Practical Application of the Immunoassay Utilizing the stability of Variable Region.

1.一种新的蛋白质相互作用检测系统（split Fv系统）构建了一种新的蛋白质相互作用检测系统split Fv（spFv）系统，该系统将一个蛋白质片段展示在丝状噬菌体上，而另一个蛋白质片段分泌到培养基中。当将该系统应用于模型抗体的VH/VL相互作用的测量时，成功地测量了相互作用中的抗原依赖性变化（开放夹心免疫测定，OS-IA）。此外，该系统可以同时展示两个结构域VH/VL，这使得能够用抗原进行生物淘选。实际上，通过构建框架区文库，我们阐明了以前未知的Fv抗原结合亲和力与VH/VL相互作用强度之间的关系，以及决定VH/VL相互作用强度的残基。2.OS-IA作为低分子量物质的非竞争性免疫分析原理 ...更多信息 在基因克隆以及基于OS-IA与各种抗半抗原抗体（双酚A、11-脱氧皮质醇、磷酸酪氨酸、赤霉素A24、骨钙素肽等）的非竞争性免疫测定中使用。对于每一种抗原，都获得了比常规竞争性免疫分析更高的灵敏度和更宽的浓度范围。此外，可以使用纯化的VH -碱性磷酸酶和例如MBP-VL的融合蛋白的组合进行实际的测定，所述融合蛋白已经使用克隆的基因表达。3.抗原特异性scFv的基于大肠杆菌的选择系统使用表达细胞表面嵌合受体的IL-3依赖性培养细胞，所述嵌合受体可以抑制抗原依赖性生长信号，阐明了一种新的基于哺乳动物细胞的抗原特异性抗体片段的选择系统。通过Cre重组酶的强制表达，被选中的细胞可以迅速转化为糖基化的scFv分泌细胞，从而导致该系统的名称（抗原介导的抗体生产者选择，ASAP）减

1.A novel system to measure protein-protein interaction (split Fv system).A novel protein-protein interaction measurement system split Fv (spFv) system was constructed which display one protein fragment on the filamentous phage while another protein fragment is secreted in the culture medium. When the system was applied to the measurement of VH/VL interaction of a model antibody, antigen-dependent change in the interaction was successfully measured (Open Sandwich immunoassay, OS-IA). In addition, the system can display the two domains VH/VL at the same time, which enables biopanning with the antigen. In fact, through the construction of the library of the framework region, we clarified the previously unknown relationship between the antigen binding affinity of Fv and the VH/VL interaction strength, and the residue that determines the VH/VL interaction.2.OS-IA as a noncompetitive immunoassay principle for the low molecular weight substancesUsing the above-mentioned SpFv system, we succe … More eded in the gene cloning of, as well as the noncompetitive immunoassay based on OS-IA with various anti-hapten antibodies (bisphenol A, 11-deoxycortisol, phosphotyrosine, gibbellerin A24, osteocalcin peptide etc.). For every antigen, a higher sensitivity and wider concentration range than those of conventional competitive immunoassays, were attained. Also, practical assays could be performed using combinations of the purified fusion proteins of VH -alkaline phosphatase and such as MBP-VL, which had been expressed using the cloned genes.3.A mammalian-based selection system for the antigen-specific scFvUsing the IL-3 dependent cultured cells expressing the cell-surface chimeric receptor that can transduce antigen-dependent growth signal, a novel mammalian cell-based selection system for antigen-specific antibody fragment was elucidated. The selected cells can be rapidly converted to glycosylated scFv-secreting cells through the forced expression of Cre recombinase, leading to the name of the system (antigen-mediated selection for antibody producers, ASAP) Less