Direct antibody rosette-forming reactions using monoclonal markers of lymphocyte subpopulations: Methodology and applications illustrated by investigations with rat pan-T antibodies of the CAMPATH series

A sensitive direct antibody rosette assay has been developed for the detection of antigens on the lymphocyte cell membrane. Indicator cells for rosette tests were prepared by chromic chloride coupling of rat or mouse monoclonal IgG or IgM anti-lymphocyte antibodies to untreated or trypsinized bovine red blood cells. The monoclonal antibodies used were reactive with a range of cell surface markers which identify various lymphocyte subpopulations, including T cell antigens, HLA class II (Ia-like antigens), Leu-7 (HNK-1) and VEP 13, a determinant of Fcγ receptors on large granular lymphocytes. Results obtained by direct rosette formation correlated well with those of parallel tests using indirect immunofluorescent antibodies staining. Several applications of the direct antibody rosetting procedure are described in further investigations with a series of pan-T monoclonal (CAMPATH) antibodies. These include the morphological examination of antibody-binding cells in cytocentrifuge smears, the separation of lymphocyte subsets by density gradient centrifugation, and the use of a rosette inhibition assay to identify monoclonal antibodies binding to the same (or closely associated) epitopes of the lymphocyte cell membrane.