T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay

The use of xanthine oxidase in immunoanalysis has never been reported. We describe here a procedure in which the xanthine oxidase dependent luminescence of luminol is enhanced in the presence of Fe-EDTA complex, providing an highly sensitive assay (3 amol of enzyme) and a long-term signal. This specific amplification has been applied to T4 and ultrasensitive TSH solid phase immunoassays, with T4-XO and anti-TSH monoclonal antibody-XO conjugates as tracers. The performances of these assays are at least equivalent to those obtained with iodinated tracers, using the same solid phases and the same calibrators. The major advantages of these immunoassays are: (1) the long-term signal which can be repeatedly recorded over several days, (2) the high detection sensitivity, (3) the long-term stability of the luminescence reagent and (4) the stability of the conjugates.