Characterization and biological activity of a rat monoclonal antibody to rat/human corticotropin-releasing factor.
To produce a rat monoclonal antibody directed to rat CRF (rCRF), female Wistar rats were actively immunized with a rCRF-bovine thyroglobulin conjugate. Immunization resulted in the formation of CRF antibodies, as indicated by binding of [125I]iodo-rCRF and attenuation of the plasma corticosterone responses to ether stress. Rat spleen cells were fused with mouse myeloma P3 cells, and a hybridoma clone (PFU 83) was selected according to its capacity to bind [125I]iodo-rCRF and inhibit rCRF-induced ACTH release from cultured rat pituitary cells. PFU 83 antibodies (IgG2a subclass) are directed to the extreme C-terminal part (amino acids 38-39) of rCRF and bind with an affinity constant of 21 nM. In vitro, PFU 83 causes a parallel shift to the right of the rCRF dose-ACTH response curve, with an apparent affinity of 10 nM. PFU 83 neither binds nor blocks the ACTH-releasing activity of oCRF. Intravenous administration of PFU 83 ascites (generated in nude mice) to Wistar rats caused a dose-dependent inhibition of ether-induced ACTH secretion. Full blockade of the ACTH response to ether was found at a dose of 10 nmol PFU 83/rat. Based on the dynamics of rCRF binding and its in vitro and in vivo effects, we conclude that the rCRF-blocking bioactivity of PFU 83 is due to binding of PFU 83 to native rCRF and formation of a biologically inactive complex. Finally, we found that PFU 83 did not affect resting or ether-induced alpha MSH secretion, indicating that CRF does not play a major role in the control of alpha MSH secretion.
van Oers
J W
JW
Department of Pharmacology, Medical Faculty, Free University, Amsterdam, The Netherlands.
Tilders
J H
JH
Berkenbosch
F
F
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D000941:Antigens / Q000276:immunology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003346:Corticotropin-Releasing Hormone / Q000276:immunology* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D007116:Immunization, Passive; D005779:Immunodiffusion; D007700:Kinetics; D008297:Male; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism; D011388:Prolactin / Q000097:blood; D051381:Rats; D011919:Rats, Inbred Strains; D000521:alpha-MSH / Q000097:blood
D000911:Antibodies, Monoclonal; D000941:Antigens; D000521:alpha-MSH; D000324:Adrenocorticotropic Hormone; D011388:Prolactin; D003346:Corticotropin-Releasing Hormone