Li R K, Cutler J E
Department of Microbiology, Montana State University, Bozeman 59717.
J Gen Microbiol. 1991 Mar;137(3):455-64. doi: 10.1099/00221287-137-3-455.
Antibody from BALB/cByJ mice immunized against a membranous fraction of Candida albicans agglutinated whole cells as well as the membranous fraction. Hybridoma techniques were used to isolate an IgM monoclonal antibody (mAb) designated 10G which agglutinated whole cells and reacted with the subcellular fraction. Yeast cells of 15 additional C. albicans strains and isolates of C. stellatoidea, C. tropicalis, C. intermedia and C. lusitaniae were also agglutinated by mAb 10G. The antigen was not detected on other fungi, including Candida krusei, C. utilis, Cryptococcus neoformans, Cr. albidus, Torulopsis glabrata, Rhodotorula spp. and Saccharomyces cerevisiae. To determine the cellular location of the epitope to which mAb 10G is specific, freeze-substitution was compared with traditional chemical fixation methods in preparation of samples for immunocolloidal gold electron microscopy (IEM). With both fixation procedures, the antigen recognized by mAb 10G was found randomly and densely concentrated on the plasma membrane on exponential-phase yeast-form cells and had a patchy distribution on the cell wall surface. Association of the antigen with the plasma membrane was confirmed by IEM of isolated membranes. On developing hyphal cells, antigen appeared first on the plasma membrane and later on the cell wall surface. Treatment of yeast cells with beta-mercaptoethanol and Zymolyase before fixation removed the antigen from the surface but left the cytoplasmic antigen undisturbed. Treatment of yeast cells or solubilized antigen with heat or proteolytic enzyme (trypsin, Pronase B, proteinase K) did not remove or destroy the antigen, suggesting a non-protein nature of the epitope.
用白色念珠菌膜组分免疫的BALB/cByJ小鼠产生的抗体，能凝集完整细胞以及膜组分。采用杂交瘤技术分离出一种IgM单克隆抗体（mAb），命名为10G，它能凝集完整细胞并与亚细胞组分发生反应。另外15株白色念珠菌菌株以及星状念珠菌、热带念珠菌、中间念珠菌和葡萄牙念珠菌的分离株的酵母细胞也能被单克隆抗体10G凝集。在其他真菌上未检测到该抗原，包括克鲁斯念珠菌、产朊假丝酵母、新生隐球菌、浅白隐球菌、光滑球拟酵母、红酵母属和酿酒酵母。为了确定单克隆抗体10G所识别的表位的细胞定位，在制备用于免疫胶体金电子显微镜（IEM）的样品时，将冷冻替代法与传统化学固定方法进行了比较。在两种固定程序中，单克隆抗体10G识别的抗原随机且密集地集中在指数生长期酵母形式细胞的质膜上，在细胞壁表面呈斑块状分布。通过分离膜的IEM证实了抗原与质膜的关联。在发育中的菌丝细胞上，抗原首先出现在质膜上，随后出现在细胞壁表面。在固定前用β-巯基乙醇和溶菌酶处理酵母细胞可去除表面抗原，但胞质抗原不受影响。用加热或蛋白酶（胰蛋白酶、链霉蛋白酶B、蛋白酶K）处理酵母细胞或溶解的抗原，不会去除或破坏抗原，这表明该表位具有非蛋白质性质。
