Coupling aminohexyl-FAD to proteins with dimethyladipimidate

We used an efficient method having general applicability to couple N6-aminohexyl-flavin adenine dinucleotide (AHFAD) to several proteins for use in an apoenzyme reactivation immunoassay system (ARIS). AHFAD is first activated with 40-fold molar excess of dimethyladipimidate, excess imidate is removed rapidly by gel filtration, the activated product is incubated with the protein, and the conjugate formed is purified. This labeling technique permits incorporation of a controlled amount of amino-label into a protein, and eliminates the possibility of self-crosslinking, which would reduce the immunoreactivity of the conjugate. Here we demonstrate the utility of such a conjugate in a totally automated ARIS assay for thyroxin-binding globulin (TBG). After a competitive protein-binding reaction, apoglucose oxidase is added to combine with free TBG-AHFAD conjugate and produce active glucose oxidase, which is measured colorimetrically in a peroxidase-linked reaction. The assay covers the clinically significant range for TBG from 0 to 60 mg/L and has a throughput of 60 reactions in 75 min. Comparison with an RIA method (x) by regression analysis yielded the equation y = 0.890x + 1.217 (r = 0.975, n = 47, Syx = 1.906 mg/L).