STUDIES ON FLUORESCENT ANTIBODY STAINING

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Abstract
Nonspecific fluorescent staining limits usefulness of the Coons fluorescent antibody technique in localizing small concentrations of antigen. In an investigation of this phenomenon, ammonium sulfate cuts of sheep immune serum were compared with serum antibody globulins separated on diethylaminoethyl (DEAE) cellulose, fluorescein isothiocyanate (FITC) was used in amorphous and crystalline state and the amount of FITC offered per gram of protein was varied as well as the duration of the coupling reaction, and the concentration of the coupled products used for staining imprints. Fluorescent globulin compounds were separated rapidly and completely from dialyzable fluorescent compounds on columns of sephadex G-50. Splenic imprints of antigen-stimulated rabbits and guinea pigs were tested for nonspecific staining, which was defined as fluorescent staining not completely inhibited by prior treatment of tissue imprints with 10-fold higher concentrations than used of the respective fluorescent reagent. Nonspecific fluorescent staining increased with the amount of fluorescein attaching per globulin molecule. By chromatography on DEAE cellulose, fluorescent gamma globulin preparations were shown to be heterogeneous with respect to the number of radicals attached. By reacting 6-8 mg of crystalline FITC per gram of gamma globulin as 2.5% protein solution (pH 9.0-9.5 for 24 hours), followed by passage through sephadex G-50 and salt gradient elution chromatography on DEAE-cellulose (0 up to 0.05 M NaCl) to eliminate the most highly coupled molecules, a fraction was obtained repeatedly which at 0.5 to 1.0 mg/ml showed specific fluorescence, and no or negligible nonspecific fluorescence. Such fractions had fluorescence protein ratios from 2.0 to 3.5 x 10-3 and could be used directly.