IDENTIFICATION OF 7 SURFACE-EXPOSED BRUCELLA OUTER-MEMBRANE PROTEINS BY USE OF MONOCLONAL-ANTIBODIES - IMMUNOGOLD LABELING FOR ELECTRON-MICROSCOPY AND ENZYME-LINKED-IMMUNOSORBENT-ASSAY
IDENTIFICATION OF 7 SURFACE-EXPOSED BRUCELLA OUTER-MEMBRANE PROTEINS BY USE OF MONOCLONAL-ANTIBODIES - IMMUNOGOLD LABELING FOR ELECTRON-MICROSCOPY AND ENZYME-LINKED-IMMUNOSORBENT-ASSAY
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DOI：
10.1128/iai.58.12.3980-3987.1990
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发表时间：
1990-12-01
影响因子：
3.1
通讯作者：
LIMET, JN
中科院分区：
文献类型：
--
作者：
CLOECKAERT, A;DEWERGIFOSSE, P;LIMET, JN
一个面板的单克隆抗体（MAbs）的7个布鲁氏菌外膜蛋白的特点。这些抗体通过用牛种布鲁氏菌或B的十二烷基硫酸钠不溶性（SDS-1）组分、细胞壁或全菌细胞免疫小鼠获得。melitensis。酶联免疫吸附试验用于筛选杂交瘤上清，并确定它们在粗糙和光滑B表面的结合。流产和B. melitensis细胞这些抗体识别的外膜蛋白（OMP）是分子量为25至27 kDa和36至38 kDa的蛋白（孔蛋白）（主要蛋白）和分子量为10、16.5、19、31至34和89 kDa的蛋白（次要蛋白）。这些外膜蛋白的表面暴露通过使用单克隆抗体和免疫金标记的电子显微镜观察。MAb在整个粗糙细菌细胞上的结合表明10-、16.5-、19-、25-至27-、31-至34-、36-至38-和89-kDa OMP暴露在细胞表面。然而，酶联免疫吸附试验结果表明，一个更好的结合的抗OMP单克隆抗体粗糙的菌株比相应的光滑的菌株，除了抗19 kDa的单克隆抗体。免疫电镜显示，光滑B.只有89-和31-至34-kDa的外膜蛋白不能被测试的单克隆抗体接近。对于粗糙B，观察到抗31-至34-kDa MA B在细胞表面的结合。粗糙型和光滑型B细胞。melitensis细胞这些结果表明空间位阻的重要性，由于存在长的脂多糖O侧链的OMP光滑布鲁氏菌菌株的可及性，并应考虑进行疫苗开发时。
A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-1) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.