title: Establishing a sensitive and specific assay for determination of glucocorticoid bioactivity.
abstract: Glucocorticoids are hormones that play a major role in energy homeostasis and stress response of the body. As drugs they are most frequently used for immunosuppressive and anti-inflammatory purposes. Glucocorticoids are exploited successfully in the treatment of a wide variety of diseases; however, some patients develop side-effects, while others fail to respond to this form of therapy. Alterations in pharmacodynamic and pharmacokinetic actions might contribute to individual differences in glucocorticoid sensitivity. Antibody-based methods such as RIA (Radioimmunoassay) and ELISA (Enzyme-linked immunosorbent assay) are routinely used to determine glucocorticoid serum levels. However, as these techniques measure the total amount of a specific glucocorticoid and do not discriminate between protein-bound and freely available (i.e. biologically active) glucocorticoids, the results do not necessarily reflect the active levels of glucocorticoid, i.e. the "glucocorticoid milieu" in a patient. Being able to determine glucocorticoid bioactivity in serum or other body fluids could help identifying glucocorticoid-sensitive or -resistant patients and help finding explanations for different responses in individual patients. For this reason, we established a glucocorticoid bioactivity assay that is based on the measurement of glucocorticoid-dependent reporter gene activity. Making use of a human T-cell leukemia line, equipped with the glucocorticoid receptor and the fluorescence protein Venus as the assay's reporter (Jurkat(GR)-MMTV-VNP), glucocorticoid bioactivity can be determined from small amounts of serum or other biologic fluids. The developed glucocorticoid bioassay is both sensitive and reproducible, without any relevant cross-reactivity with steroid hormones other than glucocorticoids and can be practically applied in daily laboratory routine.
authors.affiliation: Division of Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Austria.
authors.forename: Bastian
authors.initials: B
authors.lastname: Oppl
authors.forename: Anita
authors.initials: A
authors.lastname: Kofler
authors.forename: Siegfried
authors.initials: S
authors.lastname: Schwarz
authors.forename: Johannes
authors.initials: J
authors.lastname: Rainer
authors.forename: Reinhard
authors.initials: R
authors.lastname: Kofler
mesh_terms: D001681:Biological Assay / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D005938:Glucocorticoids / Q000032:analysis* / Q000493:pharmacokinetics*; D006801:Humans; D019169:Jurkat Cells; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid