TY - JOUR
T1 - Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41
AU - Milton, N. G.N.
AU - Hillhouse, E. W.
AU - Nicholson, S. A.
AU - Self, C. H.
AU - McGregor, A. M.
PY - 1990/10/1
Y1 - 1990/10/1
N2 - Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7.1 and 6.8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.
AB - Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7.1 and 6.8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.
UR - http://www.scopus.com/inward/record.url?scp=0025161191&partnerID=8YFLogxK
U2 - 10.1677/jme.0.0050159
DO - 10.1677/jme.0.0050159
M3 - Article
C2 - 2248688
AN - SCOPUS:0025161191
SN - 0952-5041
VL - 5
SP - 159
EP - 166
JO - Journal of Molecular Endocrinology
JF - Journal of Molecular Endocrinology
IS - 2
ER -