抗H5亚型禽流感病毒血凝素蛋白特异性单克隆抗体的研制及鉴定

以抗H5亚型禽流感病毒（AIV）JSD株鸡胚尿囊液免疫8周龄BALB/c小鼠,第4次免疫后取其脾淋巴细胞与SP2/0-Ag-14骨髓瘤细胞融合,用血凝抑制试验（HI）对杂交瘤进行筛选,经3次亚克隆后,共获得4株针对血凝素（HA）蛋白的特异性单克隆抗体,分别命名为：2D2、2C8、3D3和3D11。该4株单抗的小鼠腹水的HI效价在2^13～2^15,ELISA效价为1.2×10^5～5.1×10^5。亚类鉴定结果表明,2D2和3D11属于IgG1,2C8属于IgG3,3D3属于IgG2a亚类;特异性试验结果表明,上述4株单抗仅与H5 AIV毒株发生特异性反应,而不与其他亚型AIV以及NDV、IBV和EDSV-76等病毒反应;鸡胚中和试验结果显示,4株单抗均具有较好的中和活性。本研究成功获得4株针对H5 AIVHA蛋白的特异性单抗,为临床H5 AIV的血清学监测及鉴定提供了必需的试剂。

Development and identification of monoclonal antibodies against hemagglutinin of H5 subtype avian influenza virus

The 8-week-old female BALB/c mice were immunized with subtype H5-AIV strain JSD isolated from Jiangsu province in 2004.Spleen cells collected from immunized mice were fused with SP2/0-Ag-14 myeloma cells after the fourth immunization.Hemagglutinin inhibition(HI) test was used to screen the hybridoma cells and limited dilution method was performed to subclone the positive clones.After three cycles of subcloning,four McAbs against the hemagglutinin(HA) protein of H5-AIV were obtained,designated as 2D2,2C8,3D3 and 3D11 respectively.The obtained McAbs were proved to have high HI and ELISA titers.2D2 and 3D11 were identified to be IgG1,2C8 and 3D3 to be IgG3 and IgG2a respectively.The specificity test proved that all the McAbs were specific to AIV-H5,whereas did not react with other subtypes of AIVs,NDV,IBV and EDSV-76.The hemagglutination of H5-AIV was neutralized specifically with the developed McAbs.All the results mentioned above indicated that the obtained McAbs could replace the common chicken serum for AIV detection.They may be useful in epidemiological investigation and immunological surveillance of avian influenza in chicken farms.Furthermore,they could be used in clinical diagnosis.