ANALYSIS OF ANTIGEN-ANTIBODY REACTION IN BUFFER SOLUTION BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY

Abstract

A new immunological technique to examine the characteristics of antigen-antibody reaction in liquid phase (LBA: liquid-phase binding assay) is described. A mixture of antigen (human chorionic gonadotropin: hCG) and enzyme labeled antibody (Fab -peroxidase) was incubated and was analyzed directly by HPLC having a gel filtration column and a post-column flow through coil. Using the system, a bound form (hCG-Fab -POD) and a free form (Fab -POD) was separated by the difference of the molecular mass, and the activities of POD were detected fluorophotometrically. Based on the principle, the characteristics of antibody, equilibrium and rate constants and optimum pH were examined. This method can be used for the studies of the characteristics of antibody and also used for the assay of antigens or antibodies in buffer solution.