Isolation of B lymphocytes for HLA-DR typing using monoclonal antibodies.
Isolating sufficient B cells for DR typing is often a problem, particularly in patients with low lymphocyte and/or B cell counts. This paper describes a simple method for preparing highly purified B cells using monoclonal antibodies and complement to kill the majority of non-B cells. Mononuclear cells are isolated by standard Ficoll-Hypaque techniques and incubated at 0 degrees C for 10 min with the following monoclonal antibodies: OKT3 (pan T cell), OKT11 (pan T and null cell), and Leu11b (NK cell). Rabbit complement is added and the mixture is incubated at 37 degrees C for 20 min. Dead cells are then removed by Percoll gradient centrifugation. This procedure results in cell preparations containing 90.1 +/- 1.3% B cells as measured by two-colow immunofluorescence with an Epics C flow cytometer. A yield of 52% can routinely be obtained. In contrast, nylon wool adherence results in preparations with only 44 +/- 9.1% B cells with a yield of 31%. B cells prepared by the monoclonal antibody procedure always show stronger and clearer reactions on DR typing trays. These results are consistent with the flow cytometric analyses of the B cell preparations. In summary, this procedure has the following advantages: increased accuracy of DR typing, decreased sample size (only 1.0 X 10(7) mononuclear cells are required), reduced time of cell preparation, and reduction in the number of repeat typings necessitated by poor yields and/or results.
D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D000920:Antibody-Dependent Cell Cytotoxicity; D000954:Antigens, Surface / Q000032:analysis; D001402:B-Lymphocytes / Q000145:classification; D002469:Cell Separation / Q000379:methods*; D003165:Complement System Proteins / Q000276:immunology; D005434:Flow Cytometry; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II / Q000032:analysis*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D003165:Complement System Proteins