HOMOGENEOUS ASSAYS FOR RIBOFLAVIN MEDIATED BY THE INTERACTION BETWEENENZYME-BIOTIN AND AVIDIN-RIBOFLAVIN CONJUGATES

Homogeneous-type enzyme-linked competitive binding assays utilizing th e synthetic enzyme-biotin and avidin-riboflavin conjugates are develop ed for the detection of riboflavin as well as its binder protein, The activity of the enzyme-biotin conjugate is inhibited in the presence o f the avidin-riboflavin conjugate, and the observed inhibition is reve rsed in an amount dependent on the concentration of riboflavin binding protein (REP) added, Upon additions of free riboflavin to the mixture , activity is reinhibited in an amount proportional to the riboflavin concentration, Three different enzymes are examined as the labels: glu cose-6-phosphate dehydrogenase, adenosine deaminase, and alkaline phos phatase, The catalytic activity of these enzymes, when conjugated with biotin, is shown to be inhibited to a significant degree (>90%) by th e binding of the avidin-riboflavin conjugate, and reversed upon additi ons of REP. (C) 1995 Academic Press, Inc.