定量检测肝细胞生成素Cn的双抗体夹心ELISA检测方法的建立

摘要

Objective To establish a double antibody sandwich ELISA method for quantitive detection of hepatopoietin Cn(HPPCn).Methods Female BALB/c mice were immunized with recombinant HPPCn.The splenocytes and Sp2/0 cells were fused with PEG-1500.The positive clone was identified through indirect ELISA and then subcloned by limited dilution.The property of depurative anti-HPPCn monoclonal antibodies (mAb) was identified through immunohistochemistry and Western blotting.Then the pair bonds of the eight strains of antibodies were of detected by gliding chip method.The optimal working concentration of coating antibodies and enzyme labeled antibody were determined by square matrix titrimetry.The depurative HPPCn antigen was used as the standard substance to establish the standard curve.Results The eight strains of hybridoma cells which stably secreted anti-HPPCn antibodies were obtained and they were Ab_65-29-6,Ab_3-3-3,Ab_9-34-1,Ab_4-8-12,Ab_7-8-10,Ab_2-30-23,Ab_ll-ll-12 and Ab_12-27-23W 2-D5.The best pair bonds were Ab_2-30-23 and Ab_4-8-12 verified by the antibodies pair bond detection.The optimal working concentration of coating antibody Ab_65-29-6 and recognition antibody Ab_7-8-12 was respectively 2.5 μg/ml and 1:2000.Conclusion Monoclonal antibodies which can specificallydetect the human HPPCn protein are prepared and a double antibody sandwich ELISA method used for detecting HPPCn is established.%目的 建立定量检测肝细胞生成素Cn (HPPCn)的双抗体夹心ELISA方法.方法 利用杂交瘤方法获得8个抗HPPCn单克隆抗体(mAb),并行SDS-PAGE和Western印迹对纯化抗体特性进行鉴定.利用镀金芯片法对8个抗体进行配对检测.通过方阵滴定法确定包被抗体和酶标抗体的最适工作浓度.以纯化的HPPCn抗原为标准品建立标准曲线.结果 得到了8株稳定分泌抗人HPPCn抗体的杂交瘤细胞株,分别为Ab 65-29-6,Ab_3-3-3,Ab_9-34-1,Ab_4-8-12,Ab_7-8-10,Ab_2-30-23,Ab_1 1-11-12和Ab_12-27-23.8个抗体均能够特异结合HPPCn抗原,其中Ab_65-29-6与细胞中HPPCn结合的特异性最好.经抗体配对检测证实最佳配对组合为:Ab_2-30-23和Ab_4-8-12.包被抗体Ab_2-30-23和识别抗体Ab 4-8-12的最适工作浓度为2.5 μg/ml,最适稀释度为1∶2000,标准曲线检测的线性范围为5.0～60 ng/ml.结论 制备了可特异性检测人HPPCn蛋白的单克隆抗体,并建立了一种可用于定量检测人HPPCn的双抗体夹心ELISA方法.