Empirically optimized flow cytometric immunoassay validates ambient analyte theory

Ekins' ambient analyte theory predicts, counterintuitively, that an immunoassay's limit of detection can be improved by reducing the amount of capture antibody. In addition, it also anticipates that results should be insensitive to the volume of sample as well as the amount of capture antibody added. The objective of this study was to empirically validate all of the performance characteristics predicted by Ekins' theory. Flow cytometric analysis was used to detect binding between a fluorescent ligand and capture microparticles because it can directly measure fractional occupancy, the primary response variable in ambient analyte theory. After experimentally determining ambient analyte conditions, comparisons were carried out between ambient and nonambient assays in terms of their signal strengths, limits of detection, and sensitivity to variations in reaction volume and number of particles. The critical number of binding sites required for an assay to be in the ambient analyte region was estimated to be 0.1 VK_d. As predicted, such assays exhibited superior signal/noise levels and limits of detection and were not affected by variations in sample volume and number of binding sites. When the signal detected measures fractional occupancy, ambient analyte theory is an excellent guide to developing assays with superior performance characteristics.