抗恶性疟原虫谷氨酸脱氢酶单克隆抗体的研制与胶体金免疫层析方法的建立

Preparation of a monoclonal antibodies against Plasmodium falciparum glutamate dehydro- genase and establishment of colloidal gold-immunochromatographic assay

目的制备抗恶性疟原虫谷氨酸脱氢酶(GDH)的单克隆抗体(McAb),将其标记胶体金,建立一种诊断恶性疟的胶体金免疫层析方法.方法用基因重组的GDH蛋白免疫Balb/C小鼠,采用杂交瘤细胞技术制备McAb,测定其免疫球蛋白亚类及其亲和力.用protein-G亲和层析纯化抗体并用胶体金标记,建立胶体金免疫层析方法,选择最佳反应模式对疟疾血样进行检测.结果筛选出6株能稳定分泌抗GDH单克隆抗体的杂交瘤细胞株,单抗亚类均为IgG1,亲和常数(κ)介于1×10-8～2.82×10-10.以镜检法为参照,建立的胶体金免疫层析方法检测恶性疟的敏感性为86.66%,特异性为96.43%.结论建立的胶体金免疫层析方法检测恶性疟快速、简便、可靠,有望开发成为恶性疟快速诊断试剂盒.

Objective To prepare a monoclonal antibodies (mAbs) against glutamate dehydrogenase (GDH) of Plasmodium falciparum (FCC1/HN strain) and establish colloidal gold-immunochromatographic assay (GICA) for diagnosis of Plasmodium falciparum malaria. Methods Recombinant GDH was used to immunize Balb/C mice and the mAbs against GDH were prepared using hybridoma technique followed by identification of IgG isotype and its affinity. Protein-G affinity chromato- graphy was employed to purify the antibodies, which were labeled with colloidal gold for establishment of GICA for Plasmodium falciparum detection. Results Six mAbs were obtained and identified as IgG1(κ) of IgG isotypes with affinity constants (Kaff) ranging from 1×10-8 to 2.8×10-10. GICA had a sensitivity of 86.66% and specificity of 96.43% for Plasmodium falciparum detection compared with routine microscopic examination. Conclusion The established GICA is rapid and accurate for Plasmodium falciparum detection with such potential utility as for instant diagnosis of Plasmodium falciparum malaria.