Berger M, Gaither T A, Cole R M, Chused T M, Hammer C H, Frank M M
Mol Immunol. 1982 Jul;19(7):857-64. doi: 10.1016/0161-5890(82)90351-0.
Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.
使用生物素-N-羟基琥珀酰亚胺亚胺酯（BNHS）对纯化的人C3进行生物素化。根据BNHS的用量，每个C3分子可结合三到六个生物素分子。生物素化的C3保留了其特异性溶血活性的90%以上，并且当与绵羊红细胞结合时，仍保持其与人C3b受体结合的能力。这些功能可被抗生物素蛋白阻断。生物素化的C3在人血清中可正常裂解为C3c和C3d，用抗生物素蛋白-琼脂糖吸附表明两个片段中均存在生物素部分。荧光素偶联的抗生物素蛋白与细胞结合的生物素化C3b反应良好，可用于通过流式细胞术定量C3固定。铁蛋白偶联的抗生物素蛋白用作标记物，通过电子显微镜表征生物素化C3b在红细胞上的分布。这些结果表明，生物素化的C3和抗生物素蛋白衍生物可能是研究C3多种生物学功能的非常有用的工具。
