Chemiluminescent determination of xanthine oxidase activity using a sensitive low-light detection system

A highly sensitive and rapid chemiluminescent assay for the determination of the activity of xanthine oxidase (XOD) was developed. The chemiluminescent signal was obtained from die catalyzed oxidation of hypoxanthine, accelerated and amplified using a Fe-EDTA complex and perborate, which acts on luminol. The same luminescent mixture was previously used as detection system for immunoassays. Two different mixtures were used, which differ in their luminol and perborate content, with (CLMrho) or without (CLMb) addition of 0.1 μM rhodamine fluorophor. The response obtained from XOD standard solutions in buffer was linear from 5 to 500 U L-1 and from 0.7 to 250 U L-1 for CLMrho and CLMb respectively, at 25 °C. 5 and 0.7 U L-1 were the detection limits at 1 standard deviation level. The intra- and inter-assay relative standard deviations ranged from 6 to 12 % for both CLM. Measurements were made using the high performance, low-light level imaging Berthold luminograph LB-980 which allows simultaneous determination of several samples distributed on a microtiterplate. Various kinds of milk were analyzed for XOD content, which in pasteurized milk depends on the fat content and in the UHT milk disappears owing to the heat treatment.