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

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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.