H4亚型禽流感病毒血凝素蛋白单克隆抗体的制备

以H4亚型AIV分离株A/鸭/扬州/185/2003免疫BALB/c小鼠,取小鼠脾脏细胞与骨髓瘤细胞进行融合,经血凝抑制(HI)试验筛选并克隆后,获得能够稳定分泌抗体的单克隆细胞株:2C11、6E7、4C8和5C5。4株单抗腹水的HI效价在2~(12)～2~(15),单抗亚类均为IgG2a。4株单抗均能与感染了H4亚型AIV的MDCK细胞发生间接免疫荧光(IFA)反应。并且,均不与其他亚型AIV、NDV、IBV和EDS-76病毒发生HI反应。另外,4株单抗均能使病毒失去感染细胞的能力,显示出良好的中和特性。单抗HI反应谱表明,2C11、4C8、6E7和5C5分别可以抑制10株H4亚型AIV中9、8、7和7株病毒的血凝反应,且均不能抑制A/鸭/苏州/1/2002毒株的血凝作用。

Development of Monoclonal Antibodies Specific to Hemagglutinin of H4 Subtype Avian Influenza Virus

8-week-old female BALB/c mice were immunized with H4-AIV strain A/Duck/Yangzhou/185/2003 and the spleen cells were collected and fused with Sp2/0-Ag-14 myeloma cells.Hybridoma cells were screened by hemagglutination inhibition(HI)test and the positive clones were subjected to limited dilution for subcloning.4 mAbs against HA of H4-AIV were produced after triple subcloning and named as 2C11,6E7,4C8 and 5C5 re- spectively.All the 4 monoclonal antibodies were tested to be IgG2a subtype and the HI titers were between 1:212 and 1:215.Cross HI test and indirect immunofluorescence showed that the 4 mAbs were specific to H4-AIV as they reacted only with H4-AIV but not with NDV,IBV,EDSv-76 and other AIV subtypes.Virus neutralization test indicated that the 4 mAbs were able to inhibit the pathogenicity of H4-AIV in cell culture.The HI results re- vealed that mAbs 2C11,4C8,6E7 and 5C5 could inhibit 9,8,7 and 7 of 10 H4-AIV isolates respectively,while none of them could inhibit the A/Duck/Suzhou/1/2002 isolate,showing the antigenic difference among the HA pro- teins of the H4-AIV isolates.