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

我们发现从抗体HyHEL-10（抗鸡蛋溶菌酶抗体）可变区分离的VII和VL链在抗原存在下可以重新结合，并基于分离的VII和VL的链内相互作用，提出了新的免疫测定方法，称为Open Sandwich法（Nature Biotechnology,(1996), 14,1714 -1718）。我们的目的是阐明该方法是否适用于其他抗原-抗体免疫分析系统，并基于分离的VL和嵌合的vh酶在链内的相互作用，开发一种通用的Open Sandwich ELISA方法。结果表明，在所研究的6种抗胰岛素单克隆抗体中，抗地高辛抗体和抗np（4-羟基3-硝基苯乙酸）抗体中，有2种在抗原存在下观察到分离的VH链和VL链的重新结合。这些结果表明，这种由抗原驱动的分离VH和VL的链内相互作用普遍存在于许多抗体中。我们还构建了多个编码抗体可变区单链域和大肠杆菌碱性磷酸酶（PhoA）的基因融合体，并与大肠杆菌分泌系统合成了融合蛋白。采用固定化VL的微滴板，以VH-PhoA为检测试剂，每孔加入样品，进行Open Sandwich酶联免疫吸附试验。因此，抗原浓度测定的动态范围很宽，每一步仅需孵育和洗涤。

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.