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.