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目的制备重组人碱性成纤维细胞生长因子(recombinant human basic fibroblast growth factor,rhbFGF)单克隆抗体,鉴定其特性,建立双抗体夹心ELISA检测方法。方法以rhbFGF为免疫原,免疫Balb/c小鼠,通过细胞融合技术建立能稳定分泌抗rhbFGF杂交瘤细胞株,制备抗rhbFGF单克隆抗体,采用Ig亚类ELISA试剂盒鉴定抗体亚类,间接ELISA法检测抗体效,Western blot鉴定抗体特异性。HRP标记McAb并建立夹心ELISA检测方法。结果获得2株(分别命名2D3、5F7)可分泌特异性McAb的强阳性细胞株,腹水抗体效价在10-5以上,IgG亚类均为IgG1,轻链为K链。Western blot证明2株McAb特异性良好,双抗体夹心ELISA检测rhbFGF最低检测限达到2 ng/ml。结论成功制备高效价的抗rhbFGF单克隆抗体,建立抗rhbFGF双抗体夹心ELISA定量检测方法。
Objective To prepare recombinant human basic fibroblast growth factor (rhbFGF) monoclonal antibody to identify its characteristics and establish a sandwich ELISA. Methods Balb / c mice were immunized with rhbFGF as an immunogen. The cell lines secreting anti - rhbFGF were established by cell fusion technique. Monoclonal antibodies against rhbFGF were prepared. Antibody subclasses were identified by ELISA, and indirect ELISA Antibody detection efficiency, Western blot identification of antibody specificity. HRP labeled McAb and established sandwich ELISA detection method. Results Two strains (named as 2D3 and 5F7) secreting specific McAb were obtained. Ascites antibody titer was above 10-5, IgG subclass was IgG1, and light chain was K chain. The results of Western blot showed that McAb 2 McAb was specific and the detection limit of rhbFGF by double antibody sandwich ELISA was 2 ng / ml. Conclusion High titer anti-rhbFGF monoclonal antibody was successfully prepared and a sandwich ELISA assay was established.