The Rose Bengal assay for monoclonal antibodies to cell surface antigens: comparisons with common hybridoma screening methods.
An automated colorimetric procedure for detection of antibodies specific for cell surface antigens (1) has been compared for specificity and sensitivity to other methods of hybridoma supernatant screening. The Rose Bengal colorimetric assay (RBA) compared favourably in these respects with whole cell radioimmunoassay and indirect immunofluorescence with manual or flow cytometric analysis (FACS). A major advantage of the method is that it allows a large number of samples to be screened in a comparatively short time. Unlike other semi-automated colorimetric assays, such as ELISA, the procedure does not require cell fixation, which can destroy some antigenic determinants. The original assay of O'Neill and Parish (1) has been modified to give increased sensitivity and also to enable the detection of erythrocyte specific antibodies by elimination of the dye staining step and direct measurement of haemoglobin by spectrophotometry. The RBA allows detection of monoclonal antibodies (MoAb's) binding to only a proportion of the cells in a sample, which is an important feature when hybridoma supernatants are screened for reactivity to a minor cell population, for example against leukaemic cell samples with low percentages of blast cells.
A B
AB
Lyons
L K
LK
Ashman
D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis*; D002460:Cell Line; D003124:Colorimetry / Q000379:methods; D000939:Epitopes / Q000032:analysis; D004910:Erythrocyte Membrane / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D006801:Humans; D006825:Hybridomas / Q000032:analysis*; D007951:Leukemia, Myeloid / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods; D012395:Rose Bengal; D013347:Subcellular Fractions / Q000032:analysis; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000939:Epitopes; D012395:Rose Bengal