抗疟原虫单抗M26-32 IgM的单体化及其免疫学特性研究

Preparation of subunit and immunology characteristics of monoclonal antibody M26-32 against pan species malaria parasite

目的改造IgM类抗疟原虫单抗M26-32为单体IgM亚单位,胶体金标记天然和改造后两种M26-32用于检测疟原虫抗原。方法复苏培养M26-32杂交瘤细胞,接种BALB/c小鼠,收集含有单抗的腹水,用商品HiTrap IgM HP柱纯化腹水中的IgM抗体,并用L-半胱氨酸裂解IgM类M26-32单抗成单体IgM亚单位。经间接免疫荧光检定两种抗体的效价,并用胶体金标记,分别检测恶性疟原虫和间日疟原虫可溶性抗原。结果接种20只小鼠,共采集约80mL单抗腹水,经HiTrap IgM HP柱纯化,获得了纯度较高的M26-32单抗。经L-半胱氨酸裂解后,Native电泳显示天然IgM被裂解为单体亚单位,IFA结果显示,亚单位M26-32与天然五聚体IgM滴度无差别。经胶体金标记后,两种金标抗体均分别能检测1∶100稀释的恶性疟原虫和1∶10稀释的间日疟原虫可溶性抗原。结论建立了L-半胱氨酸裂解IgM类M26-32单抗为单体IgM亚单位及其胶体金标记的方法,为利用M26-32进一步制备相应快速诊断工具奠定基础。

The pentamer IgM rnonoclonal antibody M26-32 was modified into subunit and taken advantage of immunogold labeling of natural and modified M26-32 antibodies for detection of malaria antigens. After thawing and cultivation, M26-32 hybridoma was inoculated in BALB/c mice intraperitoneally and the ascitic fluid was collected. M26-32 was purified using HiTrap IgM Hp column, and the IgM antibody was lysed into IgM subunit with L-cysteine. After verification of titers by IFA, natural and L-cysteine lysed M26-32 antibodies were labeled by immunogold, and tested with both Plasmodium falciparum and Plasmodium vivax soluble antigens. A total of 20 mice were used and about 80 mL ascitic fluid was collected. Purified M26-32 antibody was obtained by using HiTrap IgM HP column. Native PAGE showed that L-cysteine could lyse the pentamer IgM into IgM subunit. IFA results showed that no titer difference between two types of M26-32 monoclonal antibody. After labeling with immunogold, both two types M26-32 could recognize 1 ： 100 diluted Plasmodium falciparum antigens and 1 ： 10 diluted Plasmodium vivax antigens. A method for modifying pentamer IgM M26-32 monoelonal antibody into IgM subunit by using L-cysteine have been proved, and a method for labeling both types M26-32 with immunogold have been established, providing useful information for designing malaria RDT based on M26-32 monoclonal antibody.