抗禽流感病毒M2蛋白单克隆抗体的制备及鉴定

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,2178和5G6为IgG2b.抗原捕获ELISA表明,2F8 wAb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2F8mAb的效价高、特异性强,可作为检测AIV方法的核心试剂.

Preparation and characterization of monoclonal antibodies against M2 protein of avian influenza virus

AIM: To prepare and characterize the monoclonal antibodies against M2 protein of avian influenza virus (AIV). METHODS: BALB/c mice were immunized with purified fusion protein GST-M2. Then the hybridoma cell lines secreting mAb against M2 were screened by indirect ELISA by using GST-M2 and GST as ELISA coated antigen respectively and selecting strongly positive against GST-M2 and negative against GST. RESULTS: Four hybridoma cell lines secreting anti-AIV M2 mAbs were obtained, designated 1E1, 2F8, 4E3 and 5D6. The ELISA titers of these mAbs were above 2(10)x100 and the agar gel precipitation test showed the titers of them were above 1:4. Immunoglobulin subclass tests differentiated them as IgG2a (1E1, 4E3) and IgG2b (2F8, 5D6). The Ag-Capture ELISA showed the mAb 2F8 could specifically reacted with H5 and H9 subtypes of AIV, but could not react with Newcastle disease virus (NDV) and Infectious bursal disease virus (IBDV). IFA and immunochemistry test indicated the prepared mAb 2F8 could specifically bind to MDCK cell and chicken tissue cells infected with different AIV subtypes. CONCLUSION: Four mAbs against AlV M2 were obtained in this study. The mAb 2F8 against AIV M2 could be used as the key reagent for establishment of AIV detection method.