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目的:制备抗埃博拉病毒核蛋白(EBOV NP)单克隆抗体和多克隆抗体,建立针对EBOV NP的ELISA检测方法。方法:以重组EBOV NP免疫动物并制备多克隆抗体和单克隆抗体。在此基础上,通过优化抗体浓度、包被液等条件建立检测EBOV NP的双抗夹心ELISA方法。结果:制备出了兔多克隆抗体,筛选出2株可分泌单克隆抗体的鼠源杂交瘤细胞株。Western blot实验结果表明兔多抗与鼠单抗的结合区域均为N端1~35氨基酸。通过优化,建立了针对EBOV NP的双抗夹心ELISA检测方法。其线性范围是31.2~1 000 ng/ml,最低检测限为2.6 ng/ml。结论:制备出了抗EBOV核蛋白的高特异性多克隆抗体和单克隆抗体,建立了定量检测EBOV核蛋白的方法。
Objective: To prepare anti-Ebola virus (EBOV NP) monoclonal antibody and polyclonal antibody and establish an ELISA assay for EBOV NP. Methods: Animals were immunized with recombinant EBOV NP and polyclonal and monoclonal antibodies were prepared. On this basis, a double-antibody sandwich ELISA for the detection of EBOV NP was established by optimizing the antibody concentration and coating solution. Results: Rabbit polyclonal antibodies were prepared and two murine hybridoma cell lines secreting monoclonal antibodies were screened out. Western blot results showed that the binding region of rabbit polyclonal antibody and mouse monoclonal antibody were N-terminal amino acids 1-35. Through the optimization, a double-antibody sandwich ELISA assay for EBOV NP was established. The linear range was 31.2-1 000 ng / ml with the lowest detection limit of 2.6 ng / ml. CONCLUSION: High specific polyclonal antibodies and monoclonal antibodies against EBOV nucleoprotein were prepared and a method for quantitative determination of EBOV nuclear protein was established.