副猪嗜血杆菌病原夹心ELISA检测方法的建立及应用

doi:10.7668/hbnxb.2016.S1.079

华北农学报 ›› 2016, Vol. 31 ›› Issue (S1): 463-469. doi: 10.7668/hbnxb.2016.S1.079

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副猪嗜血杆菌病原夹心ELISA检测方法的建立及应用

宋帅¹, 李春玲¹, 臧莹安², 郭海祥², 李淼¹, 杨冬霞¹, 徐志宏¹

1. 广东省农业科学院动物卫生研究所, 广东省畜禽疫病防治研究重点实验室, 广东省兽医公共卫生实验室, 粤北生猪生产及疫病防控协同创新发展中心, 广东广州 510640;
2. 仲恺农业工程学院动物科学系, 广东广州 510225

收稿日期:2016-07-31 出版日期:2016-12-28
通讯作者: 宋帅(1982-),男,河南汝州人,助理研究员,硕士,主要从事兽医微生物学研究。
作者简介:徐志宏(1965-),男,湖北荆门人,研究员,硕士,硕士生导师,主要从事兽医微生物学研究。
基金资助:
广东省公益研究与能力建设专项资金（2014B070706011；2015B070701015）；广东省农业领域科技计划项目（2013B020307002）；广州市新型畜禽疫病诊断试剂研发及产业化（201508020055）；广州市重大民生专项（201300000066）；国家“十二五”科技支撑计划项目（2015BAD12B02-4）

Application and Development of Double Antibody Sandwich ELISA for Detection Haemophilus parasuis

SONG Shuai¹, LI Chunling¹, ZANG Yingan², GUO Haixiang², LI Miao¹, YANG Dongxia¹, XU Zhihong¹

1. Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong Key Laboratory of Animal Disease Prevention, Guangdong Open Laboratory of Veterinary Public Health, North Guangdong Innovation and Development Center for Swine Farming and Disease Control, Guangzhou 510640, China;
2. Department of Animal Science, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China

Received:2016-07-31 Published:2016-12-28

摘要/Abstract

摘要： 为了建立一种准确、快速的副猪嗜血杆菌病原夹心ELISA检测方法，以副猪嗜血杆菌外膜蛋白P5的特异性单克隆抗体1E2作为捕获抗体，兔抗副猪嗜血杆菌多克隆抗体作为检测抗体，通过方阵滴定优化夹心ELISA检测方法最佳反应条件，并对该检测方法进行特异性、敏感性验证以及临床样品的检测应用。结果显示，该检测方法中单克隆抗体1E2最佳包被浓度为3.434 μg/mL，兔抗副猪嗜血杆菌多抗的最佳稀释浓度为3.350 μg/mL，用10 mg/mL明胶37℃封闭1 h优于其他封闭液，辣根过氧化物酶标记的山羊抗兔多体最佳使用浓度为1：4 000。该检测方法的特异性试验结果显示可检测出15个血清型的副猪嗜血杆菌病原，而与其他病原菌的检测结果均为阴性。敏感性试验结果显示该方法能够检测出副猪嗜血杆菌的最低菌落浓度为1×10⁶ cfu/mL。利用该检测方法对临床115份副猪嗜血杆菌可疑病料进行检测结果显示可检出83份阳性样品，检出的阳性样品数高于细菌分离及PCR鉴定方法。上述结果表明所建立的副猪嗜血杆菌病原夹心ELISA检测方法特异性强、敏感性好并可应用到临床样品的检测。

关键词: 副猪嗜血杆菌, 单克隆抗体, 夹心ELISA

Abstract: In order to develop a double antibody sandwich ELISA to detect Haemophilus parasuis quickly and accurately,which using specific monoclonal antibody 1E2 to outer membrane protein P5 of Haemophilus parasuisas the capture antibody, and the rabbit anti-Haemophilus parasuis polyclonal antibody as the detection antibody, then determined the optimal working conditions of ELISA by square matrix titrimetry,and verified for specificity, sensitivity and detected the clinical samples. The results showed that the optimal enveloped concentration of monoclonal antibody 1E2 was 3.434 μg/mL, and the best diluted concentration of polyclonal antibody was 3.350 μg/mL,and the 10 mg/mL gelatin which was used as confining liquid was better than other closed liquid, and the optimal diluted concentration of goat-anti-rabbit IgG which labeled by HRP was 1:4 000.The developed ELISA had good specificity and the results were positive to detect the 15 serovars of Haemophilus parasuis, but no cross reactions with other bacteria.The sensitivity result of the ELISA showed that the minimum detection limit was 1×10⁶ cfu/mL.When the ELISA was used to detect the suspect Haemophilus parasuis 115 clinical samples, the results showed that the 83 samples of which were positive to Haemophilus parasuis, and the number of positive samples was higher than the results of the bacteria isolation and PCR identification.The above results showed that the developed sandwich ELISA was specific and sensitive, and can be applied to the detection of clinical samples.

Key words: Haemophilus parasuis, Monoclonal antibody, Sandwich ELISA

中图分类号:

S432.4

宋帅, 李春玲, 臧莹安, 郭海祥, 李淼, 杨冬霞, 徐志宏. 副猪嗜血杆菌病原夹心ELISA检测方法的建立及应用[J]. 华北农学报, 2016, 31(S1): 463-469. doi: 10.7668/hbnxb.2016.S1.079.

SONG Shuai, LI Chunling, ZANG Yingan, GUO Haixiang, LI Miao, YANG Dongxia, XU Zhihong. Application and Development of Double Antibody Sandwich ELISA for Detection Haemophilus parasuis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(S1): 463-469. doi: 10.7668/hbnxb.2016.S1.079.

http://www.hbnxb.net/CN/Y2016/V31/IS1/463

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