检验人精浆特异蛋白P30免疫胶体金试剂条的研制

目的制备用于检验人精浆特异蛋白P30-主要是来自法医学案件的免疫胶体金层析试剂条.方法选取针对不同抗原决定簇的抗P30单克隆抗体细胞株,并制备其小鼠腹水,分离纯化单克隆抗体.制备胶体金并以纯化抗体包被,制成免疫胶体金,以免疫胶体金浸泡玻璃纤维.选取适宜的硝酸纤维素膜并于其上不同位置以未金标的另一株P30单克隆抗体和羊抗鼠IgG包被.搭建试剂条并检测其灵敏度和特异性.结果所制成的试剂条灵敏度至少可达4ng/ml;对6人份混合的人精液物质在稀释20万倍后仍出阳性结果,且无非特异性反应.结论检验人精浆特异蛋白P30的免疫胶体金试剂条可对嫌疑人精物质做出排查,有利于法医物证检验.

The study and preparation of immuno-colloidai gold test strips for detection of human seminal specific protein P30

Objective To prepare a kind of immuno-colloidal gold chromatographic test strips to detect the human seminal specific protein P 30 from evidential substances in the forensic cases.Methods The mono clonal cell strains,which produce antibodies against various epitopes of human seminal specific protein P 30 as the antigen,were selected and their relevant monoclonal antibody proteins were isolated and purified from the ascites secreted by mice injected with the cells producing antibody proteins against each epitope.Colloidal gold was prepared and coupled with one of the purified antibody proteins so that the immuno-colloidal gold was obtained.Afterwards a piece of glass fiber was saturated with the new-prepared im muno-colloidal gold and then dried.A suitable kind of nitrate fiber membrane was chosen and coated at separate two places on it with the goat or sheep ' s anti-mouse IgG and another antibodies against an epi tope on the P 30 protein different from against which the antibodies coupled with colloidal gold are.Im muno-colloidal gold test strips were fabricated and their lowest detection limit,specificities were measured.Results The detection limit of the colloidal gold test strips is at least up to4ng/ml and a positive result is stilled observed when detected to a200000-fold dilution of mixed human seminal fluid from6persons,without non-specificity,that is,the strips only having reactions with the P 30 protein.Conclusions The col loidal gold test strips detecting human seminal specific protein P 30 can be applied in the determination of suspected human seminal substances in forensic examination.