基于外膜蛋白Omp16的副猪嗜血杆菌抗体间接ELISA方法的建立

Establishment of an indirect ELISA for detection of antibodies against the Omp16 of Haemophilus parasuis

为建立检测副猪嗜血杆菌(H.parasuis)抗体方法,本研究以H.parasuis外膜蛋白Omp16为包被抗原建立了检测H.parasuis的间接ELISA方法。反应条件经优化为:抗原包被浓度为1μg/m L,血清稀释度为1:80;血清与抗原最佳作用时间为40 min,二抗稀释10 000倍,与一抗孵育30 min;TMB作用时间为30 min。利用该ELISA方法检测H.parasuis、胸膜肺炎放线杆菌、大肠杆菌和猪链球菌2型等血清,除H.parasuis为阳性外其余均为阴性,表明其特异性强。该ELISA方法可检测到最高稀释度为1∶512的阳性血清,敏感性高。重复性试验显示,其批内和批间的重复试验变异系数(CV)分别在3.62%~8.97%和4.3%~9.50%。该ELISA检测方法与进口ELISA试剂盒的阳性符合率、阴性符合率和总体符合率分别为87.8%,90.7%和88.6%。本研究建立的间接ELISA方法,为H.parasuis血清流行病学调查及疫苗免疫评价等提供了重要手段。

An indirect ELISA for detection of antibody against Haemophilus parasuis was established using outer membrane protein Omp16 of H.parasuis as coating antigen. The reaction conditions of this assay were optimized, including： （a） coating antigen at 1μg/mL, （b） serum sample at 1：80 dilution, （c） serum incubated with antigen for 40min, （d） HRP-conjugated goat anti-pig IgG antibody at 1：10,000 dilution and incubated with serum for 30 min, （e） TMB incubated for 30 min. The ELISA was highly specific and showed no cross-reaction with the positive antiserum of other porcine disease. This ELISA was highly sensitive as it was able to detect at least H.parasuis antibody in 1：512 diluted serum. This assay was also repeatability with the inter samples coefficient of variation of raw optical density values for known positive samples in different runs was 〈10% （ranged from 3.62% to 8.97%）, while intra sample coefficient of variation ranged from 4.3% to 9.50% between runs. The assay was validated by comparison with a commercial ELISA kit. The results showed that the positive coincident rate of this assay and commercial ELISA assay was 87.8%, and negative coincident rate was 90.7% and the total coincidence rate was 88.6%. The established ELISA kit could be a useful tool for detection of H.parasuis antibody, which can be used in serological epidemiology investigation.