A Solid-Phase Enzyme-Linked Assay for Vitamin B12

A new solid-phase enzyme-linked competitive binding assay for vitamin B12 (cyanocobalamin) is described. The assay is based on the competition between analyte B12 molecules and a glucose-6-phosphate dehydrogenase-vitamin B12 conjugate for a limited number of R-protein binding sites immobilized on sepharose particles. After appropriate incubation and washing steps, the enzyme activity bound to the solidphase is inversely related to the concentration of B12 in the sample. Under optimized conditions, the method can detect B12 in the range of 3 x 10-1~ x 10 -8 M (using 100 #1 sample) with high selectivity over other biological molecules.