基于重组IBDV VP3蛋白的免疫磁珠间接ELISA抗体检测方法的建立及初步应用

Establishment and preliminary application of indirect ELISA with immunomagnetic beads based on recombinant IBDV VP3 protein for the detection of antibodies against the protein

本研究旨在毕赤酵母中表达鸡传染性法氏囊病毒(infectious bursal disease virus,IBDV)的VP3蛋白,以重组VP3蛋白作为包被抗原建立IBDV血清抗体免疫磁珠间接ELISA检测方法并进行初步应用。利用毕赤酵母表达系统获得约为39 ku的重组VP3蛋白,Western-blotting检测表明,重组VP3蛋白具有良好的特异性和反应原性。免疫磁珠间接ELISA的最佳反应条件:磁珠和抗原偶联的缓冲液是50 mmol/L MES(p H 8.0),偶联时间为3 h,抗原加入量为95μg,抗原与羧基磁珠最大偶联量为80μg。待检血清和酶标二抗的稀释度分别为1∶1 600和1∶4 000,血清孵育时间为30 min,酶标二抗孵育时间为20 min。在该优化条件下,阴性、阳性临界值判定标准为0.134。特异性试验显示,对抗AIV、IBV、MDV、NDV阳性血清检测结果均为阴性。敏感性试验显示,本试验所建立的检测方法敏感性高于商品化IBDV血清抗体检测试剂盒。重复性试验显示,批内和批间重复试验的最大变异系数分别为3.8%和5.9%。稳定性试验结果显示变异系数小于7%。用建立的检测方法和商品化IBDV血清抗体检测试剂盒同时检测50份血清,结果显示两种检测方法的相对敏感性为94.6%,相对特异性为92.3%,符合率为94.0%。本试验建立的检测方法具有敏感性高、特异性强、重复性好、稳定性高特点,为其应用于临床IBDV血清抗体的检测奠定了基础。

This study aimed to express the VP3 protein of infectious bursal disease virus(IBDV) in Pichia pastoris to construct the immunomagnetic bead indirect ELISA for the detection of antibodies against the IBDV VP3 protein,with the recombinant VP3 protein as the coating antigen,and to carry out preliminary application. The recombinant VP3 protein about 39 ku in mass was obtained using the Pichia pastoris expression system,and Western-blotting detection suggested that the recombinant VP3 protein had possessed excellent specificity and immunoreactivity. The optimal reaction conditions for the immunomagnetic bead indirect ELISA were as follows:the coupling buffer solution for magnetic bead and antigen was 50 mmol/L MES(pH=8.0),the coupling time was 3 h,the antigen addition was 95μ g,and the maximum coupling amount of antigen with carboxyl magnetic bead was 80μ g.The serum and conjugated reagent were diluted at the ratios of 1 ∶ 1 600 and 1 ∶ 4 000,respectively,the serum incubation time was30 min,and the incubation time of conjugated secondary antibody was 20 min.Under those optimization conditions,the criterion for negative and positive thresholds was 0.134.The specificity test suggested that,all test results of positive serum anti-AIV,anti-IBV,anti-MDV and anti-NDV were negative.The sensitivity test suggested that,the detection method constructed in this experiment had higher sensitivity than the commercial IBDV serum antibody detection kit. The repeatability test indicated that,the maximum variation coefficients of intra-batch and inter-batch repeatability tests were 3.8% and5.9%,respectively.The stability test results demonstrated that,the variation coefficient was smaller than 7%.Subsequently,the constructed detection method as well as the commercial IBDV serum antibody detection kit was used to detect 50 serum samples at the same time,and the results suggested that,the relative sensitivity of these two detection methods was 94.0%,the relative specificity was92.3%,and the coincidence rate was 97.6%.Obviously,the detection method constructed in this experiment has high sensitivity,high specificity,good repeatability and high stability,which lay the foundation for its clinical application in detecting IBDV serum antibodies.