抗卵巢癌相关抗原单克隆抗体的制备及免疫分析方法的建立

Preparation of Monoclonal Antibodies Against Ovarian Associated Antigen and Establishment of Immunoradiometric Assay

目的 研究从卵巢浆液性乳头状囊腺癌患者的腹腔积液中 ,纯化获得携带CA 12 5抗原决定簇的卵巢癌相关抗原。以此为免疫原 ,制备单克隆抗体 ,并建立放射免疫分析方法并应用于临床中。方法 腹腔积液经高氯酸分离 ,凝胶层析及亲和胶蓝柱层析纯化CA 12 5相关抗原。应用细胞融合技术 ,抗体采用ELISA方法、免疫印记技术进行鉴定。单克隆抗体经碘原法标记放射性核素1 2 5I ,初步建立 1种碘标固相双位点夹心模式的免疫放射分析法。结果 腹腔积液中CA12 5相关抗原经纯化 ,其比活度达 1.1× 10 6 U mg。共获得 6株稳定分泌抗体的杂交瘤细胞株 ,采用 2株配对最佳的抗体 ,初步建立 1种碘标免疫放射分析法。该方法可测下限为 1.2U ml ,批内变异系数为 4.4% ,批间变异系数为 9.6% ,平均回收率为 97.0 %。初步临床应用的结果表明在正常人血清中CA 12 5相关抗原平均值为 (2 .14 7± 3 .0 92 )U ml。在 60例卵巢癌患者中 ,平均值为 (10 6.5± 187.5 )U ml ,两者比较有非常显著性差异 (P <0 .0 1)。结论 以患者腹腔积液中纯化的CA12 5相关抗原制备单克隆抗体 ,并建立放射免疫分析方法 ,初步应用于临床血清学测定中 。

Objective To study soluble ovarian associated antigen purified from ascitic fluid of a patient with ovarian adenocarcinoma and prepare the monoclonal antibody against this antigen,establish a method of Immunoradiometric assay(IRMA) and evaluate the preliminary clinical use.Methods CA125 associated antigen was purified by trichloroacetic acid precipitation followed by chromatography.Monoclonal antibodies(McAb) were generated by cell hybridization,detected by ELISA and Western blot method.Iodin labeled monoclonal antibody was used to set up a solid phase sandwich IRMA.Results The ratio of CA125 antigen was 1.1×10 6 U/mg(protein) after purification.Six stable hybridomas were obtained by cell hybridation.Two monoclonal antibodies were selected to develop a Iodio labeled IRMA.The lowest limit of measure was 1.2 U/ml,with a 4.4% of within batch CV% and 9.6% of between batch CV%.The average recovery was 97.0%.The average value of CA125 associated antigen was(2.147±3.092) U/ml in normal women and (106.5±168.5) U/ml in 60 patients with ovarian carcinoma.There was significant difference between normal individuals and cancer patients( P <0.01).Conclusion The Monoclonal antibodies can be generated by the CA125 tumor associated antigen isolated from ascitic fluid.The immunoradiometric assay,established by the self produced antibodies,is valuable for the preliminary clinical use.