Biotin-Avidin Mediated Homogeneous Enzyme-Linked Binding Assays for Riboflavin and Riboflavin Binding Protein Using Alkaline Phosphatase as the Label

The feasibility of using alkaline phosphatase (ALP) as a generic label in a homogeneous type assay is examined. In the proposed ALP-based biotin-avidin mediated assay, the activity of the ALP-biotin conjugate is inhibited in the presence of the avidin-riboflavin conjugate, and the observed inhibition is reversed in an amount proportional to the concentration of riboflavin binding protein (RBP) added. The ALP activity of the mixture is re-inhibited upon additions of free analyte riboflavin, to an extent proportional to the concentration of riboflavin added to the assay mixture. An avidin-riboflavin conjugate with a desired inhibitory effect is prepared by protecting the binding-site of avidin with 2-iminobiotin during the conjugation reaction at pH 6.5. The catalytic activity of ALP, conjugated with biotin, is inhibited to a significant degree (up to 86%) by the binding of the avidin-riboflavin conjugate, and is fully recovered upon additions of RBP.