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

1.一种新的蛋白质-蛋白质相互作用测量系统(Split Fv系统)构建了一种新的蛋白质-蛋白质相互作用测量系统Split Fv(SpFv)系统，该系统在丝状噬菌体上展示一个蛋白质片段，而在培养基中分泌另一个蛋白质片段。将该系统应用于一种模型抗体的VH/VL相互作用的测量，成功地测量了相互作用中的抗原依赖性变化(开放夹心免疫分析，OS-IA)。此外，该系统可以同时显示VH/VL两个结构域，这使得与抗原进行生物扫描成为可能。事实上，通过构建Fv的骨架区域文库，我们阐明了Fv的抗原结合亲和力与VH/VL相互作用强度之间的未知关系，以及决定VH/VL相互作用的残基。2.OS-IA作为一种用于低分子物质的非竞争性免疫分析原理，利用上述SpFv系统，我们成功地进行了…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、骨钙素等)的非竞争性免疫分析方面取得了更多进展。与传统的竞争免疫分析方法相比，每种抗原的灵敏度更高，浓度范围更广。利用纯化的VH-碱性磷酸酶融合蛋白和利用克隆基因表达的MBP-VL的组合，也可以进行实用的检测。3.基于哺乳动物的单链抗体选择系统利用表达细胞表面嵌合受体的IL-3依赖的培养细胞传递抗原依赖的生长信号，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。选择的细胞可以通过强制表达Cre重组酶而迅速转化为糖基化的ScFv分泌细胞，从而导致该系统的名称(抗原介导的抗体产生者选择，ASAP)较少

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