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

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14d可检出抗体,35d抗体滴度达峰值,并维持此滴度至143d。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。

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 antibodyagainst 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 143days 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 7with high specificity.