| 副猪嗜血杆菌间接ELISA抗体检测试剂盒的研制与应用 |
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| 引用本文： | 张鹏云,陈敏,刘明星,周红,蔺辉星,范红结.副猪嗜血杆菌间接ELISA抗体检测试剂盒的研制与应用[J].中国农业科学,2023,56(8):1606-1614. |
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| 作者姓名： | 张鹏云 陈敏 刘明星 周红 蔺辉星 范红结 |
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| 作者单位： | 南京农业大学动物医学院，南京210095 |
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| 基金项目： | 江苏省农业科技自主创新资金项目(CX(19)2020); 南京农业大学大学生创新训练项目(202117XX07); 南京农业大学大学生创新训练项目(202117XX22); 江苏高校优势学科建设工程专项资金项目(PAPD) |
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| 摘 要： | 【背景】副猪嗜血杆菌（Haemophilus parasuis,HPS）是猪的上呼吸道病原菌，引起格拉瑟氏病，主要引起断奶前后和保育阶段的猪发病，通常见于5—8周龄的青年猪，发病率一般为10%—15%。该菌有15种血清型，目前主要养猪国家流行的血清型为4型、5型、12型和13型，是严重危害养猪业发展的主要细菌性病原之一。目前国内还没有检测该菌抗体的商品化试剂盒。【目的】研制一种针对副猪嗜血杆菌的快速、敏感和特异性强的抗体检测试剂盒，为格拉瑟氏病的有效防控提供技术支持。【方法】表达并纯化OppA、DppA、HbpA 3种不同的副猪嗜血杆菌ABC转运体周质底物结合蛋白，使用副猪嗜血杆菌阴、阳性参考血清筛选以上3种蛋白中免疫反应性和反应特异性好的蛋白。以筛选的蛋白为包被抗原，建立检测HPS抗体的间接ELISA方法，优化间接ELISA反应条件，并组装试剂盒。在此基础上，确定该试剂盒的敏感性、特异性；通过对不同时间、不同猪场采集的2 000份临床猪血清样本进行检测，评价该试剂盒的实用性；在以上临床血清样本中随机选取200份，分别以研制的试剂盒、间接血凝试验以及进口商品化试剂盒进行检测，比较检测结...
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| 关 键 词： | 副猪嗜血杆菌 ABC转运体周质底物结合蛋白 间接ELISA 免疫评价 |
| 收稿时间： | 2021-12-13 |
Development and Application of Indirect ELISA Kits for Antibody Detection of Haemophilus parasuis |
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| ZHANG PengYun,CHEN Min,LIU MingXing,ZHOU Hong,LIN HuiXing,FAN HongJie.Development and Application of Indirect ELISA Kits for Antibody Detection of Haemophilus parasuis[J].Scientia Agricultura Sinica,2023,56(8):1606-1614. |
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| Authors: | ZHANG PengYun CHEN Min LIU MingXing ZHOU Hong LIN HuiXing FAN HongJie |
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| Institution: | College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095 |
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| Abstract: | 【Background】Haemophilus parasuis (HPS) is the pathogen of upper respiratory tract of pigs, causing Glaser’s disease, mainly in pigs before and after weaning and in the nursery stage, which is usually seen in young pigs aged 5-8 weeks, and the incidence rate is generally 10%-15%. There are 15 serotypes of the bacteria and the serotypes currently prevalent in major pig-raising countries are 4, 5, 12 and 13, and it is one of the main bacterial pathogens affecting the development of pig industry. At present, there is no commercial kit for detecting antibody of the bacteria in China.【Objective】The development of a rapid, sensitive and specific antibody detection kit could provide the technical support for the effective prevention and control of the disease.【Method】Three different periplasmic substrate binding proteins of OppA, DppA and HbpA were expressed and purified. The positive and negative serum was used to screen one of the above three proteins with sound immunoreactivity and specificity. Using the screened protein as the coating antigen, an indirect ELISA method for detecting HPS antibody was established, the reaction conditions of indirect ELISA were optimized, and the kit was assembled. On this basis, the sensitivity and specificity of the kit were evaluated; the practicability of the kit was evaluated by testing 2 000 clinical pig serum samples collected at different times and from different pig farms; based on the above clinical serum samples, 200 samples were selected randomly and tested with this kit, indirect hemagglutination test, and imported commercial kit, respectively, and the test results were compared to verify the compliance rate of the kit; finally, the kit developed in this study was used to detect immune and challenge pigs. The collected serum was used to evaluate the growth and decline of antibodies against the bacteria after immunization.【Result】Three proteins of OppA, DppA and HbpA were successfully expressed and purified, and it was found that OppA had the best immunoreactivity and specificity. After optimizing the reaction conditions, the coating concentration of OppA was determined to be 1 μg·mL-1, the blocking solution was 0.5% BSA-PBS solution, the sample dilution was 1% BSA-PBST solution, the sample incubation time was 30 min, the sample dilution was 1:50, the enzyme labeled secondary antibody incubation time was 30min, the substrate action time was 15min, and the cut-off value was 0.18; the sensitivity and specificity of the kit were 96.67%, and the kit could detect the HPS positive sera against HPS with common serotypes prevalent in China and no cross-reaction with positive sera of other common pathogens in pigs; the positive rate of 2 000 clinical serum samples was 34.65%; 200 serum samples were randomly selected, the coincidence rate with indirect hemagglutination test was 92.50%, and the coincidence rate with imported commercial kit was 87.00%; using the kit to detect the swine serum collected at different times after immunization and challenge, the HPS antibody fluctuation rule was in line with expectations.【Conclusion】The ELISA antibody detection kit for Haemophilus parasuis developed in this study had high specificity and sensitivity, and hads a high coincidence rate with the commercial kit and indirect hemagglutination test, so it could be used for clinical HPS antibody detection and vaccine immunity evaluation. |
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| Keywords: | Haemophilus parasuis ABC transporter periplasmic substrate binding proteins indirect ELISA immunization evaluation |
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