Evaluation of the expression of IFN-gamma in lymphocytes using a monoclonal antibody and flow cytometry.
A stable hybridoma cell line secreting specific antibodies against human interferon-gamma (IFN-gamma) and designated IGMB-14 has been established. It belongs to the IgG1, kappa subclass and it reacts in Western blot with the 28 kDa and 56 kDa polypeptides present in two different affinity purified IFN-gamma preparations. Peripheral blood mononuclear cells (PBMC) from a healthy individual, stimulated in vitro by PHA, were analysed for IFN-gamma production both when viable and following fixation. The presence of cytoplasmic or surface IFN-gamma was visualized by an indirect immunofluorescence 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-gamma molecules in lymphocytes at day 1 after PHA stimulation and surface IFN-gamma at day 2. IFN-gamma 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-gamma staining by pretreatment of MAb IGMB-14 with IFN-gamma. The presence of surface IFN-gamma 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-gamma-producing cells in vitro and, in addition, for evaluating the presence of PBMC expressing IFN-gamma on their surface during a viral disease.
D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D006825:Hybridomas / Q000032:analysis; D007371:Interferon-gamma / Q000276:immunology / Q000302:isolation & purification*; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D007371:Interferon-gamma