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目的 制备抗SARS病毒N蛋白的单克隆抗体并研究其初步应用。方法 用基因重组N蛋白免疫小鼠 ,取免疫后的鼠脾细胞与骨髓瘤细胞融合 ,筛选分泌抗SARS病毒N蛋白单克隆抗体细胞株。将阳性细胞株接种小鼠腹腔制备单克隆抗体腹水并对抗体进行纯化 ,分析纯化抗体的相对亲和力。选择亲和力较高的抗体制备检测SARS病毒抗原的酶联免疫诊断试剂 ,并对其敏感性和特异性进行分析。结果 共获得 11株单克隆抗体细胞株 ,其中 3株单抗与N蛋白具有较高的亲和力 ,4株纯化单抗与N蛋白反应很弱 ,其余 4株单抗介于两者之间。用亲和力较高的单抗制备检测SARS病毒抗原的诊断试剂 ,其敏感性可达 31PFU ml,而且与其他呼吸道病毒无交叉反应。结论 该试剂特异性较好 ,可用于SARS病毒抗原的检测 ,其敏感性仍需用临床急性期样品进行评价。
Objective To prepare anti-SARS virus N protein monoclonal antibody and to study its preliminary application. Methods Mice were immunized with genetically modified recombinant protein N and murine spleen cells were fused with myeloma cells to screen and secrete monoclonal antibody against SARS virus N protein. The positive cell lines were inoculated intraperitoneally in mice to prepare monoclonal antibody ascites and the antibodies were purified, and the relative affinity of the purified antibodies was analyzed. The higher affinity antibodies were selected to prepare ELISA reagents for detecting SARS virus antigens and their sensitivity and specificity were analyzed. Results A total of 11 monoclonal antibody cell lines were obtained, of which 3 monoclonal antibodies had high affinity with N protein, 4 monoclonal antibodies reacted weakly with N protein, and the other 4 McAbs were in between. The diagnostic reagent for detecting SARS virus antigens was prepared with higher affinity monoclonal antibody, the sensitivity was up to 31 PFU ml, and there was no cross-reaction with other respiratory viruses. Conclusion The specificity of the reagent is good and can be used for the detection of SARS virus antigen. The sensitivity of the reagent still needs to be evaluated in clinical acute phase samples.