重组人碱性成纤细胞生长因子单克隆抗体的制备、鉴定及初步应用

Preparation,identification and preliminary application of monoclonal antibodies against recombinant human basic fibroblast growth factor

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。

This study aimed to prepare and identify monoclonal antibodies against recombinant human basic fibroblast growth factor,which will be used in the establishment of a sandwich ELISA method.Balb/c mice were immunized with rhbFGF,and then positive clones of McAbs were obtained after cell fusion and hybridoma selection.The ascites titers,the subclasses type,and the specificity of McAbs were tested by Ig subtype ELISA kit,indirect ELISA method and Western blot analysis,respectively.Moreover,optimization of the sandwich enzyme-linked immunosorbent assay was studied.As results,two of positive hybridomas were screened out,named 2D3 and 5F7 whose ascites titers of McAbs were over 1：105.It proved that the Ig subtypes of 2D3 and 5F7 McAbs were IgG1,and the light chain was kappa.Western bolt identification suggested that either McAbs of 2D3 or 5F7 could react specifically with rhbFGF.The results indicated that two of high titer,specific McAbs against rhbFGF are achieved and can applied to the development of a quantitative double-antibody sandwich ELISA method to detect rhbFGF.