A Radioassay for Serum B 12 Using Unsaturated

I N THE PAST SEVERAL YEARS a number of radioassay procedures for serum vitamin B12 have been described. Each method is based on the fact that unlabeled crystalline B12 will competitively inhibit isotopically labeled B12 (tracer B12) from binding to a specific B12 binding protein(s). Basic to each procedure is the reaction of B12 (labeled and unlabeled) with a limited quantity of binding protein followed then by the separation and quantitation of the free and protein bound B12 fractions. By virtue of this competition between the two forms of the vitamin for the binding sites of the protein, the fraction of tracer B12 bound is inversely proportional to the concentration of unlabeled B12 in the reaction mixture. The first data obtained regarding serum B12 concentration with a radioassay system employing intrinsic factor ( IF) as the B12 binding protein was reported from this laboratory1’2 and the protein bound and free B12 fractions were separated by precipitation of the IF-B12 complex with zinc sulfate and barium hydroxide. In the procedure described by Barakat and Ekins,3’4 plasma was used as a source of binding protein, and the free and protein bound B12 was separated by equilibrium dialysis. Grossowicz et al5. also employed the B12 binding proteins of blood, but separated the bound and free B12 fractions with charcoal. Lau and co-workers6 used IF as the binding protein and separated the bound from the free B12 by adsorbing the latter on protein coated charcoal. Frenkel et al.7 more recently described an assay using normal serum as the source of binding protein, and DEAE cellulose for separating the bound and free B12. Although IF would appear to be most suited for use in such a radioassay because of its specific B12 binding property, experience in this laboratory with low concentrations of a number of partially purified preparations of this protein has revealed many disadvantages. Since the level of tracer B12 generally employed in the radioassay was in the order of 60 pg, the quantity of IF needed to bind 70-80 per cent of this tracer quantity was exceedingly small and such low concentrations of the IF preparations were quite unstable. Consequently, the B12 binding capacity of each IF preparation had to be checked just prior to use. Additionally, this variable binding capacity required