Detection by immunofluorescence of surface molecules present in low copy numbers. High sensitivity staining and calibration of flow cytometer.

Receptors for lymphokines and growth factors are present on cell surfaces often at concentrations of 100-500 copies per cell. Although conventional immunofluorescence cannot detect such low levels, cell membrane antigens present at these concentrations can be detected using an optimally set up flow cytometer together with a three-layer immunofluorescence technique, consisting of monoclonal antibody reacted with selected batches of biotinylated horse anti-mouse immunoglobulin and phycoerythrin-streptavidin. In this study we purified and radiolabelled a number of monoclonal antibodies, determined the specific radioactivity by self-displacement analysis, and used the radiolabelled antibody in experiments where the number of molecules of antibody bound per cell and the fluorescence intensity were measured on the same sample. This permitted us to determine the sensitivity of the fluorescence procedure in molecules per cell, using several different antibody/target cell combinations. The method was consistently capable of detecting fewer than 100 molecules of antibody bound per cell.