RADIOASSAY OF SERUM VITAMIN B12 BY QUANTITATING THE COMPETITION BETWEEN Co57B12 AND UNLABELED B12 FOR THE BINDING SITES OF INTRINSIC FACTOR*

The extremely low concentration of vitamin B,2 in normal and B.2-deficient blood has, heretofore, necessitated the use of microbiologic methods for its assay.Such bioassays are not only indirect and time consuming, but also require particular bacteriologic skills.In an earlier report from this laboratory (1) preliminary data were pre- sented describing a simplified method for assaying this vitamin based on the competition between unlabeled vitamin B12 and isotopically labeled vitamin B12 for the binding sites of intrinsic factor.This investigation was prompted by the obser- vation that the electrophoretic mobility of Co60B1, could be altered by prior incubation with intrinsic factor (IF).Similar findings had been made by Schilling and Deiss (2) using human gastric juice instead of IF. Figure 1 is a diagrammatic il- lustration of radioelectroplhoretograms of free and IF-bound Co6OB12.Whereas free Co60B12 has essentially no electrophoretic mobility (Figure 1A), when bound to IF, it migrates toward the anode (Figure 1B).If the IF is incubated with a mixture of Co60BJ, and unlabeled B12, less of the Co60B,, will be bound because of the competition now present for the binding sites of IF.Two peaks of radioactivity appear, the free unbound Co60B,, at the origin and that bound to the IF (Figure 1C) .These findings are similar to those reported by Berson and co-workers (3) on the binding of in- sulin-I131 by specific anti-insulin antibodies in studies employing paper electrophoresis.As they exploited their observations in developing an im- munoassay for insulin (4, 5), so it seemed possible that the inverse relationship between the ratio of bound to free Co60B,2 and total concen-