副猪嗜血杆菌Apd-ELISA抗体检测试剂盒的制备和初步应用

doi:10.11843/j.issn.0366-6964.2020.09.020

摘要/Abstract

摘要： 旨在对副猪嗜血杆菌（Haemophilus parasuis，HPS）的黏附素蛋白（autotransporter passenger domain，Apd）ELISA抗体检测方法进行参数优化、临界值确定和应用评价，获得性能稳定的副猪嗜血杆菌病抗体检测试剂盒。首先通过猪的免疫攻毒试验获得HPS阴、阳性对照血清，优化Apd-ELISA的反应参数；然后用背景确认的阴、阳性血清确定其临界值，并对封闭液及酶标板的包装方式进行选择；最后用Apd-ELISA试剂盒对临床样品进行检测并与荷兰Biocheck的OppA-ELISA试剂盒进行比较。结果表明，抗原包被质量浓度为0.5 μg·mL^-1、被检血清以1：200倍稀释后反应45 min以及二抗稀释度为1：15 000时，Apd-ELISA的反应背景下降，区分度提高。根据背景确认的27份阳性和40份阴性血清的OD_{630 nm}值以及临界值计算公式（S-N）/（P-N），得到Apd-ELISA的阴阳性临界值是0.33。证实用封闭液K封闭和真空包装的酶标板可以在50℃保存4 d（相当于在4℃保存22个月）。用Apd-ELISA试剂盒检测1 179份临床血清，表明母猪、后备和育肥猪以及仔猪的HPS血清阳性率分别为83.76%、61.09%和27.48%。与荷兰的Biocheck试剂盒进行比较，发现Apd-ELISA与Biocheck试剂盒（OppA-ELISA）对HPS临床阴性血清和疫苗免疫血清检测的符合率为100%，对HPS临床阳性血清的符合率仅为4.76%（6/126）。然而，Apd-ELISA与OppA Western blot的阳性符合率可达到73.02%（92/126）。本研究获得了能有效区分HPS阴、阳性血清和稳定保存的Apd-ELISA抗体检测试剂盒，可用于HPS灭活疫苗免疫后的抗体检测和副猪嗜血杆菌病的血清抗体检测。

关键词: 副猪嗜血杆菌, ELISA试剂盒, 抗体检测, 临界值, 封闭液

Abstract: The present study aimed to develop an ELISA for detecting antibodies of Haemophilus parasuis by optimization of reaction conditions, determination of its cutoff value and evaluation of its application with autotransporter passenger domain (Apd) as the coating antigen. Firstly, the porcine sera from immunization and challenge experiments were used as HPS-positive and -negative control sera to optimize the Apd-ELISA reaction parameters and conditions. Next, the cutoff value was determined based on testing of the serum samples with confirmed background, and then the blocking solutions and the packing methods of plates were selected. Finally, Apd-ELISA was used to detect clinical serum samples and then compared with the Biocheck OppA-ELISA kit from Netherland. With the coating antigen at 0.5 μg·mL^-1, the tested sera being diluted at 1:200 and incubation for 45 min and HRP-labeled goat anti-pig second antibody being diluted at 1:15 000, Apd-ELISA displayed the improved discriminative capability and the reduced background signal. The cutoff value was determined to be 0.33 by calculation formula (S-N)/(P-N) and the OD_{630 nm} value of the 27 positive porcine sera and 40 negative porcine sera with the confirmed background. The blocking solution K and the vaccum package can preserve ELISA plates for 4 days at 50 ℃ (comparable to 22 months at 4 ℃). Detection of 1 179 clinical sera with Apd-ELISA indicated that the positive rates were 83.76%, 61.09% and 27.48% in sows, fattening pigs and piglets, respectively. Compared with the OppA-ELISA kit from Biocheck (OppA-ELISA), Apd-ELISA showed 100% identity to the results of OppA-ELISA kit in clinically negative sera and experimentally positive sera from the pigs immunized with the inactivated vaccine, but 4.76%(6/126)identity to the results in the sera from clinically positive sera. However, Apd-ELISA displayed 73.02%(92/126)identity to the results of OppA-Western blot in the sera from clinically and experimentally infected pigs. The present study obtained the Apd-ELISA kit that can effectively distinguish HPS-positive and -negative sera and can be stably preserved, which would be applied for antibody monitoring among pigs immunized with HPS-inactivated vaccine and subjected to infection of H. parasuis.

Key words: Haemophilus parasuis, ELISA kit, antibody detection, cutoff value, blocking solutions

中图分类号:

S852.613

田杨, 刘云宝, 马辉, 潘其聪, 肖静, 陈焕春, 蔡旭旺, 徐晓娟. 副猪嗜血杆菌Apd-ELISA抗体检测试剂盒的制备和初步应用[J]. 畜牧兽医学报, 2020, 51(9): 2227-2237.

TIAN Yang, LIU Yunbao, MA Hui, PAN Qicong, XIAO Jing, CHEN Huanchun, CAI Xuwang, XU Xiaojuan. Development and Preliminary Application of Apd-ELISA Kit for Antibody Detection of Haemophilus parasuis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2227-2237.

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