THE EFFECT OF HYDROGEN ION CONCENTRATION ON FLUORESCENT LABELLED ANTIBODIES.

Abstract

3 - Aminotetramethylrhodamine, lissamine rhodamine B (sulforhodamine B, B. C. 12145 National Aniline Division, Buffalo, New York), 5-dimethylaminonaphthalene-1-sulfonyl chloride and amino fluorescein were employed for conjugation of horse antirabbit gamma globulin for the detection of rabbit antibodies to rat kidney glomeruli. For comparative purposes labelled antibodies having comparable number of residues/mole protein were employed. The possibility of selectively quenching fluorescent labelled antibodies with change of the hydrogen ion concentration was investigated. Quenching of the fluorescence for fluorescein could be accomplished by dropping the pH of the medium to 3; partial reactivation of fluorescence occurred when the medium was made alkaline. Fluorescein fluoresced well from pH 11 to 7; appreciable quenching occurred at pH 5 and 4. From pH 4 complete reactivation occurred when the medium was made alkaline. Tetramethylrhodamine fluoresced well from pH 11 to 4; some loss of fluorescence was seen at pH 3. Lissamine Rhodamine B fluoresced best at pH 7 and 6. 5-Dimethylaminonaphthalene-1-sulfonyl chloride was a poor label and did not give the intensity of the other dyes at all pH ranges. At pH 2 all showed irreversible quenching. Fluorescein- and tetramethylrhodamine-labelled antibodies used simultaneously on a single kidney section showed yellow fluorescence when both the orange of tetramethylrhodamine and green of fluorescein reacted at the same antigenic sites. Changes in the hydrogen ion concentration can be employed to selectively quench the fluorescence of fluorescein but not the tetramethylrhodamine-labelled antibodies.