双单抗夹心ELISA方法检测EPF活性

目的 建立双单抗夹心酶联免疫吸附试验法用于测定早孕因子(EPF)。方法筛选可稳定分泌抗EPF单克隆抗体的细胞株，制备腹水型单抗(McAb)并分离纯化，用改良碘化钠法进行辣根过氧化物酶(HRP)标记单抗，分析McAb的结合位点，通过抗体配对试验选择合适的捕获和检测McAb，建立双McAb-ELISA检测方法。结果筛选出二株稳定分泌抗EPF抗体的单克隆细胞株；两株单抗识别不同的抗原表位；双McAb夹心ELISA法测EPF活性灵敏度可达0．01udml，线性测定范围在0．01～10ug/ml，2份同批血清标本分别重复8次测定，平均批内变异系数为7．7％，2份不同批血清分别重复6次测定，平均批间变异系数为5．8％。建立的双McAb-ELISA法灵敏度90．2c％和特异度为92．8％。结论 建立的双McAb夹心ELISA法，有优异的灵敏度和特异度，稳定性强，而且简便、快速，适用于血清EPF检测，并有一定的临床应用前景。

Establishment of EPF -McAb-ELISA in Measurement of EPF activity

Objective To develop a double monoclonal antibodies sandwhich ELISA assay for the detection EPF. Methods The hybridoma cell lines that secrete monoclonal antibodies against EPF were screened. Ascitic fluid containing the monoclonal antibodies against EPF was obtained by intraperitoneal injection of the hybridoma cells. Purified monoclonal antibodies were labeled with HRP by sodium iodide method. Capture and sandwich McAbs were selected by sandwich ELISA matched with each other. A double EPF-McAb sandwich ELISA method for measurement EPF activity was then established. Results Two hybridoma cell lines that secrete monoclonal antibodies against EPF were obtained. The lowest threshold of detection was 0.01ug/ml, and the calibration curves for EPF were linear ranged between 0.01-10.0ug/mL, when two serum plasma samples from one batch were assayed repeatedly for 8 times and another 2 samples from other batch were assayed repeatedly for 6 times ,the average intra-assay and the inter-assay CV were 7.7% and 5.8% respectively. The sensitivity and specificity of this method were 90.2% and 92.8%. Conclusion This method is simple and rapid with high sensitivity and stability,which is valid for measurement of the serum EPF activity.