DETECTION OF THIOREDOXIN IN HUMAN SERUM AND BIOLOGICAL SAMPLES USING A SENSITIVE SANDWICH ELISA WITH DIGOXIGENIN-LABELED ANTIBODY

Thioredoxin is a low molecular weight, redox active protein important in cellular proliferation, signal transduction and antioxidant functio n. Thioredoxin is secreted by normal as well as neoplastic cells and i s potentially involved in paracrine cell communication as suggested by its co-cytokine activity. Thus, the thioredoxin level in biological f luids, cells and tissue homogenates could be an important indicator of physiological or pathophysiological conditions. Hence, an accurate an d sensitive measurement is of paramount importance in studies involvin g thioredoxin. We present here an ultrasensitive enzyme linked immune- absorbent assay (ELISA) for human thioredoxin using digoxigenin-labell ed goat polyclonal anti-human thioredoxin. The assay could detect a mi nimum level of 15 pg/ml thioredoxin in human serum, cell culture media , and in cell and tissue samples. The assay was optimized for concentr ation of both antibodies, blocking agent, plates, incubation time and reaction volumes. Excellent linearity and reproducibility were obtaine d. The assay was applied to different baboon tissues and human serum s amples. The intrassay coefficient of variation (CV) was between 6.0 to 14 and the interassay CV was from 1.6 to 11.1 Excellent parallelism o f standards with serum samples, tissue homogenates or cell lysates was obtained. More than 90% recovery of human thioredoxin was observed in 10% human serum. The assay is easy to use, rapid, reproducible, but a bove all it is a quantitative, specific and sensitive way to measure t hioredoxin in a variety of biological specimens. (C) 1998 Elsevier Sci ence B.V.