猪SIgA直接ELISA检测方法的建立及应用

采集猪肠黏液并分离纯化出猪分泌型免疫球蛋白A(SIgA),制备兔抗猪SIgA IgG抗体,经HRP标记,并优化直接ELISA检测猪SIgA的条件,建立了检测猪SIgA的直接ELISA方法。结果显示,筛选的最佳反应条件:黏液蛋白包被浓度为10μg/mL,兔抗猪SIgA IgG-HRP酶标抗体的工作浓度为1∶200,反应时间为60min。包被SIgA浓度标准曲线的线性范围为90～1 400ng/mL,回收率为85.85%～133.88%,变异系数为13.12%～17.67%。采用该方法对猪肺炎支原体灭活疫苗免疫与攻毒后的仔猪肺冲洗液和肠黏液中的SIgA进行检测;结果显示,攻毒后免疫猪肺冲洗液中SIgA含量平均为205.81ng/mL,显著高于攻毒对照猪的147.56ng/mL和空白对照猪的124.37ng/mL(P<0.05);免疫猪肠黏液中SIgA含量平均为67.94ng/mL,与对照猪无明显差异。结果证实,建立的猪SIgA直接ELISA方法具有经济、适用和准确的特点。

Establishment and application of a direct ELISA for detection of pig SIgA

In this study,secreting immunoglobulin A(SIgA) from pig intestinal mucosa was isolated and purified.IgG of rabbit anti-pig SIgA was obtained by immunizing rabbit and labeled with horseradish peroxidase(HRP).A direct ELISA for the detection of pig SIgA was established through optimization as follows:the concentration of coated mucin was 10μg/mL,the working concentration of rabbit anti-pig IgG-HRP was 1∶200 and the reaction time was 60min.The standard linear interval range of coating concentration of SIgA was from 90 to 1400ng/mL.This method could give recoveries from 85.85% to 133.88%,variation coefficient from 13.12% to 17.67%.The detection of SIgA from lung and intestinal mucus of the vaccinated piglets with inactivated Mycoplasma hyopneumoniae(Mhp) vaccine and challenged with a wild Mhp strain(MY-99),showed that SIgA in lung fluid was 205.81ng/mL after challenged with MY-99,which was significantly higher than that of vaccinated group(147.56ng/mL) and blank control group(124.37ng/mL)(P<0.05).The result confirmed that the direct ELISA method was economical,convenient and accurate for SIgA detection.