Homogeneous enzyme-linked competitive binding assay for riboflavin

A rapid and precise homogeneous enzyme-linked competitive binding assay for riboflavin (vitamin B₂) is described. The method utilizes a malate dehydrogenase/3-carboxymethylriboflavin conjugate in conjunction with soluble riboflavin binding protein. In the absence of the vitamin, the catalytic activity of the enzyme/riboflavin conjugate is inhibited up to 71% by the binding protein. In the presence of riboflavin, activity is regained in an amount dependent on the riboflavin concentration. The detection limits of the dose/response curves are dependent on both the degree of conjugation (average number of 3-carboxymethylriboflavins per enzyme molecule) and the reagent ratio (conjugate/binder) used in the assay tube. Under optimized conditions, a detection limit of 3 ng ml^?1 of riboflavin can be achieved with high selectivity over other vitamins and biomolecules. While malate dehedrogenase activity is inhibited to some degree by components of human urine, use of riboflavin standards prepared in a diluted urine matrix enables the method to be utilized for direct determination of urinary riboflavin.