Detection of Two Antibodies in Single Plasma Cells by the Paired Fluorescence Technique

Abstract

The concept that a single antibody-forming cell may have the capacity to synthesize more than one antibody simultaneously was tested. This problem was approached by using a single antigen on which two determinant groups could be detected. Guinea pigs were hyperimmunized with a column-purified human γ-globulin preparation. Animals showing antibodies that reacted with a single band to the whole antigen but with two bands to the papain-digested antigen by immunoelectrophoresis were sacrificed and the spleen sections studied to see if any single cell was responding with two antibodies to both fragments of the whole antigen. A multiple antibody response in individual cells was detected using the sensitive indirect paired fluorescence technique. From a total of 482 antibody-containing cells counted, 30% had antibodies directed toward the CP-I fragment and 24% to the CP-II fragment. It appeared that about 45% of the cells contained antibodies directed to both the CP-I and CP-II fragments. The gradation of colors caused by the combination of tetramethylrhodamine and fluorescein conjugates in the multiple antibody-producing cells and the selective quenching studies of fluorescence at pH 4.0 indicate that different cells contained different amounts of one type of antibody over the other.