Simultaneous quantitation of two antigens in mixture in individual cells by microimmunofluorimetry

Double immunofluorescence has so far only been used qualitatively. In present work the possibility of quantitative double immunofluorescence was evaluated in model systems with the ultimate goal to enable simultaneous measurements of different antigens in mixture in biological objects. In one test system a conjugate of fluorescein isothiocyanate (FITC) was studied in combination with conjugates of dimethyl-amino-naphthalene-sulphonyl-chloride (DANSC) or with tetramethyl-rhodamine-isothiocyanate (TRITC) in solutions. In another test system FITC + DANSC antibody conjugates were analyzed after reaction with antigen mixtures in insolubilized form. Fluorescence intensities were measured in a microspectrofluorimeter using narrow band excitation and emission filters of appropriate types for different fluorochromes. Fading was no problem in this system. A formula was developed to calculate the antibody proportions from the fluorescence contribution of either fluorochrome in the region of optimal fluorescence of other fluorochrome. Experimental values obtained from analyses of FITC + DANSC and FITC + TRITC combinations in solutions agreed excellently with theoretically expected values. In the second model system polyacrylic beads were coated with different proportions of two antigens, one labelled with 125I and the other with 131I. After radioactivity measurements the beads were stained with appropriate antisera conjugated with FITC and DANSC, respectively. "Double fluorescence" intensities of individual beads were measured and a very strong correlation was found between the radioactivity and the fluorescence values. The results indicate that quantiation of double fluorescence is possible and meaningful for measurements of two different antigens in mixture on individual particles.