Theory of a sequential addition competitive binding immunoassay based on high-performance immunoaffinity chromatography.

This study examined the theory and behavior of a chromatographic competitive binding immunoassay with sequential injection of sample and a labeled analyte analog. Based on nonlinear chromatographic theory, an equation was derived to describe the calibration curve for this assay in a system with adsorption-limited kinetics and homogeneous binding sites. This equation related assay response (B/Bo) to the column's binding capacity, the moles of analyte and labeled analog injected, and the flow rate/adsorption kinetics of the system. There was good agreement between this equation and experimental data for the binding of human serum albumin (HSA) to an immobilized anti-HSA antibody column. It was found that the amount of labeled analog injected, when applied in excess vs binding sites in the column, had little or no effect on the relative response or position of the calibration curve. The position of the curve was determined mainly by the number of binding sites on the column; however, this position could be shifted over several orders of magnitude by varying the flow rate used for analyte injection. By varying both the injection flow rate and labeled analog load, this method could be designed for use as either a screening test or quantitative assay.