Solid phase enzyme-linked competitive binding assay for riboflavin

Abstract

A new solid-phase enzyme-linked assay for riboflavin (vitamin B2) is described. The assay is based on the competition between analyte vitamin molecules and a glucose-6-phosphate dehydrogenase-3-carboxymethylriboflavin conjugate for a limited number of riboflavin-binding protein sites immobilized on Sepharose particles. Significant improvements in conjugate catalytic activity and thus detectability are achieved by optimizing the reaction conditions used to covalently link 3-carboxymethylriboflavin to the enzyme. Optimization experiments include studying the effects of reaction pH and organic solvent composition. Final assay detection limits and the sensitivity of the dose-response curves are dependent on the ratio of conjugate to binding protein sites utilized in an equilibrium assay protocol. Selectivity of the method correlates well with that predicted based on the known association constants of riboflavin-binding protein with flavin analogs. The assay is shown to offer adequate detection limits and selectivity for direct measurement of riboflavin in urine, infant formula, and vitamin capsules.

A solid-phase enzyme-linked assay for vitamin B12

“…However, the rate of binding is significantly slowed after the first 2 h and thus, in subsequent competitive binding assays, we chose a 2.5 h time period for all incubations between the conjugate, immobilized binder, and standards or samples. Interestingly, in related work [5], we observed that the overall kinetics of conjugate binding are enhanced when the solidphase has lower levels of protein coverage. Therefore, in the present effort, we purposely prepared our solid-phase reagent using high mass ratios of Sepharose beads to R-protein.…”

Biosensor-Based Determination of Riboflavin in Milk Samples

“…Any remaining DMF, DCC, or sulfo-NHS was removed by ethanol, a solvent in which the activated ester product is not soluble. The activated ester (powder form) prepared in this manner was stable for at least 6 months when stored below 0 °C …”

Riboflavin in Nutrition, Food Processing, and Analysis - A Review

“…Efforts to develop high-affinity antibodies selective for riboflavin have met with limited success (Monroe, 1984), although recently Wang et al (2013) have claimed development of a sensitive and specific polyclonal antibody for riboflavin. Natural binders are utilized in most assays, such as riboflavin-binding protein from chicken egg white (Fazekas, Menendez, & Rivlin, 1974;Bashor & Tillotson, 1978;Geun & Meyerhoff, 1988).…”