Optimization of post-column chemiluminescent detection for low-molecular-mass conjugates of acridinium esters

In this study, various factors are examined that affect the post-column chemiluminescence detection of low-molecular-mass compounds labelled with acridinium esters, using 9-phenyl acridinium ester (PAE) as the initial model. Reaction conditions examined included the effect of hydroxide or hydrogen peroxide concentration, ionic strength and surfactant content on the degree and length of light production by the acridinium ester label. Based on these results, a system was developed for the post-column detection of acridinium ester conjugates in HPLC. The final post-column reactor had optimum conditions similar to those for a benchtop luminometer but gave light production over slightly longer periods of time and had a broader range of reagent concentrations that gave a maximum response. The detection limit of this system for PAE was 7 x 10(-19) mol per injection, with linear and dynamic ranges that extended up to 3 x 10(-15) and 3 x 10(-13) mol, respectively. Some preliminary work was conducted examining the use of this system in chromatography-based competitive binding immunoassays with a thyroxine-acridinium ester conjugate being used as the label. The estimated limit of detection was 2.5 x 10(-17) mol (or roughly 10(-12) M for a 25 microliters sample) based on the retained fraction of the conjugate.