目的 :制备抗艰难梭菌A毒素的单克隆抗体 (mAb)并鉴定其特性。方法 :用纯化的艰难梭菌A毒素免疫BALB/c小鼠 ,将免疫小鼠的脾细胞与骨髓瘤细胞Sp2 / 0融合 ,采用间接ELISA筛选杂交瘤细胞。用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析 ;用Westernblot检测mAb的特异性。结果 :得到 6株杂交瘤细胞株 ,5C10株细胞分泌的mAb为IgG2a ,4B5和 8A1株细胞分泌的mAb为IgG1,其他 3株细胞mAb (2H7、3E9和 6G8)均分泌IgM。中和试验表明 ,所有的mAb均无中和活性。腹水mAb的效价均在 10 -4以上 ,其中mAb 2H7、6G8、5C10、4B5和 8A1具有共同的表位 ,而mAb 3E9识别的位点与其他 5株不同。mAb 8A1和 4B5的相对亲和力>10 5,其他 4株mAb的相对亲和力 >10 4。在非变性条件下 ,PAGE后Westernblot的结果显示 ,6株mAb均可与相对分子质量 (Mr)为 5 5× 10 4的A毒素产生反应 ;而在变性条件下 ,还原与非还原SDS PAGE后Westernblot均显示 ,6株mAb均可与Mr 为 5× 10 4～ 2 4× 10 4的A毒素产生反应。结论 :6株杂交瘤细胞株均能分泌抗艰难梭菌A毒素的特异性mAb ,为艰难梭菌A毒素的研究提供了有利的工具 Objective: To prepare monoclonal antibodies against C. difficile A toxin (mAbs) and to characterize them. Methods: BALB / c mice were immunized with purified C. difficile toxin. The splenocytes of immunized mice were fused with Sp2 / 0 of myeloma cells. The hybridoma cells were screened by indirect ELISA. The titer, relative affinity and epitope analysis of mAb ascites were detected by ELISA; the specificity of mAb was detected by Western blot. RESULTS: Six hybridoma cell lines were obtained. The mAb secreted by 5C10 cells was IgG2a. The mAb secreted by 4B5 and 8A1 cells was IgG1. The other 3 mAbs (2H7, 3E9 and 6G8) secreted IgM. Neutralization tests showed that all mAbs had no neutralizing activity. The titer of ascites mAb was more than 10 -4, of which mAb 2H7, 6G8, 5C10, 4B5 and 8A1 shared the same epitope, while mAb 3E9 recognized the same locus as the other 5 strains. Relative affinity of mAb 8A1 and 4B5> 10 5, relative affinity of the other 4 mAbs> 10 4. Under non-denaturing conditions, the result of PAGE after Westernblot showed that all 6 mAbs could react with A toxin with relative molecular mass (Mr) of 5 5 × 10 4; while under denaturing conditions, Western blot showed that all 6 mAbs could react with Mr toxin A of 5 × 10 4 ~ 2 4 × 10 4. CONCLUSION: All six hybridoma cell lines can secrete specific mAbs against Clostridium difficile toxin A, which provides an advantageous tool for the study of C. difficile A toxin