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利用人工合成的高粱花叶病毒P3蛋白的多肽片段作为抗原,制备多克隆抗体,利用免疫印迹对抗体的特异性进行鉴定,表明抗体具有高度特异性。通过方阵滴定法确定抗原和抗体的最佳工作浓度,并分别对包被时间、二抗工作浓度、孵育条件、底物显色时间等进行优化,从而建立了高粱花叶病毒(Sr MV)的间接ELISA高通量检测方法。通过测试确定了阴阳性结果判定的临界值,评估了该方法的灵敏度以及与RT-PCR检测法的符合度。所建立的间接ELISA检测方法的抗原、一抗、二抗最佳稀释度分别为1:4、1:600和1:5 000;最佳抗原包被条件为4℃、12 h;抗原抗体最佳孵育条件为37℃、60 min;二抗最佳孵育条件为37℃、45 min;最佳显色条件为37℃、12 min;此检测方法检测速度快、灵敏度高、特异性强,与RT-PCR法的符合度为87.00%。
The polyclonal antibody was prepared by using the peptide fragment of artificial sorghum mosaic virus P3 protein as the antigen, and the specificity of the antibody was identified by immunoblotting, indicating that the antibody is highly specific. The optimum working concentration of antigen and antibody was determined by square titration. SrMV (Sorghum Mosaic Virus) was established by optimizing the coating time, working concentration of secondary antibody, incubation conditions and substrate color time. Indirect ELISA high throughput assay. Through the test to determine the threshold value of the positive and negative results to assess the sensitivity of the method and the RT-PCR detection of compliance. The optimal dilutions of the antigens, primary antibodies and secondary antibodies established by indirect ELISA were 1: 4, 1: 600 and 1: 5 000, respectively. The optimal antigen coating conditions were 4 ℃ and 12 h. The optimal incubation conditions were 37 ° C for 45 min and 37 ° C for 12 min. The detection method was rapid, sensitive and specific The coincidence of RT-PCR method was 87.00%.