A duplexed microsphere-based fluorescent immunoassay

Microsphere-based immunoassays are described for the simultaneous measureme nt of the clinically important drugs digoxin and theophylline. Competitive immunoassays were performed using haptenized microspheres and antibodies la beled with horseradish peroxidase. Enzyme-catalyzed reporter deposition (CA RD) resulted in immunofluorescence signal amplification. Two encoding dyes were used to differentiate analytical signals from microspheres containing assays for the two analytes, An epifluorescence microscope and a CCD camera interfaced with a computer were utilized to measure fluorescence signals o f individual microspheres. The microspheres from a duplexed assay were moun ted on microscope slides as well as inserted into wells etched into the dis tal ends of optical imaging fibers. Fluorescence images from both formats w ere captured. In the experiments using microscope slides, the immunoassays were successfully duplexed and only marginal interferences at high analyte concentrations were observed. Preliminary results suggest that simultaneous determination of the two analytes using a fiber-based sensor-array format is feasible, but requires further development before precise quantitative a nalyses are possible. (C) 2001 Academic Press.