Development of high-performance immunoaffinity chromatography

The theory and behavior of a chromatographic competitive binding immunoassay with sequential injection of sample and labeled analyte were examined. An equation was derived relating assay response (B/B$\rm\sb{o}$) to the column\u27s binding capacity, the moles of analyte and labeled analyte 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 analyte injected, when applied in excess versus binding sites in the column, had little effect on the calibration curve. The position of the calibration curve was determined mainly by the number of binding sites on the column, but could be shifted over several orders of magnitude by varying the flow rate used for analyte injection. A fully automated HPLC method was developed for the analysis of atrazine in water. This method used a high-performance immunoaffinity column for the extraction of atrazine and other triazines from samples, followed by separation of the retained compounds with an on-line reversed-phase column. This technique used only 250 $\mu$L of sample, required minimal sample pretreatment and showed no significant interferences from the sample matrix or several common pesticides tested. Atrazine plus all of its major degradation products could be determined in 20 min with this system. The calibration curve for atrazine was linear over two orders of magnitude and had a lower limit of detection of 0.1 $\mu$g/L. The within-day precision was $\pm$5.4% for samples containing 1.1 $\mu$g/L atrazine. The results of this method showed good correlation with those obtained by GC/MS or GC/NPD. By using different immunoaffinity columns and elution conditions, this method could be adapted for use in the determination of other compounds of environmental interest. The feasibility of coupling Flow Injection Analysis to the affinity column waste stream was demonstrated using the determination of urinary catecholamines and creatinine as an example. This effectively doubled the throughput of the high- performance affinity chromatographic technique. Accuracy and precision of this method compared favorably with standard methods. These studies lay the foundation for the rapid, fully automated determination of analytes in complex matrices by the use of High-performance immunoaffinity chromatography