Evolution of visual pigment and differentiation of membrane structure : Production of monoclonal antibody and electron microscopic immnunohistochemistry

1.抗七鳃鳗-视网膜匀浆单抗的研制我们研制了10个新的单抗。用光镜和电子显微镜免疫组织化学方法检测它们在视网膜切片上的结合部位。W26、W27、W39：与短光感受器细胞(SPC)的外节特异性结合。2)W226、W231、W312、W425：与短光感受器细胞(SPC)的外节、内节(除SPC和轴突的椭圆体外)结合。3)W15：与SP3的外节和内节结合)W38：与SP5的所有区域结合)W318：与视网膜神经层结合。以下3种单抗专用于参与光接收过程的分子组分。1)W39：对于七色紫质，分子量约为40kDa2)W312，W425：针对Prey arrestin、W4252.免疫金定量电子显微镜的建立，标本用4%多聚甲醛固定，包埋在LR-白色中。与常规固定相比，视网膜神经层有相当大的清洗和破坏痕迹，感光细胞外段变形。然而，就光感受器细胞而言，偶然保存下来的外节被不同程度地夹杂着细小的金粒，这使得我们能够进行定量检查。结论制备了一系列抗七鳃鳗视网膜抗体，并用于识别视网膜细胞中的蛋白质、糖结合物和脂类。这些抗体显然可以作为不同物种的相应细胞之间关系的探针。

1.Production of monoclonal antibodies against lamprey-retinal homogenate.We have developed 10 new monoclonal antibodies. Their binding sites were detected on the sectioned retina by optical and electron microscopic immunohistochemistry. Molecular weight of antigens were estimate by western blotting.1) W26, W27, W39 : Specifically bound to the outer segment of short photoreceptor cell (SPC).2) W226, W231, W312, W425 : To the outer segment, inner segment except for ellipsoid of SPC and axon.3) W15 : To the outer and inner segment of SPC.4) W38 : To the all region of SPC.5) W318 : To the neural layr of retina.Following 3 monoclonal antibodies were specialized for a molecular component involved in photoreceiving procedure.1) W39 : For lamprey rhodopsin, molecular weight is about 40kDa.2) W312, W425 : For lamprey arrestin, molecular weight is about 49kDa.2.Establishment of quantitative immunogold electron microscopy.The specimen was fixed with 4% paraformaldehyde and embedded in LR-white. There were considerably remakable washing out and trace of destruction in the neural layr of retina, and deformation of outer segment of photoreceptor cells as compared with conventional fixation. As far as photoreceptor cells are concerned, however, fortuitously preserved outer segments were found as assosiated variously with fine gold particles, allowing our quantitative examinatoin.ConculusionA series of anti-lamprey retinal antibodries were produced and used for recognition of protein, glycoconjugate and lipids in retinal cells. These antibodies are apparently useful as probes for relationship between corresponding cells of different species.