Gaur V P, de Leeuw A M, Milam A H, Saari J C
Department of Ophthalmology, School of Medicine, University of Washington, Seattle 98195.
Exp Eye Res. 1990 May;50(5):505-11. doi: 10.1016/0014-4835(90)90039-w.
Monoclonal antibodies to performic acid-oxidized cellular retinoic acid-binding protein (CRABP) from bovine retina were prepared by fusion of spleen cells from immunized mice with mouse myeloma cells. Five antibodies were studied in detail. It was established by ELISA that the antibodies react with CRABP and oxidized CRABP, but not with other oxidized or unmodified retinoid-binding proteins. Competitive ELISA demonstrated that the antibodies react with heat-denatured antigen but not with native protein. Western blotting and immunostaining, following sodium dodecyl sulfate gel electrophoresis, provided evidence for recognition of a single component in retinal supernatants whose staining is prevented by preabsorption of the antibody with heat-denatured CRABP. The insoluble fraction from a retinal homogenate contains residual CRABP and two weakly-reacting components, whose staining is not affected by preabsorption of the antibody with antigen. Each antibody produces the same staining pattern on cryostat sections of rat retina by indirect immunofluorescence. Amacrine somata on both sides of the inner plexiform layer are labeled, as well as processes forming laminae within this layer. These results suggest that retinoic acid may play a functional role in the inner retina.
通过将免疫小鼠的脾细胞与小鼠骨髓瘤细胞融合，制备了针对来自牛视网膜的过甲酸氧化细胞视黄酸结合蛋白（CRABP）的单克隆抗体。对五种抗体进行了详细研究。通过酶联免疫吸附测定（ELISA）确定，这些抗体与CRABP和氧化型CRABP反应，但不与其他氧化或未修饰的类视黄醇结合蛋白反应。竞争性ELISA表明，这些抗体与热变性抗原反应，但不与天然蛋白反应。十二烷基硫酸钠凝胶电泳后的蛋白质印迹法和免疫染色法证明，视网膜上清液中的一种单一成分可被识别，用热变性CRABP预先吸收抗体可阻止其染色。视网膜匀浆的不溶性部分含有残留的CRABP和两种反应较弱的成分，用抗原预先吸收抗体不会影响它们的染色。通过间接免疫荧光法，每种抗体在大鼠视网膜的低温切片上产生相同的染色模式。在内网状层两侧的无长突细胞胞体以及该层内形成薄片的突起都被标记。这些结果表明视黄酸可能在内视网膜中发挥功能作用。
