【摘要】 目的建立产生抗人游离前列腺特异抗原单克隆抗体(f-PSA m Ab)的细胞株,获得抗体后建立抗人f-PSA化学发光免疫分析法(CLIA)并进行应用。方法采用杂交瘤技术获得2株稳定分泌抗f-PSA m Ab的细胞株,将细胞株用转瓶扩大培养并将上清液采用亲和纯化法获得抗体,ELISA测定抗体效价、特异性及表位,表面等离子共振法测抗体亲和力;建立双抗体夹心CLIA,采用标准曲线定量法对该体系进行分析性能评估,同时对426例临床血清样本进行检测[其中130例的总PSA水平在(4～10) ng/mL,为"诊断灰区"],评价该试剂与罗氏试剂的检测一致性。结果获得抗人f-PSA m Ab的杂交瘤细胞株(f-10-1、f-14-1)。用f-10-1和f-14-1建立的CLIA的检测范围为(0. 1～30) ng/mL,灵敏度为0. 05 ng/mL,检测结果的相对偏差均在±5%内,与癌胚抗原(CEA)、甲胎蛋白(AFP)及结合态PSA(c-PSA)无交叉反应,该试剂与罗氏试剂相关系数达到0. 99,灰区样本阳性符合率、阴性符合率、总符合率均高于90%。结论成功制备了抗人f-PSA m Ab,建立了特异性定量检测人f-PSA的双抗体夹心CLIA。更多还原

【Abstract】 Objective Construction of monoclonal antibody cell lines against human free prostate specific antigen( f-PSA),and establish double antibody sandwich CLIA for human f-PSA. Methods Two hybridoma cell lines secretinganti-f-PSA m Ab were obtained by hybridoma technique. The cell lines were expanded by spinner bottles and purified byaffinity purification,from which the antibodies were tested for antibody titers,specificity,epitopes by ELISA and affinity bysurface plasmon resonance. Establishment,analysis and performance evaluation of a double m Abs sandwich CLIA systemusing standard curve quantitative method. In total 426( 130 gray area) clinical specimens were used for system comparisonbetween our assay and Roche f-PSA assay. Results anti-f-PSA( f-10-1、f-14-1) m Abs were obtained. The CLIA systemwith detection range of 0. 1-30 ng/mL,sensitivity of 0. 05 ng/mL,relative detection deviation of ± 5% and not cross-reactwith the tumor marker AFP,CEA and c-PSA. The correlation coefficient of our reagent with Roche’s was 0. 99. The positivecoincidence rate,negative coincidence rate and total coincidence rate of gray area specimens were all higher than 90%.Conclusion m Abs against human f-PSA were successfully prepared,and the double m Abs sandwich CLIA for specificquantitative detection of human f-PSA was established.更多还原