猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用

为建立一种检测口蹄疫病毒（foot-and-mouth disease virus，FMDV）特异性IgA抗体的检测方法，本研究以原核表达系统表达纯化的FMDV结构蛋白VP1作为包被抗原，以鼠抗猪IgA单克隆抗体为二抗，辣根过氧化酶标记的羊抗鼠IgG抗体为三抗，建立猪A型FMDV特异性IgA抗体间接ELISA检测方法。确定抗原包被浓度为3.50 μg/mL，二抗与三抗的最佳稀释度为1∶10 000，二抗和三抗作用时间均为30 min。所建立的方法与抗猪瘟病毒、猪繁殖与呼吸道综合征病毒等病原的特异性IgA抗体间无交叉反应，A型口蹄疫感染样品的阳性检出率在90%以上，批内和批间重复性试验的变异系数介于3.16%~9.76%。该方法为监测FMDV特异性IgA抗体水平变化规律及猪的黏膜免疫效果评价及口蹄疫的早期诊断提供了一种新方法。

Development and Primary Application of Indirect ELISA Method for Detecting the IgA Antibody against Foot-and-mouth Disease Virus Type A in Porcine

An indirect ELISA for detecting the IgA antibody against porcine foot-and-mouth disease virus (FMDV) type A was developed by using purified FMDV structural protein VP1 as coating antigen, mouse anti-pig IgA monoclonal antibody as second antibody and HRP-conjugated goat anti-mouse IgG as third antibody.The concentration of coating antigen was optimized as 3.50 μg/mL,the dilution and reaction time of second antibody and third antibody were optimized as 1:10 000 and 30 min, respectively.There was no cross-reactivity with anti-CSFV, PRRSV and other pathogen specific IgA antibodies.The positive detection rate of FMDV type A infectedsamples was above 90%.The coefficient variation of intra-and inter-assay was ranged from 3.16% to 9.76%.The ELISA method described in this study was proved to be specific and rapid for the detection of FMDV specific IgA antibody.It was potential to be applied for detection the level of FMDV specific IgA and evaluate the effect of mucosal immunity.Besides,it provided a new method for clinical diagnosis of foot-and-mouth disease.