旋毛形线虫P53重组蛋白抗体间接ELISA检测方法的建立

Development of an indirect ELISA for the detection of antibody against P53 recombinant protein of Trichinella spiralis

以纯化的旋毛形线虫P53排泄分泌(ES)重组蛋白作为包被抗原,建立了检测旋毛形线虫P53ES蛋白抗体的间接ELISA方法,并确定了间接ELISA的最适反应条件:抗原包被浓度为2μg/mL(100μL),血清稀释度为1∶200,辣根过氧化物酶标记羊抗鼠IgG抗体稀释度为1∶10000,抗原和血清于37℃反应1.5h,血清和二抗于37℃反应1h,底物于37℃显色10min。应用该方法对试验小鼠血清进行检测,结果表明,建立的间接ELISA方法具有较好的特异性和重复性,适用于检测特异性P53血清抗体。

Using the purified recombinant P53 excretory-secretory（ES） protein of Trichinella spiralis as coating antigen,an indirect ELISA was developed for the detection of antibody against P53 ES protein of T. spirali.s. In the indirect ELISA, the optimal concentration of the recombinant P53 for coating was 2μg/mL（100μL）,the optimal dilution of serum sample was 1 = 200,the optimal dilution of the goat antimice IgG labeled with HRP was 1：10 000,the serum sample should be incubated with P53 protein at 37℃ for 1.5 h,the HRP-labeled goat anti-mice IgG should be incubated with the serum sample at 37℃ for 1 h, and the substrate should be incubated at 37℃ for 10 min. The murine serum samples were examined by the indirect ELISA to monitor antibody titer. The results revealed that the indirect ELISA had good reproducibility and specificity,and was suitable to detect the specific antibody against P53 ES antigen of T. spiralis.