Evaluation of the expression of IFN-γ in lymphocytes using a monoclonal antibody and flow cytometry

Abstract A stable hybridoma cell line secreting specific antibodies against human interferon-gamma (IFN-γ) and designated IGMB-14 has been established. It belongs to the IgG1, κ subclass and it reacts in Western blot with the 28 kDa and 56 kDa polypeptides present in two different affinity purified IFN-γ preparations. Peripheral blood mononuclear cells (PBMC) from a healthy individual, stimulated in vitro by PHA, were analysed for IFN-γ production both when viable and following fixation. The presence of cytoplasmic or surface IFN-γ was visualized by an indirect immunofluoresence assay using monoclonal antibody (MAb) IGMB-14 and a single laser FACS-III fluorescence-activated cell sorter. The staining permitted the detection of newly synthesized cytoplasmic IFN-γ molecules in lymphocytes at day 1 after PHA stimulation and surface IFN-γ at day 2. IFN-γ was expressed on almost all the CD4 + lymphocytes as shown by a double staining technique. The specificity of the reaction was confirmed by Western blots and abolishing IFN-γ staining by pretreatment of MAb IGMB-14 with IFN-γ. The presence of surface IFN-γ was also visualized on freshly isolated PBMC from two patients suffering from measles and AIDS but not on PBMC from a healthy individual. The experiments showed that this immunofluorescent method is useful for the detection, enumeration, and phenotypic characterization of IFN-γ-producing cells in vitro and, in addition, for evaluating the presence of PBMC expressing IFN-γ on their surface during a viral disease.