Kabir S
Department of Immunology, Karolinska Institute, Stockholm, Sweden.
J Med Microbiol. 1991 Mar;34(3):167-73. doi: 10.1099/00222615-34-3-167.
Monoclonal antibodies (MAbs) were raised against the major, 46-48-Kda outer-membrane proteins of Vibrio cholerae O1. The hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) with cell-surface proteins of V. cholerae O1 as the coating antigen. Four hybridomas, which secreted anti-V. cholerae cell-surface-protein antibodies, were subcloned by limiting dilution and obtained as ascites in vivo. A MAb of the IgG1 subclass was isolated in good yield from the murine ascites by affinity chromatography with recombinant protein G-Sepharose 4B. It gave positive reactions, as determined by ELISA, against cell-surface proteins prepared from both biotypes (classical and El Tor) and both serotypes (Ogawa and Inaba) of V. cholerae O1. The MAb did not have any reactivity towards V. cholerae lipopolysaccharide preparations. Immunoblotting studies were performed on cell-surface proteins separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE) and also by two-dimensional (2D) electrophoresis with iso-electric focusing in the first dimension followed by SDS-PAGE in the second dimension. When proteins were separated by 1D SDS-PAGE, only one band at 46-48 Kda reacted with the MAb. This protein appeared to consist of two narrowly-spaced and cross-reactive bands when a nitrocellulose blot, obtained by 2D SDS-PAGE, was exposed to the MAb.
制备了针对霍乱弧菌O1主要的46 - 48千道尔顿外膜蛋白的单克隆抗体（MAb）。以霍乱弧菌O1的细胞表面蛋白作为包被抗原，通过酶联免疫吸附测定（ELISA）筛选杂交瘤克隆。4个分泌抗霍乱弧菌细胞表面蛋白抗体的杂交瘤通过有限稀释法进行亚克隆，并在体内获得腹水。通过用重组蛋白G - 琼脂糖4B进行亲和层析，从鼠腹水中以高产率分离出一种IgG1亚类的单克隆抗体。通过ELISA测定，该单克隆抗体对由霍乱弧菌O1的两种生物型（古典生物型和埃尔托生物型）和两种血清型（小川型和稻叶型）制备的细胞表面蛋白均产生阳性反应。该单克隆抗体对霍乱弧菌脂多糖制剂没有任何反应性。对通过一维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（1D SDS - PAGE）分离的细胞表面蛋白以及通过二维（2D）电泳（第一维进行等电聚焦，第二维进行SDS - PAGE）分离的细胞表面蛋白进行免疫印迹研究。当蛋白通过1D SDS - PAGE分离时，只有一条46 - 48千道尔顿的条带与该单克隆抗体发生反应。当通过2D SDS - PAGE获得的硝酸纤维素印迹与该单克隆抗体反应时，这种蛋白似乎由两条间隔很近且交叉反应的条带组成。
