Application of a long-term enhanced xanthine oxidase-induced luminescence in solid-phase immunoassays

The use of xanthine oxidase (XO) as a label in immunoanalysis has not been previously reported. This can be explained by the difficulties encountered in XO assays (poor sensitivity and versatility) and the competitive inhibition of the enzyme by allopurinol, a widely used hypouricemic agent. We demonstrate here that both difficulties can be circumvented. (i) The XO-dependent luminescent signal related to the oxidation of luminol is dramatically enhanced in the presence of iron-EDTA complex and sodium perborate in alkaline buffer. The mechanism of this enhancement is consistent with an O2-driven Fenton reaction, leading to the production of highly reactive OH radical. (ii) Residual inhibition of solid-phase bound XO by serum allopurinol and its metabolites is spontaneously reversible and can be prevented by the presence of folic acid or azahypoxanthine in the incubation buffer. With these two problems solved, XO can be classified as a choice label in immunoanalysis with the following properties: (i) high detection sensitivity (3 amol label), (ii) long-term luminescent signal (several days), (iii) versatile preparation and stability of conjugates, and (iv) long-term stability of the luminescence reagent. As an example of application, some data concerning total IgE and direct 17 beta-estradiol assays are described. Several other luminescent immunoassays of large and small molecules have been developed using XO conjugates as tracer (free and total T4, ultrasensitive thyroid stimulating hormone, CA 19.9, prolactin, hCG, specific IgE, anti-toxoplasma, and anti-chlamydia IgG), thus proving that XO can be classified as a universal label.