Double immunofluorescence staining of pokeweed mitogen differentiated plasma cells--a sensitive assay to ascertain purity of anti-human Ig reagents as each cell produces only one isotype.
A simple, but highly sensitive assay is described for the determination of the specificity and purity of heterologous antibodies to human immunoglobulin isotypes. In this assay, cytocentrifuge cell preparations of normal pokeweed mitogen differentiated plasma cells are stained initially with an FITC conjugate of an antibody to 1 isotype and then restained with a TRITC conjugate of an antibody to another isotope. No double staining of intracytoplasmic immunoglobulin in plasma cells was observed when highly purified and specific reagents were used. However, some reagents from commercial laboratories, which used immunodiffusion assays and enzyme immunoassay techniques (ELISA) to determine specificity of antisera, were found to lack specificity by the double immunofluorescence technique. This technique of obtaining cultured plasma cells with a wide variety of isotypes is reproducible and utilizes reagents that are easily available. Hence, one need not depend on panels of myeloma plasma cells or spleen cells which can be difficult to obtain.
D000888:Antibodies, Anti-Idiotypic / Q000032:analysis*; D000918:Antibody Specificity; D003593:Cytoplasm / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007126:Immunoglobulin Allotypes / Q000096:biosynthesis / Q000276:immunology; D008213:Lymphocyte Activation; D010950:Plasma Cells / Q000276:immunology*; D011043:Pokeweed Mitogens / Q000494:pharmacology
D000888:Antibodies, Anti-Idiotypic; D007126:Immunoglobulin Allotypes; D011043:Pokeweed Mitogens