Solid-Phase Cobalamin Assay Using Cobalamin-Binding Protein from Dog Stomach

Abstract

Ninety-five samples from 42 animals were studied for the presence of a cobalamin-binding protein fulfilling the criteria of occurring in major quantities and being homogeneous regarding size and binding constant. The binder from dog stomach thus found was made insoluble by coupling to bromoacetyl-cellulose and used for solid-phase assay of serum cobalamins. Equilibrium between free and bound radioactive cobalamin was unaffected by centrifugation of the samples, thus allowing the straightforward use of the Scatchard equation for calculation of results. The precision of the method was 0.05 to 0.10 at concentrations above 100 pmolxl⁻¹, the sensitivity was about 30 pmolxl⁻¹, the accuracy could be based on aqueous solutions of cyanocobalamin, and the reference interval was as for the Lactobacillus leichmannii method, i.e. 160–480 pmolxl⁻¹.

Key Words:

• Cobalamin
• cobalamin-binding proteins
• solid-phase assay
• vitamin B₁₃ quantitation