A rapid staining procedure for two-color analysis of lymphocyte antigen expression.
Two color immunofluorescence analysis of lymphocyte cell surface antigen expression using an unconjugated plus a biotinylated monoclonal antibody (mAb) requires four incubation steps: (1) unconjugated mAb; (2) fluorochrome-labelled goat anti-mouse Ig; (3) biotinylated mAb; (4) fluorochrome-labelled avidin or streptavidin. We describe a time-saving modification of this procedure which requires only two incubation steps: (1) simultaneous unconjugated and biotinylated mAbs; (2) fluorochrome-labelled avidin/streptavidin followed by fluorochrome-labelled goat anti-mouse Ig. The slightly delayed (5 min) addition of the goat anti-mouse Ig prevents it from binding to the mAb which has already interacted with avidin/streptavidin. Both procedures yield identical results with a variety of different mAbs.
Institute of Immunology, University of Heidelberg, Germany.
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Pechhold
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Kabelitz
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007074:Immunoglobulin G / Q000145:classification; D008214:Lymphocytes / Q000276:immunology*; D051379:Mice; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D007074:Immunoglobulin G