LIQUID-PHASE BINDING ASSAY OF HUMAN CHORIONIC-GONADOTROPIN USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A new enzyme immunoassay technique (LBA: liquid-phase binding assay) to examine the characteristics of liquid-phase antigen-antibody reactions is described. Antigen (human chorionic gonadotropin: hCG) and peroxidase (POD)-labeled anti-hCG monoclonal antibody (Fab'-POD) solutions were mixed, incubated, and analyzed directly by gel filtration high-performance liquid chromatography with postcolumn enzyme activity measurement. Using the system, bound (hCG-Fab'-POD) and free (Fab'-POD) forms of enzyme-labeled antibody were separated by their molecular mass difference, and the POD activity of the conjugate was determined fluorophotometrically. All analytes became bound upon addition of excess Fab'-POD, because reaction conditions in a liquid phase could be easily altered. Thus, hCG molecule could be measured via the activity of one POD molecule. And the liquid-phase antibody reaction was very fast and quantitative. On the basis of this stoichiometric relationship, equilibrium and rate constants, optimum pH, and temperature effects were easily examined. The method is simple and convenient for examination of the antigen-antibody reaction and is applicable for antigen assays requiring an accurate definition of concentrations.