Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies.
Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.
Sacks
D B
DB
Department of Pathology, Brigham & Women's Hospital, Boston, Massachusetts.
Porter
S E
SE
Ladenson
J H
JH
McDonald
J M
JM
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000276:immunology* / Q000378:metabolism; D002417:Cattle; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011863:Radioimmunoassay
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002147:Calmodulin; D002118:Calcium