PRODUCTION AND FLOW CYTOMETRIC APPLICATION OF A MONOCLONAL ANTI-GLUCOCORTICOID RECEPTOR ANTIBODY

Detection and monitoring the expression and level of intracellular glu cocorticoid receptor (GCR) is necessary in many clinical and experimen tal situations. Binding of radioactive steroids (H-3 dexamethasone) to the cytosolic fractions of cells has been recently used. However, it is an expensive, time-consuming technique difficult to use in routine diagnostics. In this article we describe a novel, simple method for GC R detection, using a FITC-conjugated anti-GCR monoclonal antibody (mAb ) for flow cytometric measurements in permeabilized cells. The monoclo nal antibody was raised against a conserved sequence (150-176 amino ac ids) of the regulatory part of the receptor. Synthetic peptide (called APTEK-26) fragment of the receptor conjugated to different carriers ( TG, BSA) was used for immunization and screening of the hybridomas. Th e a-GCR 8E9, 3C8 and 5E4 clones (IgG1) were further characterized by i mmunoserological methods for their reactivity against overlapping synt hetic peptide fragments of the receptor and by Western blot technique on cytosolic fraction of HEP G2 cells (containing the GCR). Furthermor e the mAbs could be used for the FAGS based detection of GCR, despite its low number of antigen structure within the cells. Solving the prob lem of nonspecific binding of the secondary antibodies we used our hig h affinity IgG1 a-GCR mAbs directly labeled with the fluorescent dye F ITC. The fluorescent labeling of the GCRs in HEP G2 cell line and huma n peripheral blood mononuclear cells (PBMC) were demonstrated by flow cytometric analysis after fixation with 4% paraformaldehyde and permea bilization with saponin. Competition with molar excess of unlabelled a ntibodies and with the GCR peptide fragment confirmed the specific bin ding of the 8E9 and 5E4 mAbs to the GCRs. Monitoring the GCR level by flow cytometry would be useful in clinical diagnostics, e.g., in stero id-treated patients and in steroid-resistant states. (C) 1998 Elsevier Science B.V. All rights reserved.