López-Ribot J L, Martínez J P, Chaffin W L
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Infect Immun. 1995 Jun;63(6):2126-32. doi: 10.1128/iai.63.6.2126-2132.1995.
Using polyclonal antibodies (PAbs) raised against the Candida albicans C3d receptor (CR2; PAb anti-CR2) and the 58-kDa fibrinogen-binding mannoprotein (mp58; PAb anti-mp58) as well as ligand interactions, we have studied the relationship between these two receptors. In an indirect immunofluorescence assay with germ tubes, greater intensity was observed on the mother blastoconidium when PAb anti-CR2 was used, whereas greater intensity was localized to the hyphal extension when PAb anti-mp58 or binding of soluble fibrinogen was used. No competition or change in the fluorescence pattern was observed in dual-labeling experiments with PAb anti-CR2 and either fibrinogen or PAb anti-mp58. Binding competition also was not observed in an enzyme-linked immunosorbent assay using the components present in a beta-mercaptoethanol extract from the cell wall of germ tubes. In immunoblots, PAb anti-CR2 recognized three different discrete bands with apparent molecular masses of 21, 40, and 66 kDa in the beta-mercaptoethanol extracts from the cell wall, whereas a different, single, broader band with an apparent molecular mass of 58 kDa was detected with PAb anti-mp58. However, when nondenaturing conditions were used to separate the materials present in the cell wall extracts, no reactivity could be detected on Western blots (immunoblots) with PAb anti-mp58. When PAb anti-CR2 was used for analysis, a single band migrating in the area corresponding to approximately 40 kDa was detected. These observations suggest a higher molecular weight for mp58 and one or more of the components detected with PAb anti-CR2 in their native state.
我们使用针对白色念珠菌C3d受体(CR2;抗CR2多克隆抗体)和58 kDa纤维蛋白原结合甘露糖蛋白(mp58;抗mp58多克隆抗体)产生的多克隆抗体以及配体相互作用,研究了这两种受体之间的关系。在对芽管进行的间接免疫荧光测定中,使用抗CR2多克隆抗体时,母芽生孢子上观察到更强的荧光强度,而使用抗mp58多克隆抗体或可溶性纤维蛋白原结合时,更强的荧光强度定位于菌丝延伸部位。在抗CR2多克隆抗体与纤维蛋白原或抗mp58多克隆抗体的双重标记实验中,未观察到荧光模式的竞争或变化。在使用芽管细胞壁β-巯基乙醇提取物中的成分进行的酶联免疫吸附测定中,也未观察到结合竞争。在免疫印迹中,抗CR2多克隆抗体在细胞壁β-巯基乙醇提取物中识别出三条不同的离散条带,表观分子量分别为21、40和66 kDa,而抗mp58多克隆抗体检测到一条不同的、单一的、更宽的条带,表观分子量为58 kDa。然而,当使用非变性条件分离细胞壁提取物中的物质时,在免疫印迹(Western印迹)上用抗mp58多克隆抗体检测不到反应性。当使用抗CR2多克隆抗体进行分析时,检测到一条迁移到对应于约40 kDa区域的单一条带。这些观察结果表明,mp58以及抗CR2多克隆抗体在天然状态下检测到的一种或多种成分具有更高的分子量。