Schoepe H, Wieler L H, Bauerfeind R, Schlapp T, Potschka H, Hehnen H R, Baljer G
Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universität, Giessen, Germany.
Microb Pathog. 1997 Jul;23(1):1-10. doi: 10.1006/mpat.1996.0123.
Three murine monoclonal antibodies (MAbs 3B4, 1E8, 1F9) were produced by fusion of X63-Ag8.653 myeloma cells and splenocytes of mice immunized with glutaraldehyde-inactivated alpha-toxin of Clostridium perfringens. All MAbs belonged to the immunoglobulin G (IgG) class and possessed a kappa light chain. All the MAbs were specific for alpha-toxin of C. perfringens as demonstrated by immunoblotting experiments performed with culture supernatants of C. perfringens, C. bifermentans, C. sordellii, and Bacillus cereus. Competition analysis in an ELISA revealed that the MAbs recognized different epitopes on the alpha-toxin molecule. In an immunoblot assay based on a recombinant protein expressed in Escherichia coli, the binding site of MAb 1E8 but not those of MAbs 3B4 and 1 F9 were mapped to the COOH-terminal fragment of alpha-toxin (aa 248-370). To prove the neutralizing potential of the MAbs, alpha-toxin was preincubated with MAbs and subsequently tested for its lecithinase activity in an egg yolk diffusion turbidity (EYDT) assay, its hemolytic activity in a hemolysis test, and its lethal effect on mice after intraperitoneally administration. When the MAbs were tested individually, neutralization was only seen in the EYDT assay, where MAb 3B4 completely abolished the lecithinase activity of alpha toxin. However, when MAbs 3B4 and 1 E8 were used in combination, they acted synergistically and inhibited the lysis of rabbit erythrocytes in vitro. The same mixture of MAbs was also able to completely neutralize the lethal effect of three LD50 of alpha-toxin on Balb/c mice. Our results suggest that the alpha-toxin molecule contains several domains which are differently involved in the various activities of the toxin. We conclude that the hemolytic domain(s) of alpha-toxin is (are) identical with or very closely located to the domain(s) that cause the mouse lethal effect. The lecithinase activity may be involved in the mechanisms of hemolysis and mouse lethality but appears not to be the only determinant.
通过将X63 - Ag8.653骨髓瘤细胞与用戊二醛灭活的产气荚膜梭菌α毒素免疫的小鼠脾细胞融合，制备了三种鼠单克隆抗体（单克隆抗体3B4、1E8、1F9）。所有单克隆抗体均属于免疫球蛋白G（IgG）类，且具有κ轻链。用产气荚膜梭菌、双酶梭菌、索氏梭菌和蜡样芽孢杆菌的培养上清液进行免疫印迹实验表明，所有单克隆抗体对产气荚膜梭菌α毒素具有特异性。酶联免疫吸附测定（ELISA）中的竞争分析显示，这些单克隆抗体识别α毒素分子上不同的表位。在基于大肠杆菌中表达的重组蛋白的免疫印迹分析中，单克隆抗体1E8的结合位点被定位到α毒素的COOH末端片段（氨基酸248 - 370），而单克隆抗体3B4和1F9的结合位点未被定位到该片段。为了证明单克隆抗体的中和潜力，将α毒素与单克隆抗体预孵育，随后在蛋黄扩散浊度（EYDT）测定中检测其卵磷脂酶活性，在溶血试验中检测其溶血活性，并在腹腔注射后检测其对小鼠的致死作用。当单独检测单克隆抗体时，仅在EYDT测定中观察到中和作用，其中单克隆抗体3B4完全消除了α毒素的卵磷脂酶活性。然而，当联合使用单克隆抗体3B4和1E8时，它们具有协同作用，并在体外抑制兔红细胞的裂解。相同的单克隆抗体混合物也能够完全中和三个半数致死剂量（LD50）的α毒素对Balb/c小鼠的致死作用。我们的结果表明，α毒素分子包含几个结构域，这些结构域在毒素的各种活性中发挥不同的作用。我们得出结论，α毒素的溶血结构域与导致小鼠致死作用的结构域相同或非常接近。卵磷脂酶活性可能参与溶血和小鼠致死的机制，但似乎不是唯一的决定因素。
