抗牛型结核杆菌单克隆抗体的制备、鉴定及其纯化抗原的研究

Production and characterization of monoclonal antibodies against mycobacterium bovis and preliminary study on the purified antigen

用牛结核杆菌超声抗原及纯蛋白衍生物(PPD)免疫BALB/c小鼠,经融合、筛选和克隆化得到9株稳定分泌单克隆抗体(McAb)的杂交瘤细胞株,应用抗原蛋白酶消化试验、抗原过碘酸氧化试验、加成试验及免疫印迹分析等,表明9株McAbs所识别抗原决定簇均对蛋白酶敏感,其中2株的抗原决定簇既存在于蛋白质中,又存在于脂类中。9株McAbs所识别的抗原分子量均为30kDa。4株均作用于同一抗原决定簇,其它McAb各作用于另外互不相同的抗原决定簇。免疫电镜观察,McAb 1A3所作用的蛋白质抗原主要分布在细胞内,少量分布于细菌胞壁。9株McAbs除1株外均显示出很强的特异性,其中4株仅与牛型结核茵反应阳性。利用McAb 1A3通过亲和层析柱纯化牛型结核杆菌PPD后,得到亲和层析纯抗原Ag 1A3,它仅与牛型结核杆菌免疫血清反应阳性,与其它分枝杆菌免疫血清反应阴性;对牛型结核杆菌致敏的豚鼠皮肤反应阳性,而与人型及BCG致敏豚鼠皮肤反应阴性。

In the present experiment, nine hybridoma cell lines were established by fusion of cells from BALB/c mice immunized with mycobacterium bovis sonicated antigen and PPD. The cell lines were capable of secreting monoclonal antibodies ( McAbs ) against mycobacterium bovis and the antigenic determinants recognized by 9 McAbs were shown to be sensitive to protease by antigen digestion test, antigen oxidation test, ELISA and Immunoblot analysis. The results indicated that antigenic determinants recognized by two McAbs existed not only in protein but also in lipid. The antigens recognized by the nine McAbs had a molecular weight of 30 kD and four McAbs recognized the same antigenic determinants. Immunoelectromicroscopy showed that the protein antigen recognized by McAb lA3 distributed mainly in cell and sparsely in the cell wall of mycobacterium. ELISA analysis demonstrated that all McAbs except one was of high specificity and four McAbs bound specifically to mycobacterium bovis. All McAbs were not reactive with nonmycobacterium tested. Affinity chromatography using immunoabsorbents prepared from McAblA3 was employed for the purification of antigens from PPD of mycobacterium bovis and purified antigen lA3 ( Ag1A3 ) was obtained. ELISA analysis showed that AglA3 reacted only to sera immunized with mycobacterium bovis, but not to other mycobacterium. Delayed cutaneous hypersensitivity (DCH) reactions to small concentrations of Ag1A3 was elicited in guinea pigs sensitized to mycobacterium bovis, but not elicited in those sensitized to mycobacterium tuberculosis H37Rv and mycobacterium bovis BCG.