Development of microchromatography-based immunobiosensor

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Various methods have been developed for immunoassay. The most popular method is enzyme-linked immunoadsorbent assay (ELISA), in which antibody is immobilized on the solid phase. In this study, free antigen-antibody complex was measured by using size exclusion chromatography (SEC). For comparison, factors affecting the precision and the reproducibility of 96-well microplate ELISA were examined experimentally. The items tested were antibody concentration, time for antibody immobilization, concentration of contaminant proteins, methods for washing, and etc.Antibody and antigen-protein were successfully separated by micro-SEC (4.6 mm diameter and 30 cm length). However, it was found that quite large antibody-antigen complexes are formed during SEC separation even with monoclonal antibody. The present method is also suitable for characterizing monoclonal antibody properties.