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Study on molecular design of artificial antibody and its application to immunosensor

人工抗体分子设计及其在免疫传感器中的应用研究

基本信息

批准号：
09450301
负责人：
NAGAMUNE Teruyuki
金额：
$ 9.02万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

We found that separated VII and VL chains from variable region of antibody HyHEL-10 (anti-Hen Egg Lysozyme antibody) could re-associate in the presence of antigen and demonstrated new immunoassay method, named Open Sandwich method, based on the intrachain interaction of separated Vii and VL (Nature Biotechnology, (1996), 14, 1714-1718). We aimed to elucidate whether this method is applicable to other antigen-antibody immunoassay system and to develop a general Open Sandwich ELISA method based on the intrachain interaction of separated VL and chimeric VH-enzyme.It was shown that re-association of separated VH and VL chains in the presence of antigen could be observed in two of six anti-insulin monoclonal antibodies investigated, anti-digoxin antibody and anti-NP (4-hydroxy 3-nitrophenyl acetic acid) antibody. These results indicate that such intrachain interaction of separated VH and VL driven by antigen exists generally in many antibodies. We also constructed several gene fusions encoding the mono-domain of antibody variable regions and E.coli alkaline phosphatase (PhoA) and produced the fusion proteins with E.coli secretion system. Open Sandwich ELISA was performed using microtiter plate with immobilized VL, and VH-PhoA as detection reagent which was added to each well with samples. As a result, antigen concentration was determined with wide dynamic range of measurement by only one-each step of incubation and washing.

我们发现，来自抗体HyHEL-10（抗鸡卵溶菌酶抗体）的可变区的分离的VII和VL链可以在抗原存在下重新结合，并证明了基于分离的VII和VL的链内相互作用的新的免疫测定方法，称为开放三明治法（Open Sandwich method）（Nature Biotechnology，（1996），14，1714-1718）。我们的目的是阐明这种方法是否适用于其他抗原抗体免疫分析系统，并建立一个通用的开放夹心ELISA方法的基础上分离的VL和嵌合VH-酶链内的相互作用，表明分离的VH和VL链在抗原存在下的重新联合可以在两个被研究的抗胰岛素单克隆抗体中观察到，抗地高辛抗体和抗NP（4-羟基3-硝基苯乙酸）抗体。这些结果表明，这种由抗原驱动的分离的VH和VL的链内相互作用普遍存在于许多抗体中。我们还构建了编码抗体可变区单域的基因与大肠杆菌碱性磷酸酶（PhoA）的融合体，并利用大肠杆菌分泌系统产生融合蛋白。使用具有固定化VL的微量滴定板和作为检测试剂的VH-PhoA进行开放夹心ELISA，将VH-PhoA加入到具有样品的每个孔中。结果，仅通过孵育和洗涤各一个步骤，以宽的测量动态范围测定抗原浓度。