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目的:制备抗禽流感病毒M2蛋白的单克隆抗体(mAb)并进行特性鉴定。方法:利用纯化的融合蛋白GST-M2免疫BALB/c小鼠,然后以GST-M2和GST分别作为ELISA抗原进行筛选,选择GST-M2抗原检测强阳性、GST抗原检测阴性的杂交瘤细胞进行克隆,建立能稳定分泌抗AIV M2 mAb的杂交瘤细胞株。mAb的效价采用间接ELISA和琼脂扩散试验(AGP)测定,用夹心ELISA测定Ig亚类,Western blot、抗原捕获ELISA、间接免疫荧光及免疫组化染色法检测mAb的特性。结果:得到4株分泌抗禽流感病毒M2蛋白的mAb的杂交瘤细胞株1E1、2F8、4E3和5D6,小鼠腹水中抗体的ELISA效价大于210×100、AGP效价大于1∶4。抗体亚类鉴定1E1和4E3为IgG2a,2F8和5G6为IgG2b。抗原捕获ELISA表明,2F8 mAb能与H5、H9亚型AIV发生特异性反应,而不能与鸡新城疫病毒(NDV)和鸡传染性法氏囊病毒(IBDV)反应。IFA和免疫组化试验表明,2F8 mAb能够与感染了禽流感病毒的MDCK细胞以及鸡体组织细胞发生特异性结合。结论:本研究获得4株抗禽流感病毒M2蛋白的mAb,其中2F8 mAb的效价高、特异性强,可作为检测AIV方法的核心试剂。
Objective: To prepare and characterize the monoclonal antibody (mAb) against avian influenza virus M2 protein. Methods: BALB / c mice were immunized with the purified fusion protein GST-M2 and then screened by GST-M2 and GST as ELISA antigens respectively. GST-M2 antigen was used to detect the hybridoma cells which were strongly positive and negative for GST antigen. , To establish a hybridoma cell line that can stably secrete anti-AIV M2 mAb. The titer of mAb was determined by indirect ELISA and agar diffusion assay (AGP). The Ig subclasses were determined by sandwich ELISA. Western blot, antigen capture ELISA, indirect immunofluorescence and immunohistochemistry were used to detect the mAb. RESULTS: Four hybridoma cell lines, 1E1, 2F8, 4E3 and 5D6 secreting anti-avian influenza virus M2 protein, were obtained. The antibody titer of ascites in mouse ascites was more than 210 × 100, and AGP titer was greater than 1: 4. Antibody subclasses identified 1E1 and 4E3 as IgG2a, 2F8 and 5G6 as IgG2b. Antigen capture ELISA showed that 2F8 mAb reacted specifically with H5 and H9 subtype AIV, but not with NDV and IBDV. IFA and immunohistochemistry showed that 2F8 mAb could specifically bind to MDCK cells infected by avian influenza virus and chicken body cells. Conclusion: In this study, four mAbs against avian influenza virus M2 protein were obtained. Among them, the 2F8 mAb was highly potent and specific and could be used as a core reagent in the detection of AIV.