Detection of Low Numbers of Lymphocyte Surface Membrane Molecules Using Concentration Immunofluorescence Analysis (CIA)

Abstract

Using viable lymphocytes in a concentration immunofluorescence assay (CIA), conditions were ascertained which allow detection of low numbers of surface membrane molecules. Utilizing 3 layer labeling [monoclonal antibody (MAb) specific for the membrane molecule, biotin-conjugated antibody specific for the MAb, and R-phycoerythrin-strepavidin], large numbers of lymphocytes in the assay wells, and the Pandex Fluorescence Concentration Analyzer, a fluorescence signal significantly above background was generated by as few as 8x10⁷ molecules among 4x10⁵ lymphocytes. Experiments using membrane molecules (FCγR II, Ia antigens, Ly-39) which differ considerably in their level of expression indicated that comparable signals were generated by equivalent numbers of labeled molecules in a cell population irrespective of the number of molecules on an individual cell. Thus, CIA is theoretically capable of detecting membrane molecules whose expression is as low as 2x10² per cell. CIA should be useful in the assay of cytokine receptors and other lymphocyte membrane molecules expressed at low levels, and in the development of MAb specific for these molecules.