肝癌组织γ-谷氨酰转移酶单克隆抗体制备及ELISA方法的建立

目的应用纯化的人肝癌组织中蔓陀罗凝集素(DSA)强结合的γ-谷氨酰转移酶(GGT)制备单克隆抗体(McAb),并建立ELISA检测方法。方法应用亲和色谱法纯化肝癌组织中DSA强结合的GGT;采用单克隆抗体技术获得其McAb;protein A-Sepharose亲和色谱法纯化McAb,过碘酸钠法标记HRP法后建立ELISA方法。结果获得5株能特异性识别DSA强结合GGT的McAb细胞株,其中3株细胞所分泌McAb具有较高的特异性,应用所获得McAb进行HRP标记后,建立的ELISA方法,其检测灵敏度为10μg/L,批内及批间平均变异系数为8.9%和11.5%。结论成功制备了DSA强结合的GGT McAb杂交瘤细胞系,并建立了ELISA免疫学检测方法,为临床应用打下基础。

Preparation of monoclonal antibody against γ-glutamyltransferase of primary hepatic cancer tissue strongly bound to datura stramonim(DSA) lectin and establishment of ELISA method

Purpose To prepare monoclonal antibody(McAb) against γ-glutamyltransferase(GGT) of primary hepatic cancer tissue strongly bound to datura stramonim(DSA) lectin and establish an ELISA method.Methods The GGT from primary hepatic cancer tissue strongly bound to DSA Lectin(DSA-GGT)was purified by Lectin affinity chromatography and the McAb was obtained by monoclonal antibody technique;the McAb was purified by the protein A-sepharose affinity chromatography and an ELISA examination method was established after the McAb labelled to horseradish peroxidase(HRP) by sodium periodate method.Results Results 5 strains which can produce the McAb specifically recognizing γ-glutamyltransferase strongly bound to DSA wereacquired,and the specificity of McAbs was high;the sensitivity of ELISA method which was built up after the purified McAb effectively labeled with HRP was 10 μg/L.The average intra assay and inter assay precision were 8.9% and 11.5% respectively.Conclusion the McAb hybridoma cell lines against γ-glutamyltransferase strong binding with DSA were successfully prepared and the examination method of the ELISA was successfully built up,which lay the foundation for the clinical application.