CHEMILUMINESCENT DETERMINATION OF XANTHINE-OXIDASE ACTIVITY USING A SENSITIVE LOW-LIGHT DETECTION SYSTEM

A highly sensitive and rapid chemiluminescent assay for the determinat ion of the activity of xanthine oxidase (XOD) was developed. The chemi luminescent signal was obtained from the catalyzed oxidation of hypoxa nthine, accelerated and amplified using a Fe-EDTA complex and perborat e, 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 (CLMr ho) or without (CLMb) addition of 0.1 mu M rhodamine fluorophor. The r esponse 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 resp ectively, at 25 degrees C. 5 and 0.7 U L(-1) were the detection limits at 1 standard deviation level. The intra- and inter-assay relative st andard deviations ranged from 6 to 12% for both CLM. Measurements were made using the high performance, low-light level imaging Berthold lum inograph LB-980 which allows simultaneous determination of several sam ples distributed on a microtiterplate. Various kinds of milk were anal yzed for XOD content, which in pasteurized milk depends on the fat con tent and in the UHT milk disappears owing to the heat treatment.