检测胸膜肺炎放线杆菌7型抗体的竞争ELISA方法的建立与应用

doi:10.11843/j.issn.0366-6964.2015.10.016

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (10): 1829-1837.doi: 10.11843/j.issn.0366-6964.2015.10.016

检测胸膜肺炎放线杆菌7型抗体的竞争ELISA方法的建立与应用

李树清¹，陈志飞¹，张强¹，吴建祥²，刘雨潇³，王巧全¹，林颖峥¹，宋青¹，唐智芳¹，陆承平^4*

(1.上海出入境检验检疫局，上海 200135；2.浙江大学，杭州 310058；3.上海交通大学农业与生物学院，上海 200240；4.南京农业大学，南京 210095)

收稿日期:2015-01-26 出版日期:2015-10-23 发布日期:2015-10-23
通讯作者: 陆承平， E-mail: lucp@njau.edu.cn
作者简介:李树清（1964-），女，四川眉山人，研究员，硕士，从事进出口动物检疫工作，Tel：021-38620591, E-mail：lisq@shciq.gov.cn
基金资助:
上海出入境检验检疫局科研专项（HK004-2008）

Development and Application of Competitive ELISA for Detecting Antibody against Actinobacillus pleuropneumoniae Serotype 7

LI Shu-qing¹，CHEN Zhi-fei¹，ZHANG Qiang¹，WU Jian-xiang²，LIU Yu-xiao³，WANG Qiao-quan¹，LIN Ying-zheng¹，SONG Qing¹，TANG Zhi-fang¹，LU Cheng-ping^4*

(1.Shanghai Entry-Exit Inspection and Quarantine Bureau of the People′s Republic of China，Shanghai 200135，China；2.Zhejiang University，Hangzhou 310058，China；3.School of Agriculture and Biology，Shanghai Jiaotong University，Shanghai 200240，China；4.Nanjing Agricultural University，Nanjing 210095，China)

Received:2015-01-26 Online:2015-10-23 Published:2015-10-23

摘要/Abstract

摘要：

针对猪传染性胸膜肺炎（App）最主要的流行血清型7型，建立该型特异的抗体检测方法，并应用于出入境检疫。使用App 7型参考菌株制备单克隆抗体，基于辣根过氧化物酶标记的单抗，建立检测App 7型抗体的竞争ELISA方法，用于检测出入境临床采集及免疫接种的猪血清样品，并与商品化进口试剂盒进行比较。结果显示制备的单抗为IgM，与App 7型参考菌株之外的其他14个血清型以及27株其他相关菌株均无交叉反应。建立的竞争ELISA方法只能检测App 7型抗体，特异性好。用该方法检测App 7型参考菌株免疫猪的血清，免疫后14 d可检出抗体，35 d抗体滴度达峰值，并维持此滴度至143 d。使用建立的竞争ELISA检测免疫猪及临床血清样品260份，结果与法国IDvet试剂盒检测符合率为97.2%，与加拿大Biovet试剂盒检测符合率为97.9%～98.2%。建立的竞争ELISA方法可以应用于App 7型抗体的检测。

Abstract:

The study was conducted to develop the specific method for detecting antibody against Actinobacillus pleuropneumoniae(App) serotype 7(S7)，the most prevalent serotype all over the world，and apply to the inspection and quarantine for antibodies of imported and exported pigs.Hybridoma cell line secreting monoclonal antibody(McAb) against App S7 was screened，and the McAb was purified from mouse ascitic fluid and labeled with HRP.The competitive ELISA（cELISA）was developed and its specificity and stability were tested.The sera collected from pigs immunized with reference strain of App S7 or field samples were assayed by this cELISA and other commercial ELISA kits parallelly.The McAb，characterized as IgM，can react specifically with reference strain of App S7，and no cross-reaction with other serotype strains and 27 other strains.The developed cELISA was also specific for detecting antibody against App S7.The antibody against App S7 from the immunized pig can be detected by this method at 14 days post injection，the antibody titer reached and kept at the peak from 35 to 143 days post immunization.The data detected from the samples of 260 clinical sera and the immunized swines，showed that the cELISA have 97.2% coincidence with IDvet test kit and 97.9%-98.2% consistent with Biovet test kit.This cELISA can be used to detect antibody against Actinobacillus pleuropneumoniae serotype 7 with high specificity.

中图分类号:

S852.619

李树清，陈志飞，张强，吴建祥，刘雨潇，王巧全，林颖峥，宋青，唐智芳，陆承平. 检测胸膜肺炎放线杆菌7型抗体的竞争ELISA方法的建立与应用[J]. 畜牧兽医学报, 2015, 46(10): 1829-1837.

LI Shu-qing，CHEN Zhi-fei，ZHANG Qiang，WU Jian-xiang，LIU Yu-xiao，WANG Qiao-quan，LIN Ying-zheng，SONG Qing，TANG Zhi-fang，LU Cheng-ping. Development and Application of Competitive ELISA for Detecting Antibody against Actinobacillus pleuropneumoniae Serotype 7[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(10): 1829-1837.

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检测胸膜肺炎放线杆菌7型抗体的竞争ELISA方法的建立与应用

Development and Application of Competitive ELISA for Detecting Antibody against Actinobacillus pleuropneumoniae Serotype 7

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