Consfrudaon of sensitive environment monitoring devices using a noncompetitive immunodetection system

本研究的目的是通过使用一种新的免疫测定原理，利用抗体Fv区与抗原结合后的稳定性（开放夹心免疫测定，OS-IA），建立一个全面、灵敏的低分子量分析物检测系统。为此，我们克隆了两种抗新烟碱类杀虫剂抗体、一种真菌毒素抗体和一种低分子量肽bp - c7抗体的Fv基因，并成功构建了OS-IA体系并对其进行了灵敏的非竞争性检测。特别是在最后一个系统中，通过基于微芯片的检测系统实现了从微量血清样本的快速检测。通过这个过程，我们认识到优化噬菌体展示格式对于选择适合OS-IA的抗体的重要性。除了已经开发的分裂Fv系统外，还开发了基于ere重组酶的scFv-OS转换系统，以及能够构建和选择10^8大小的更大库的Fab-OS转换系统。虽然无细胞选择系统的计划建设尚未完成，但我们认为很快就可以实现对OS-IA更大、更集中的文库的高效选择，这将使各种小分析物（如环境污染物）和蛋白质修饰（如磷酸化和截断）的敏感检测成为可能。作为一个意外的结果，我们开发了一种新的方法，通过细胞核中的前mrna的“反式”剪接在抗体产生细胞中表达抗体融合蛋白。作为模型系统，在COS-1细胞中成功表达了OS-IA中不可缺少的vh -酶融合mRNA和相应的蛋白。基于这一原则以及其他原则，设想了从已建立的杂交瘤资源中快速制备这种融合蛋白而不需要基因克隆的方法。

The objective of this study was to make a comprehensive and sensitive detection system of low molecular weight analytes through the use of a novel immunoassay principle that utilizes the stabilization of antibody Fv region upon binding with antigen (open sandwich immunoassay, OS-IA). To this end, we cloned the Fv genes of the antibodies for two anti-neonicotinoid pesticides, a mycotoxin, and a low molecular weight peptide BGP-C7, and succeeded in the construction of OS-IA system and sensitive noncompetitive detection thereof. Especially in the last system, rapid detection from a minute amount of serum sample was attained by a microchip-based detection system.Through this process, we realized the importance of optimized phage display format for the selection of antibodies that are suitable to OS-IA. In addition to split Fv system already developed, an scFv-OS conversion system based on ere recombinase was developed, as well as an Fab-OS conversion system which enabled construction and selection of larger library of 10^8 size. While the scheduled construction of cell-free selection system is yet to be done, we think efficient selection of larger and focused library for OS-IA is soon to be realized, which will enable sensitive detection of various small analytes such as environmental pollutants and protein modification such as phosphorylation and truncation.As an unplanned result, we developed a novel method to express antibody fusion proteins in antibody producing cells through ''trans'' splicing of the pre-mRNAs in the cell nucleus. As a model system, a VH-enzyme fusion mRNA and corresponding protein that is indispensable in OS-IA were successfully expressed in COS-1 cells. Elucidation of rapid fabrication methods of such fusion proteins from established hybridoma resources without the need of gene cloning is envisaged based on this principle as well as other principles.