Technical Factors Affecting the Preparation of Fluorescent Antibody Reagents

Abstract

Interference due to ammonium sulfate contamination of globulin, resulting from the serum fractionation procedure, was eliminated by dialyzing 5- to 10-ml samples of the antiglobulin in dialysis tubing 20/32 in. in diameter for 4 hr against 2 liters of 0.85% sodium chloride. Contamination of Brucella abortus antiglobulin by 0.08 M or greater concentrations of ammonium sulfate caused interference with the biuret estimation of the protein and its subsequent conjugation to fluorescein isothiocyanate. Conjugates prepared in the presence of graded concentrations of the salt showed proportionally greater fluorescence and greater light absorption values prior to dialysis than did conjugates prepared in the absence of this salt. The results suggested that ammonium sulfate reacted with free fluorescein isothiocyanate to form an unstable dialyzable complex which was more brilliant than the conjugate alone. Ammonium sulfate-contaminated conjugates from which salt and free dye were removed by dialysis fluoresced weakly and showed poorly staining titers as compared to salt-free controls. Solutions of fluorescein isothiocyanate stored in the dark at 4°C for 70 days show a marked reduction in fluorescence intensity, whereas the light absorbing component remained stable. On the other hand, Brucella melitensis antiglobulin labeled with fluorescein isothiocyanate and similarly stored for 74 days showed no significant changes in fluorescence intensity. The colorant concentration of the conjugate was similar in stability to that of the fluorescein isothiocyanate solution.