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目的:对比G200凝胶过滤、反向亲和层析、DEAE离子交换等不同的方法纯化孕妇血清中的妊娠相关蛋白A(PAPP-A),为下一步的临床应用研究奠定基础。方法:把正常男性血清过G200凝胶过滤纯化后免疫家兔得到兔抗人抗体,连接溴化氰活化G25凝胶制备反向免疫层析柱。收集足月孕妇血清,离心后依次应用G200凝胶过滤、反向亲和层析及离子交换层析柱纯化。用ELISA检测试剂盒检测各个洗脱峰的PAPP-A活性,SDS-PAGE检测纯化蛋白的纯度。结果:稀释兔抗人血清抗体至浓度为1∶16,双向扩散有明显沉淀线,可以用于制备抗体,蛋白A亲和层析洗脱pH3.0洗脱峰,G200凝胶过滤第一个洗脱峰,反向亲和层析的0.1 mol/LPBS洗脱峰,离子交换层析的0.45 mol/L NaCl洗脱峰中PAPP-A活性均较高。SDS-PAGE结果显示,孕妇血清用G200凝胶过滤处理后杂蛋白条带较多,反向亲和层析或者DEAE分别处理后同样存在杂蛋白条带,三种纯化方法结合应用杂蛋白条带明显减少,Western blot鉴定显示三种方法结合应用后纯化蛋白样品条带单一。结论:本研究用三种层析方式相结合的方法得到纯度较高的PAPP-A抗原,鉴定显示可以到达制备单克隆抗体(mAb)的要求,为mAb的制备以及酶联免疫试剂盒的建立奠定了基础,使PAPP-A在临床应用方面前景广阔。
OBJECTIVE: To purify PAPP-A from maternal serum by different methods, such as G200 gel filtration, reverse-phase affinity chromatography and DEAE ion exchange, which laid the foundation for further clinical application. Methods: Rabbit anti-human antibodies were obtained by immunizing rabbits with normal male serum after G200 gel filtration and purification. Reverse immunochromatographic column was prepared by linking cyanogen bromide activated G25 gel. Collect full-term pregnant women serum, followed by centrifugation G200 gel filtration, reverse affinity chromatography and ion exchange chromatography purification. The activity of PAPP-A in each elution peak was detected by ELISA kit, and the purity of purified protein was detected by SDS-PAGE. Results: The rabbit anti-human serum antibody was diluted to a concentration of 1:16, and two-dimensional diffusion was obvious precipitation line, which can be used for the preparation of antibody. Protein A affinity chromatography eluted at pH 3.0, The highest peak of PAPP-A activity was found in the eluted peak of 0.45 mol / L NaCl by ion exchange chromatography. SDS-PAGE results showed that pregnant women serum G200 gel filtration treatment more hybrid protein bands, reverse affinity chromatography or DEAE treatment after the same presence of hybrid protein bands, three purification methods combined with the application of hybrid protein bands Significantly reduced, Western blot identification showed that the combination of three methods after purification of protein samples with a single band. Conclusion: In this study, the purity of PAPP-A antigen was obtained by a combination of three chromatographic methods. The identification of mAb showed that the preparation of monoclonal antibody (mAb) can be achieved. The preparation of mAb and the establishment of enzyme-linked immunosorbent assay kit Laid the foundation for the future of PAPP-A in clinical application.