A Radioassay for Serum B₁₂ Using Unsaturated Transcobalamin I as the B₁₂ Binding Protein

A radioisotopic assay for serum vitamin B₁₂ is described based on the fact the unlabeled B₁₂ will competitively inhibit the binding of Co⁵⁷B₁₂ to a specific B₁₂ binding protein, transcobalamin I. This protein is stable in dilute solution and at the concentration of Co⁵⁷B₁₂ used for the assay, its binding capacity increases much less than IF or normal serum as the concentration of B₁₂ increases in the reaction mixture. Because of the stability of the binding capacity of stored frozen TC-I containing serum, only a single standard curve need be established with each lot of binding protein. When a limited quantity of TC-I is incubated with 60 pg of CO⁵⁷B₁₂ and crystalline B₁₂ concentrations ranging from 10 to 160 pg/ml, a linear standard curve is obtained when the reciprocal of the fraction of Co⁵⁷B₁₂ bound to TC-I is plotted as a function of unlabeled B₁₂ concentration. This permits use of a simple arithmetic expression to determine the B₁₂ concentration of unknown extracts after experimentally determining the fraction of Co⁵⁷B₁₂ bound to TC-I. The serum B₁₂ concentration of 25 normal subjects ranged from 154 to 674 pg/ml while the range in 15 B₁₂ deficient patients was 0-126 pg/ml. The B₁₂ concentration in 4 patients with chronic myelogenous leukemia was greater than 1000 pg/ml. The mean variance of 15 serum extracts frozen and reassayed several weeks later was 7.4 per cent. The range of recovery of B₁₂ added to serum extracts was 84-118 per cent.