A study of the true competitive protein binding of current vitamin B12 radioassays

The use of purified intrinsic factor in vitamin B12 radioassays has greatly reduced the misdiagnosis of pernicious anaemia in recent years, but anomalies still occur. Observations made on current vitamin B12 radioassays suggest that many methods fail to convert all serum cobalamins to cyanocobalamin and thus are not true competitive protein binding assays. No KCN in the extraction buffer resulted in an average 30% reduction in the vitamin B12 level in normal sera. High concentrations of KCN caused a significant increase in the vitamin B12 level in serum from most vitamin B12 deficient patients. This increase could have caused a misdiagnosis in 7 of 12 patients studied. The specificity of (57Co) cyanocobalamin binding to both transcobalamin II (TCII) in a pool of normal sera and to intrinsic factor in a commercial kit binder reagent was 99%. We conclude that the KCN concentration in the extraction mixture should be between 2 and 5 mg/l.