Characterization of a monoclonal antibody to human serum vitamin D binding protein (Gc globulin): recognition of an epitope hidden in membranes of circulating monocytes.
We have developed a murine hybridoma cell line that secretes a monoclonal antibody directed to the serum human vitamin D binding protein (hDBP), a 58,000-dalton alpha-globulin with a high avidity for 25-hydroxycholecalciferol and globular actin. This immunoglobulin G1 kappa-light chain antibody was produced by the fusion of the spleen cells from BALB/c mice, immunized with purified hDBP, with SP2/0-AG4 myeloma cells. The antibody was easily removed from the supernatant of hybridoma cultures or mouse ascites fluid by Protein A affinity chromatography. Apparent serum monospecificity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing gels transblotted to nylon membranes and overlayed with purified MAK 89 antibody and radioiodinated Protein A. The affinity of the antibody is high [dissociation constant (Kd) = 2.6 X 10(-11) M]. Parallel displacement of tracer by hDBP and human serum was observed. The sera from various species displaced the hDBP tracer in the following potency: monkey more than cat more than dog more than guinea pig. RIAs for DBP from several species are feasible with this antibody. This antibody does not, in contrast to polyclonal anti-hDBP antiserum, bind to viable monocytes. However, the MAK 89 antibody does bind to the membranes of well washed, fixed, and permeant circulating monocytes. Surface membrane radioiodination of monocytes and immunoprecipitation of the detergent lysates with the antibody demonstrates a protein with molecular weight equivalent to hDBP. The epitope recognized, therefore, appears to be hidden in the viable cells, suggesting an intimate and intricate association of the hDBP and monocyte plasma membrane.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002415:Cats; D002520:Cercopithecidae; D004285:Dogs; D000939:Epitopes / Q000276:immunology; D006168:Guinea Pigs; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D009000:Monocytes / Q000276:immunology*; D013045:Species Specificity; D014809:Vitamin D-Binding Protein / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D008565:Membrane Proteins; D014809:Vitamin D-Binding Protein