Optimization and development of a high-performance liquid chromatography-based one-site immunometric assay with chemiluminescence detection

Various practical and theoretical considerations were examined in the creation and optimization of a high-performance liquid chromatography (HPLC)-based one-site immunometric assay. This method used an HPLC analyte analog column and post-column chemiluminescence detection. The specific analyte chosen as the model for this study was l-thyroxine (also known as T₄). In this technique, a sample containing thyroxine was first combined with an excess of anti-T₄ antibody Fab fragments that had earlier been conjugated with chemiluminescent acridinium ester labels. After incubation, the mixture was injected onto a column that contained immobilized T₄. The amount of thyroxine in the original sample was then determined by measuring the labeled Fab fragments that appeared in the non-retained fraction, or the decrease in excess Fab fragments that were bound to and later eluted from the column. Items considered in creating this assay included the preparation of acridinium ester-labeled Fab fragments, the detection of these fragments with a post-column reactor, and the creation of a suitable immobilized analog column for capturing excess labeled Fab fragments. The final method could measure T₄ in standards at clinically-relevant concentrations and provided a response within 1.5 min of sample injection, following a 20-45 min incubation with the labeled Fab fragments. Possible applications of this method include its use in clinical chemistry and the screening of proteomic or combinatorial libraries.