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目的 制备抗SARS冠状病毒的单克隆抗体 ,用于SARS的实验室诊断。方法 灭活SARS冠状病毒 (PUMC0 1)全病毒免疫BALB/C小鼠 ,取小鼠脾细胞与骨髓瘤细胞 (NS 1)融合 ,用酶联免疫吸附试验 (ELISA)、间接免疫荧光法 (IFA)及免疫印迹法对所获得的细胞克隆进行筛选和鉴定 ,并用其中一株 (M2 )杂交瘤诱生的腹水单克隆抗体对SARS患者尸检肺组织切片进行免疫组织化学染色。结果 共筛选出 6株杂交瘤细胞 ,ELISA及IFA法证实其分泌的单克隆抗体与SARS冠状病毒有特异性反应 ,与其他常见呼吸道病原体均无交叉反应。免疫双扩散方法鉴定 1株杂交瘤细胞(M2 )为免疫球蛋白IgG3型 ,其余 5株均为IgG1型。免疫印迹试验显示 1株杂交瘤细胞 (M 2 )分泌的抗体与相对分子质量 6 80 0 0的蛋白有特异性反应 ;4株杂交瘤细胞分泌的抗体与相对分子质量 2 70 0 0的蛋白有特异性反应 ,1株在免疫印迹上未见到结果。SARS患者尸检肺组织病理切片免疫组织化学染色阳性 ,在肺泡上皮细胞、支气管上皮细胞及巨噬细胞的胞浆均可见阳性颗粒。结论 我们制备的 6株单克隆抗体是抗SARS冠状病毒的特异性单克隆抗体 ,用于SARS尸检肺组织免疫组化染色 ,结果阳性。
Objective To prepare anti-SARS coronavirus monoclonal antibody for the laboratory diagnosis of SARS. Methods BALB / C mice were immunized with whole virus of SARS coronavirus (PUMC0 1). The mouse spleen cells were fused with myeloma cells (NS 1). The immunofluorescence assay (IFA) ) And Western blotting were used to screen and identify the obtained cell clones. Immunohistochemical staining was performed on autopsy lung tissue sections of SARS patients using monoclonal antibody of ascites induced by one (M2) hybridoma. Results A total of 6 hybridoma cells were screened out. ELISA and IFA confirmed that the monoclonal antibodies secreted by them reacted with the SARS coronavirus and did not cross-react with other common respiratory pathogens. One hybridoma cell (M2) was identified as immunoglobulin IgG3 by immunodouble diffusion method, and the other five were IgG1. Western blotting showed that the antibody secreted by one hybridoma cell (M 2) reacted specifically with a protein of relative molecular mass of 80,000. The antibodies secreted by the four hybridoma cells and the protein with a relative molecular mass of 270,000 Specific reaction, 1 strain in the Western blot did not see the results. SARS patients were autopsy lung tissue biopsy immunohistochemical staining positive, alveolar epithelial cells, bronchial epithelial cells and macrophages in the cytoplasm showed positive particles. Conclusions The six monoclonal antibodies we prepared are specific anti-SARS coronavirus monoclonal antibodies for the immunohistochemical staining of SARS autopsy lung tissue with a positive result.