O型口蹄疫病毒VP1蛋白竞争ELISA诊断方法的初步建立

利用表达的重组融合蛋白pGEX-6P-1-VP1作为抗原,用PET-32a-VP1蛋白免疫家兔获得的阳性血清作为FMDV阳性血清的竞争抗体,建立了检测FMDV抗体的竞争ELISA,并进行了条件优化,利用统计学方法计算竞争ELISA的阳性临界值。结果显示,重组蛋白抗原的最适包被质量浓度为1μg·mL~(-1),待检血清、酶标抗体和高免血清的最佳稀释度分别为1∶40、1∶2000和1∶500;与液相阻断ELISA试剂盒相比较,该方法的敏感性为91.3%,特异性为95.1%,符合率为96.7%。结果表明,该方法敏感性好、特异性强、重复性好,可用于O型FMDV血清抗体水平的检测。

Establishment of Competitive ELISA Diagnostic Method for Foot-and-mouth Disease Based on VP1 Protein in Serotype OVirus

Competitive ELISA was developed for detecting the antibodies against the foot-and-mouth disease virus (FMDV)with the purified recombinant pGEX-6P-1-VP1 protein as the antigen and the rabbit-anti-VP1 serum as the competitive antibody.The positive threshold of the competitive ELISA was calculated using statistical methods.The results showed that (a)the dose of the coating antigen protein was 1ug/ml;(b)the working concentration of the detected serum sample was 1 ∶ 40dilution,and the enzyme-labeled goat anti-rabbit lgG was 1 ∶ 2000;(c)the working titer of specific rabbit serum to N protein was 1 ∶500;and,(d)comparing to the liquid-phase blocking ELISA,the sensitivity and specificity of the competitive ELISA were 91.3% and 95.1%,respectively,and the coincident rate between the two methods was 96.7%.It appeared that this newly developed diagnostic method could be applied for the detection of the serum antibodies against serotype O FMDV with high sensibility,specificity and repeatability.