A solid-phase enzyme-linked assay for vitamin B 12

Abstract

A new solid-phase enzyme-linked competitive binding assay for vitamin B 12 (cyanocobalamin) is described. The assay is based on the competition between analyte B 12 molecules and a glucose-6-phosphate dehydrogenase-vitamin B 12 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 solid-phase is inversely related to the concentration of B 12 in the sample. Under optimized conditions, the method can detect B 12 in the range of 3×10 −10 −1×10 −8 M (using 100 μ l sample) with high selectivity over other biological molecules.