Homogeneous apoenzyme reactivation immunoassay for thyroxin-binding globulin in serum

In this automated apoenzyme reactivation immunoassay system (Ames Optimate) for thyroxin-binding globulin (TBG), the sample and N6-aminohexylflavin adenine dinucleotide-labeled TBG react sequentially with antiserum. Then apoglucose oxidase is added to combine with the free fraction and generate glucose oxidase activity, which is measured colorimetrically. The assay requires 100 microL of sample and covers the clinically significant range for TBG (less than 2.5 to 55 mg/L). The first result is obtained in 16 min; assay of 29 samples and their blanks is completed in less than 1 h. The lower limit of detection is about 2.5 mg/L. Between-assay CVs (n = 9) were less than 9%, within-assay CVs (n = 5) were less than 6%, and analytical recovery of TBG was 103-112%. Reagents are stable at 4 degrees C for at least five months. Results by this method for serum TBG (y) compared well with those determined by radioimmunoassay (x): y = 1.029x--0.352 (r = 0.990, n = 49, Syx = 1.165 mg/L). In addition, with 39 other sera the ratio of total thyroxin (by RIA) to TBG compared well with free thyroxin measured by equilibrium dialysis (r = 0.930) and the free thyroxin index (r = 0.970).