Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies.
BACKGROUND
Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards.


METHODS
Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors.


RESULTS
Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 x 10(4)-1.5 x 10(5); CD8, 5.0 x 10(5)-2.1 x 10(6); CD3, 6.6-7.8 x 10(5). Binding constants were highest for unlabeled CD4 antibody, 2. 7 x 10(10)-2.1 x 10(12) M(-1), and then unlabeled CD3 antibody, 1.1 x 10(10)-1.9 x 10(11) M(-1). FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies.


CONCLUSIONS
Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.
Advanced Technology, Beckman Coulter, Inc., Miami, FL 33196-2500, USA. olav.siiman@coulter.com
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Burshteyn
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology*; D015704:CD4 Antigens / Q000276:immunology*; D016827:CD8 Antigens / Q000276:immunology*; D003227:Conjugation, Genetic; D003911:Dextrans; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D011956:Receptors, Cell Surface / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D003911:Dextrans; D000939:Epitopes; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011956:Receptors, Cell Surface