抗鸡γ-干扰素单克隆抗体的制备及定量ELISA方法的建立

Generating a monoclonal antibody against chicken interferon gamma for the establishment of quantitative ELISA

将原核表达重组鸡γ-干扰素（rChIFN-γ）以100μg/只剂量免疫8周龄BALB／c小鼠，4次免疫后进行细胞融合，以昆虫细胞表达重组鸡γ-干扰素为检测抗原，筛选出阳性克隆株，经有限稀释，获得1株稳定分泌抗鸡γ-干扰素单克隆抗体的杂交瘤细胞株，分泌抗体亚型为IgG1型，轻链为K链，命名为G1株。Western blot检测显示，该单克隆抗体与原核表达和昆虫细胞表达的rCbIFN-γ均具有良好的免疫反应原性。昆虫细胞表达rChIFN-γ经该G1单克隆抗体作用后，失去抑制水疱性口炎病毒（VSV）在鸡胚成纤维细胞（CEF）复制的能力？利用纯化的单克隆抗体和昆虫细胞表达rChIFN-γ建立相对定量ELISA检测方法，取吸光度值（Y）对昆虫细胞表达rChIFN-γ的抗病毒活性单位对数（X）作回归曲线，回归方程为Y=0．1164^＊X-0．1281，回归系数R=0.9539，具有良好的线性关系.

BALB/c mice were immunized four times with recombinant chicken interferon-gamma expressed in E.co/i at 100 μg per mice and cell fusion was conducted according to standard procedure. Positive hybridoma clones were screened by indirect ELISA. A hybridoma that stably secreted specific monoclonal antibody against chicken gamma interferon was developed. The monoclonal antibody, named Gl strain belonged to IgG1 isotype and had K chain. Western blot showed that G1 strain reacted strongly with recombinant chicken interferon-gamma expressed either in baculovirus expression systerm or in E.coli. However the recombinant chicken interferon-gamma could not inhibit the replication of the VSV＊GFP in CEF cells. Relative quantitative ELISA was successfully established using the monoclonal antibody and recombinant chicken interferon-gamma expressed in si9 cell.