Competitive protein binding analysis of vitamin B 12 using vitamin B 12-free serum as a standard diluent

Abstract

Vitamin B 12 (B 12) was assayed using pooled human serum as binding agent and DEAE-cellulose for separation of bound from free B 12. It was shown that the assayed B 12 concentration was dependent on a factor not recognised before, namely the amount of binding serum. This effect could be corrected by adding B 12-free serum to the standard solutions. Stability of pooled human serum was improved by previous separation of the large lipoproteins. The assay procedure was simplified by predosing of DEAE-cellulose and [ 57Co]B 12, extraction with glutamic acid to avoid centrifugation and using filtration as the method for separation of bound from free B 12. The correlation with the Lacto-bacillus leichmannii bioassay was determined. The influence of several drugs and hormones on the assayed B 12 concentration was investigated. Ranges for normal and B 12-deficient sera were established.