Measurement of cyanocobalamin in serum by a specific radioimmunoassay.

Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was estimated to be 8.6 X 10(9) and 9.6 X 10(6) L/mol, respectively. The association constant for adenosylcobalamin, methylcobalamin, and hydroxocobalamin was indirectly determined, and values of 2.5 X 10(5), 1.7 X 10(9), and 2.3 X 10(9) L/mol, respectively, were obtained. Photolysis in the presence of potassium cyanide rendered each of the three cobalamins equivalent to cyanocobalamin in immunoreactivity. The mean concentration of cobalamin in normal human sera and cobalamin-deficient sera measured as cyanocobalamin by radioimmunoassay using this anticobalamin antibody was significantly lower than the concentration measured in the same extracts by competitive ligand-binding radioassays using intrinsic factor and transcobalamin I. These findings, although indirect, support the proposition that there may be factor(s) in normal and cobalamin-deficient sera that falsely elevate the concentration of true cobalamin if the radioassay uses R protein as the binder. However, the lower concentration of serum cobalamin measured by radioimmunoassay compared with the intrinsic factor radioassay also indicates that this "purported" factor(s) reacts to some extent with intrinsic factor but not with the cobalamin antibody.