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)的可变区分离出的VII和VL链可以在抗原存在下重新结合，并展示了一种新的免疫分析方法，称为开放夹心法，该方法基于分离的VII和VL的链内相互作用(Natural Biotech，(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，并建立一种基于分离的VL和嵌合VH酶的链内相互作用的通用开放式夹心ELISA法。结果表明，在所研究的六种抗胰岛素单抗、抗地高辛抗体和抗NP(4-羟基-3-硝基苯乙酸)抗体中，有两种在有抗原存在的情况下可以观察到分离的VH和VL链重新结合。这些结果表明，在许多抗体中普遍存在由抗原驱动的分离的VH和VL的链内相互作用。我们还构建了多个编码抗体可变区和大肠杆菌碱性磷酸酶(PhoA)单区基因的融合蛋白，并与大肠杆菌分泌系统进行了融合表达。采用固定VL的微孔板，以VH-PhoA为检测试剂，每孔加样品，进行开放式夹心ELISA检测。因此，只需一步孵育和洗涤即可在宽动态测量范围内测定抗原浓度。

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.