抗AIV(H9)独特型单克隆抗体的制备及其免疫原性的研究

Preparation of the anti-AIV(H9)-idiotypic monoclonal antibody and the investigation on its immunogenicity

用H9N2亚型AIV作为抗原免疫接种SPF鸡制备高免血清,用饱和硫酸铵法从该血清中提取免疫球蛋白G(IgG)。以该IgG制备弗氏佐剂疫苗免疫SPFBALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS-1在聚乙二醇(PEG)作用下融合,采用间接ELISA检测法,筛选获得3株(2H1D1、2H1G4、3H2D7)分泌特异性单克隆抗体的杂交瘤细胞。3株融合细胞培养上清液中的单克隆抗体效价均为2-4,小鼠腹水中的单抗效价是细胞培养上清液中的300～500倍。经检测表明,3株细胞系产生的单克隆抗体仅与相应的鸡抗H9N2亚型AIV血清发生特异性反应。用2H1D1分泌的抗独特型单克隆抗体制备疫苗与抗AIV灭活疫苗同时免疫SPF鸡收获的高免血清与AIV在MDCK细胞上进行中和实验,中和效价分别为10-1.8/10μl、10-1.6/10μl。由此表明,该抗独特型抗体疫苗具有良好的免疫原性。

The SPF chickens were inoculated with avian influenza virus, subtype H9N2 to prepare the high titer serum antibodies with the subsequent purification with precipitation by saturated （NH4）2SO4. Thus prepared IgG emulsified with Freund＇s adjuvant was used as antigen to immunize SPF BALB/c mice, and the spleen cells of the immunized mice were fused with myeloma cells NS-1 by PEG to prepare the hybridoma cells. By using this procedure, three strains of hybridoma cells secreting the specific monoclonal antibodies could be detected by indirect ELISA assay, and they were designed as 2H1D1, 2H1D4, 3H2D7 respectively. The antibody titer of the monoclonal antibody in the supernatant in the cell cultures of these 3 strains of hybridoma cells was 2-4, and that from the ascetic fluid was 300-500 times than that of the supernatant of the cell cultures as demonstrated by indirect ELISA. It was also found that the monoclonal antibodies thus prepared could react specifically with chicken anti-AIV subtype H9N2 serum. Two high titered anti-idiotypic antibodies secreted by the 2H1D1 showed excellent neutralization reaction upon AIV on MDCK cells, and the PD50 were 10^-1.8/10 μl and 10^-1.6/10μl respectively. It demonstrates that the anti-idiotypic antibody vaccine shows superior immunogenicity and can induce more neutral antibodies in SPF chickens than the inactive vaccine.