2021年11月30日保藏于中国典型培养物保藏中心(简称CCTCC，地址：中国.武汉.武汉大学)，保藏号为CCTCC C2021104。并且该培养物的存活性已由该保藏中心(CCTCC)于2021年12月6日检测完毕，结果为存活。
2.2杂交瘤细胞上清和腹水效价的测定
将阳性杂交瘤细胞培养上清和腹水分别进行10倍倍比稀释，用建立的间接ELISA方法检测，同时设立鼠阳性和阴性血清对照。P/N值≥2.1时抗体的最大稀释度即为其抗体效价。
表1杂交瘤细胞培养上清和腹水抗体效价
Figure PCTCN2022136408-appb-000001
2.3单克隆抗体的间接免疫荧光鉴定(IFA)
用CVI-988感染CEF细胞，并用1B7单抗进行间接免疫荧光试验，设未感染CEF细胞为空白对照(MOCK)。试验结果显示：1B7单抗可与CVI-988结合产生特异性的绿色荧光(见图1)，并且其与病毒的gB蛋白发生特异性结合(见图2)。
2.4单克隆抗体的抗体亚类鉴定按照SBA ClonotypingTM System/HRP(Cat.NO5300-05)抗体亚类试剂盒说明书进行，对筛选的单克隆抗体1B7株进行抗体亚型鉴定，结果显示杂交瘤细胞1B7分泌的单克隆抗体的抗体亚类为IgG2a，且其轻链为к(kappa)链，结果见图3。
实施例2马立克氏病毒阻断ELISA抗体检测方法的建立
1.马立克氏病毒阻断ELISA抗体检测操作方法
⑴用PH9.6的碳酸盐缓冲液包被纯化的CIV-988抗原，抗原按照1：800倍稀释，4℃过夜。次日取出后，用0.05％Tween-20PBS(PBST)洗涤三次，每次3min；⑵用5％脱脂乳的PBS封闭酶标板，37℃2h，洗涤3次，方法同上；⑶加入抗体稀释液稀释10倍的待测血清、阴性和阳性对照，每孔100μL 37℃孵育1h，洗涤方法同上。⑷每孔100μL 1B7单克隆抗体， 37℃孵育1h，洗涤3次；⑸每孔加入2000倍稀释的HRP羊抗鼠IgG，100μL每孔，37℃孵育1h，洗涤3次；⑹每孔加入TMB底物显色液100μL，室温避光显色10min；⑺每孔加入2M H 2SO 4 50uL终止反应，酶标仪读取吸光度值OD450nm,计算阻断率，阻断率(％)＝(阴性对照的吸光度值-待测血清的吸光度值)/阴性对照的吸光度值×100％。
2.最适抗原包被浓度和血清稀释度的优化
将检验合格的纯化抗原用包被液稀释，然后进行2倍倍比稀释。依次按1：200、1：400、1：800、1：1600、1：3200、1：6400、进行稀释，分别加入到96孔ELISA酶标板中，每个稀释度8孔，每孔100μL。4℃放置15～18小时；弃去包被液，加入PBST洗涤液，放置3分钟后弃去洗涤液，洗涤3次后，加入封闭液，每孔100μL，20～25℃放置1h，弃去封闭液。将阳性血清和阴性血清都按照1：200、1：400、1：800和1：1600稀释，分别加入到不同抗原浓度包被的酶标板孔中，每孔100μL。血清加入后在37℃孵育1h，弃去血清稀释液，PBST洗涤3次；每次3min；加入1:2000稀释的羊抗鼠IgG酶标二抗(0.8mg/ml)，每孔100μL，37℃下放置1h，同上洗涤3次后，弃去洗涤液，加入TMB底物溶液，每孔100μL，室温避光放置10min，每孔加入50μL终止液。在酶标仪上测定各孔的OD450nm值。当阴性血清的OD450nm值约为1.5、阴性血清OD450nm值和阳性血清OD450nm比值最大时的抗原浓度确定为最佳抗原包被浓度。
3.最佳封闭液与封闭时间的确定
以上述2确定的最适浓度的抗原包被酶标板(100μl/孔)，置于4℃，作用15～18小时，洗涤3次，分别加入3％脱脂乳、5％脱脂乳、0.1％明胶、1％牛血清封闭和商品化封闭液，封闭1小时，按照上述操作程序，确定最适封闭液。
4.单抗最佳稀释度和工作时间的确定
按照上述2确定的最适浓度的抗原包被酶标板，用上述3确定的封闭液和条件进行封闭。取阳性血清和阴性血清按2确定的稀释度和时间进行作用，单抗分别做1:500、1:1000、1:2000和1:4000稀释，每孔加入0.1ml，37℃孵育1小时，按照上述1操作程序，确定单抗最佳稀释度。
5.酶标抗体最佳工作浓度和工作时间的确定
按照上述2确定的最适浓度的抗原包被酶标板，用上述3确定的封闭液和条件进行封闭，血清的稀释度和作用时间按照上述2确定的进行，单抗按照上述4确定的条件进行，羊抗鼠酶标抗体分别作1:1000、1:2000、1:4000、1:8000倍稀释，37℃1小时，按照上述2操作程序，确定酶标抗体最佳工作浓度和工作时间。
6.底物显色时间的确定
按照上述2确定的最适浓度的抗原包被酶标板，用上述3确定的封闭液和条件进行封闭，血清的稀释度和作用时间按照上述2确定的进行，单抗按照上述4确定的条件进行，酶标抗体按照上述5确定的条件进行，加入底物TMB，室温避光显色，显色时间分别为5、10、15和20分钟，按照1.6.1操作程序，确定最佳显色时间。
7.阻断ELISA方法的临界值判定
在阻断ELISA方法建立后，检测150份来自于临床的阴性血清，计算阻断率和临界值，确定方法的结果有效性成立条件和判断标准。临界值＝阴性样品平均抑制率+2倍或3倍标准偏差。
8.结果：阻断ELISA反应条件的确定
8.1抗原最佳包被浓度和血清最佳稀释度的确定
将CVI-988纯化病毒检验合格的纯化抗原用包被液稀释，然后进行2倍倍比稀释。依次按1：200、1：400、1：800、1：1600、1：3200、1：6400进行稀释,共6个稀释度的抗原，阳性血清与阴性血清分别以1：5、1：10、1：20和1：40稀释进行ELISA试验，各个稀释度包被病毒与阳性血清和阴性血清的OD 450nm值见表2。从表2的结果可以看出最佳抗原稀释度为1：800，待测血清的工作浓度为1：10。
表2抗原最佳包被浓度和血清最佳稀释度的选择
Figure PCTCN2022136408-appb-000002
Figure PCTCN2022136408-appb-000003
注：“P”代表阳性血清；“N”代表阴性血清；“P/N”代表阳性血清与阴性血清比值。
8.2待检血清作用时间的确定
使用最佳条件包被和封闭板ELISA板后，加入最佳稀释浓度的阴阳性血清，37℃分别作用0.5h、1h、1.5h和2h，对阴阳性血清进行阻断ELISA试验，各个时间阴阳性血清阻断结果见表3。从表的结果可以看出，血清作用时间为1h时，阳性对照血清的阻断率最高，所以选择1h为最佳血清作用时间。
表3血清最佳作用时间选择
Figure PCTCN2022136408-appb-000004
8.3单克隆抗体工作浓度的确定
使用最佳条件包被和封闭板ELISA板后，加入最佳稀释浓度的阴阳性血清，37℃分别作用1h，将单克隆抗体1B7进行1:100、1:1000和1:2000倍稀释后进行阻断ELISA试验，试验结果见表4。从结果可以看出，单抗1B7进行1:2000稀释时，阻断率最高，所以选择1:2000作为单抗最佳稀释浓度。
表4单抗(腹水)最佳稀释浓度的选择
Figure PCTCN2022136408-appb-000005
8.4底物显色时间的确定
用最适浓度的抗原包被酶标板，用确定的封闭液和条件、血清的稀释度、单抗稀释度和酶标抗体进行阻断ELISA试验，加入底物TMB，室温避光显色，显色时间分别为5、10、15和20分钟时，室温显色10分钟时阻断率最高，因而确定最佳显色时间为室温10分钟。
表5 TMB底物溶液最佳显色时间的确定
Figure PCTCN2022136408-appb-000006
Figure PCTCN2022136408-appb-000007
8.5阻断ELISA判定标准的确定
对150份鸡阴性血清检测结果进行统计学分析，其平均阻断率为4.8％±7.2％(平均值±SD)。阴性和阳性阻断率的临界值为19.2％(平均值+2SD)和26.4％(平均值+3SD)，确定了本发明方法的判定标准为当阻断率PI≥26.4％时判为鸡马立克氏病毒抗体阳性，PI≤19.2％时判定鸡马立克氏病毒抗体阴性，19.2％<PI<26.4％时判为可疑，需重复检测1次，如果仍低于26.4％，则判为鸡马立克氏病毒抗体阴性。
实施例3马立克氏病毒阻断ELISA抗体检测方法的评估
1.特异性试验
用建立的阻断ELISA检测方法对禽新城疫病毒(NDV)、H5、H7、H9亚型禽流感病毒(AIV)、禽呼肠孤病毒(ARV)、禽腺病毒4型(FADV-4)、鸡传染性喉气管炎病毒(ILTV)阳性血清和2份鸡阴性血清1:10稀释进行检测，结果如表6所示，根据表6试验结果，该方法与上述病毒(NDV、AIV、ARV、FADV-4和ILTV)的阳性血清没有交叉反应性，只特异性的检测CVI-988抗体，具有很好的特异性。
表6阻断ELISA特异性试验结果
Figure PCTCN2022136408-appb-000008
注：“+”表示抗体阳性，“—”表示抗体阴性。
2.敏感性试验
将鸡马立克氏病毒阳性血清进行1:2～1:1024倍比稀释，用建立的阻断ELISA方法和间接免疫荧光(IFA)试验平行进行检测。结果显示：待检血清IFA效价为1:64，而阻断ELISA的抗体效价为1:512(见表7)，阻断ELISA抗体效价比中和试验敏感8倍，具有很好的敏感性。
表7阻断ELISA敏感性试验结果
Figure PCTCN2022136408-appb-000009
Figure PCTCN2022136408-appb-000010
注：“+”表示抗体阳性，“—”表示抗体阴性。
3.批内批间重复性试验
用同一批制备的马立克氏病毒抗原包被不同的ELISA板，取3份不同抗体水平的血清和3份阴性血清，在同一条件中按阻断ELISA方法测定，每份血样做5孔平行试验，对结果进行统计学分析。用3个不同批次制备并纯化的病毒包被不同的ELISA板，取6份上述血清，在同一条件按阻断ELISA方法测定，对结果进行统计学分析。
结果：用同批次抗原对3份阳性血清和3份阴性血清进行检测，统计学分析检测结果显示其批内变异系数在0.964％～6.786％之间(见表8)，小于10.0％，说明同一样品在同一批试验中的变异程度很小，具有良好的重复性。用3个不同批次制备并纯化的病毒包被的ELISA板进行批间重复性试验，其批间变异系数在1.067％～8.065％之间(见表9)，小于10.0％，说明同一样品在不同批次抗原试验中的变异程度很小，具有很好的重复性。综上，本发明方法具有良好的批内批间重复性。
表8批内重复性试验
Figure PCTCN2022136408-appb-000011
表9批间重复性试验
Figure PCTCN2022136408-appb-000012
实施例4马立克氏病毒ELISA抗体检测试剂盒的组成和应用
1.试剂盒的组成
本发明试剂盒用于检测鸡血清中马立克氏病毒抗体。试剂盒中含有如下表11所列成分。
表11试剂盒成分
Figure PCTCN2022136408-appb-000013
2.用法与判定
2.1试剂准备 使用前，将试剂盒中的抗原包被板、样品稀释液、10倍浓缩洗涤液、阳性对照血清、阴性对照血清、单克隆抗体、羊抗鼠酶标抗体、TMB底物溶液和终止液等恢复至室温(20～25℃)并混匀待用。10倍浓缩洗涤液在使用前用去离子水或蒸馏水稀释10倍。
2.2样品准备 将待测血清用样品稀释液按1:10稀释后进行测定；若测定待测血清的抗体滴度，则先用样品稀释液按1:5稀释血清后，再进行2倍倍比稀释，对各稀释度进行测定。
2.3操作步骤
2.3.1加样与孵育 取抗原包被板，分别将稀释好的待检血清、阴性和阳性对照血清加入到抗原包被板孔中，阴性和阳性对照血清各2孔，0.1ml/孔。轻轻振匀孔中样品，用封板膜密封包被板，置37℃孵育1小时。
2.3.2洗涤 去除封板膜，弃去孔中液体，每孔加入0.3ml洗涤液，作用3分钟，弃去洗涤液，重复洗涤3次。
2.3.3与单克隆抗体作用 每孔加入单克隆抗体0.1ml，用封板膜密封包被板，置37℃孵育1小时。
2.3.4洗涤 按2.3.2中方法洗涤。
2.3.5与酶标抗体作用 每孔加入羊抗鼠酶标抗体0.1ml，用封板膜密封包被板，置37℃孵育1小时。
2.3.6洗涤 按2.3.2中方法洗涤。
2.3.7显色 每孔中加入TMB底物溶液0.1ml，轻摇2秒，室温(20～25℃)下避光显色10 分钟。
2.3.8终止 每孔加入终止液0.05ml，置酶标仪上测定各孔OD 450nm值。
3.判定
3.1结果有效性 当阴性对照孔OD 450nm平均值≥0.8，且阳性对照孔的阻断率(PI值)≥40％，判定检测结果有效。
3.2阻断率的计算阻断率(PI)用以下公式计算：
PI＝(1-样品孔平均OD 450nm值/阴性对照孔平均OD 450nm值)×100％
3.3结果判定
当样品的阻断率(PI)≥26.4％，判为鸡马立克氏病毒抗体阳性；
当样品的阻断率(PI)≤19.2％，判为鸡马立克氏病毒抗体阴性；
当样品的阻断率(PI)在19.2％～26.4％，判为可疑；应再重复检测1次，若PI值仍＜26.4％，则判为鸡马立克氏病毒抗体阴性。
ELISA判定为阳性样品的最高稀释倍数，即为该血清样品的ELISA抗体滴度。
4.注意事项
(1)所有的试剂应在2～8℃贮存，使用前恢复至室温，使用后放回2～8℃。
(2)被检血清发生严重溶血或腐败时勿用于检测。
(3)不同批号的试剂盒组份不得混用，不同试剂使用时应防止交叉污染。
(4)待检血清较多时，应先稀释完所有待测血清，再加入到抗原包被板上，使反应时间一致。
(5)TMB底物溶液和终止液对皮肤有刺激性，且不能暴露于强光或接触氧化剂。
(6)稀释10倍浓缩洗涤液时若发现有结晶，应在室温或者放置28～37℃水浴条件下10～15分钟，待结晶溶解后使用。
(7)所有的废弃液应在丢弃前合理处理以免污染环境。
(8)操作过程中的移液体积、时间和洗涤次必须精准。
5.规格:96孔/板×1板/盒。
6.贮藏与有效期:2～8℃保存，有效期为12个月。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (11)

  1. 一种抗马立克氏病毒单克隆抗体。
  2. 根据权利要求1所述的单克隆抗体，其特征在于，所述单克隆抗体与马立克氏病毒的gB蛋白特异性结合。
  3. 根据权利要求2所述的单克隆抗体，其特征在于，所述单克隆抗体由保藏号为CCTCC NO：C2021104的杂交瘤细胞株产生。
  4. 一种分泌产生权利要求1所述抗马立克氏病毒单克隆抗体的杂交瘤细胞株。
  5. 根据权利要求4所述的杂交瘤细胞株，其特征在于，该杂交瘤细胞株的保藏号为CCTCC NO：C2021104。
  6. 一种马立克氏病毒抗体检测试剂盒，其特征在于，包含权利要求1所述的单克隆抗体。
  7. 根据权利要求6所述的马立克氏病毒抗体检测试剂盒，其特征在于，还包含马立克氏病毒抗原包被的ELISA酶标板、阳性对照血清、阴性对照血清、样品稀释液、10倍浓缩洗涤液、马立克氏病毒单克隆抗体、酶标二抗、显色液、终止液、封板膜和试剂盒说明书。
  8. 根据权利要求7所述的马立克氏病毒抗体检测试剂盒，其特征在于，所述试剂盒采用阻断ELISA方法检测马立克氏病毒抗体，包括以下步骤：
    用纯化的马立克氏病毒抗原包被ELISA酶标板；
    将待测血清样品与包被抗原作用后，依次加入权利要求1所述的单克隆抗体、酶标二抗、显色液和终止液；
    利用酶标仪读取吸光度值OD 450nm,并按公式：阻断率(％)＝(阴性对照孔平均OD 450nm值-样品孔平均OD 450nm值)/阴性对照孔平均OD 450nm值×100％，计算待测血清样品的阻断率，得检测结果。
  9. 根据权利要求8所述的马立克氏病毒抗体检测试剂盒，其特征在于，所述检测结果的判定标准为：阻断率≥26.4％时为马立克氏病毒抗体阳性；阻断率≤19.2％时为马立克氏病毒抗体阴性；19.2％＜阻断率＜26.4％时为可疑，需重复检测，若重复检测结果阻断率仍小于26.4％，则判定为马立克氏病毒抗体阴性。
  10. 权利要求1-3任一项所述单克隆抗体在制备诊断或治疗马立克氏病的产品中的应用。
  11. 权利要求6-9任一项所述马立克氏病毒抗体检测试剂盒在制备诊断马立克氏病的产品中的应用。
PCT/CN2022/136408 2021-12-17 2022-12-03 抗马立克氏病毒单克隆抗体、其杂交瘤细胞株及检测试剂盒应用 WO2023109535A1 (zh)

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