Development of a theoretical model for chromatographic-based competitive binding immunoassays with simultaneous injection of sample and label

This study examined the theory and behavior of an HPLC-based chromatographi c competitive binding immunoassay with the simultaneous injection of sample and a labeled analyte analogue. Equations based on nonlinear chromatograph ic theory were derived to describe the calibration curve for this assay in a system with adsorption-limited kinetics and homogeneous binding sites. Th ese equations related the assay response (B/B-o) to the column's binding ca pacity, the moles of injected analyte or labeled analogue, and the now rate /adsorption kinetics of the system. There was good agreement between the pr edicted theoretical response and experimental data obtained for the binding of human serum albumin (HSA) to an immobilized anti-HSA antibody column. T his theory was also successful in describing the changes that occurred in t he calibration curve when the now rate or amount of labeled analogue applie d to the column was varied. A comparison was made between the results of th is study and previous theoretical work that examined the behavior of a rela ted, sequential injection competitive binding method. On the basis of the r esults reported in this work, several general guidelines were developed for the design and optimization of simultaneous injection methods for use in s uch areas as clinical testing, pharmaceutical analysis, and environmental m onitoring.