Generalization of the Immunoassay Utilizing the Stability of Antibody Variable Region

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The objective of the study was to elucidate a rapid selection system to obtain a Fv that is suitable to the novel immunoassay based on the stabilization of Fv by bound antigen (Open Sandwich ELISA). To this end, a novel filamentous phage display system termed 'spFv system' in which antigen binding ability of Fv as well as VH/VL interaction can be easily evaluated. Based on a model experiment using HyHEL-10, which is a known antibody to be suitable to Open Sandwich ELISA, the principle was shown to work by simply changing the host strain used. Also, a model selection of HyHEL-10 against another nonbonding antibody was shown to work. Along with this, the analysis of important amino acid residue(s) in the determination of VH/VL interaction strength was performed by phage display of human antibody model. As a result, H95 of CDR H3 was shown to be a key determinant of VH/VL interaction.To attain higher sensitivity of homogeneous Open Sandwich immunoassays, novel methods utilizing the energy transfer from a luciferase to a fluorescent protein, as well as enzymatic complementation of two enzyme fragments were engineered. As a result, 10 to thousand folds improvement in sensitivity was attained compared with previous homogeneous assays.