摘要目的 探讨亲和层析法纯化抗前胃泌素释放肽(PGRP)单克降抗体(MAb)的效果,为该法纯化其他抗体提供数据依据.方法 采用蛋白A-琼脂糖亲和层析法纯化含有MAb的腹腔积液,并用SDS-PAGE和酶联免疫吸附(ELISA)法分别检测其纯度和效价,用流式细胞术和免疫组织化学法榆测其对小细胞肺癌细胞株NCI-H446的组织切片的生物功能.结果 亲和层析法纯化前小鼠腹腔积液蛋白质质量浓度平均为23.62 mg/ml;纯化前、后蛋白总量分别为148.79和146.67 mg,回收率为98.58%.腹腔积液中MAb质量浓度平均为5.21 mg/ml;纯化的MAb纯度达95%以上.纯化后抗体免疫学活性均高于纯化前,提高了6.90～15.40倍.结论 蛋白A-琼脂糖亲和层析法可快速、高效地从小鼠腹腔积液中纯化抗PGRP MAb,且抗体具有较高的纯度,免疫学活性明显提高,能够选择性的和小细胞肺癌细胞的PGRP结合.

abstractsObjective To explore the effect of purification on monoclonal antibody (MAb) against PGRP by Protein A-Sepharose affinity chromatography, and to provide some based data for the purification of other antibody using the same method. Methods The ascites which include MAb was purified by Protein A-Sepharose affinity chromatography. The purity and activity of MAb was tested by SDS-PAGE and ELISA. The biological function was identified by flow cytometer and immunohistochemistry. Results The average concentration of protein in ascites before purification is 23.62 mg/ml. Before and after purification, the total protein is 148.79 mg and 146.67 mg, respectively. The recovery coefficient of protein is 98.58%. The concentration of MAb in ascites is 5.21 mg/ml averagely. The MAb purity is more than 95 %. The immunoactivity of purified antibody is higher than that of unpurified antibody. Conclusion The purity of MAb against PGRP purified by Protein A-Sepharose affinity chromatography is very high. The immunoactivity of purified antibody is higher than that of unpurified antibody. So the ProteinA-Sepharose affinity chromatography is a rapid, convenient and reliable method for the purification of MAb Against PGRP.