Generation, characterization and utilization of anti-human growth hormone 1-43, (hGH1-43), monoclonal antibodies in an ELISA.
The major isoform of hGH is a polypeptide of 191 amino acids. Human GH1-43 is an amino terminal segment of hGH1-191 which comprises the first 43 amino acids. This peptide is a potent regulator of glucose homeostasis. To facilitate our understanding of the physiological regulation of hGH1-43 an assay to measure its levels in biological fluids and extracts is needed. This communication describes the development of anti-hGH1-43 monoclonal antibodies and their use in the development of an indirect competitive ELISA for the quantification of hGH1-43. Hybridomas were produced by the fusion of FOX-NY myeloma cells with spleen cells taken from a mouse immunized with hGH1-43. The hybridomas were screened for production of antibodies to hGH1-43 by antibody capture ELISA. Hybridomas which produced antibodies reactive to hGH1-43 were cloned by limiting dilution. Three monoclonal hybridomas, CCL-1, CCL-2, and CCL-3 were subsequently obtained. These hybridomas secreted antibodies that were highly reactive towards hGH1-43 but minimally reactive towards hGH1-191. The isotypes of the mAbs secreted by CCL-1, CCL-2 and CCL-3 were all IgG1 kappa as shown by isotype specific antibody capture analysis. An indirect competitive ELISA with a detection limit that ranged from 1 to 10 ng/ml was developed using mAbs from monoclonal hybridoma CCL-3. Dose-response curves for competing hGH1-191, hPRL, and hPL indicated minimal cross-reactivity of mAbs with these hormones and conversely, a high degree of specificity for hGH1-43. Dose-response curves for dilutions of human serum and pituitary extract were parallel to the standard. The availability of a sensitive assay for the measurement of hGH1-43 will help us answer questions regarding the biosynthesis, regulation of secretion, and role of hGH1-43 in the control of glucose homeostasis.
López-Guajardo
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CC
Division of Life Sciences, The University of Texas at San Antonio, 78249, USA.
Armstrong
L S
LS
Jordan
L
L
Staten
N R
NR
Krivi
G G
GG
Martinez
A O
AO
Haro
L S
LS
D000818:Animals; D000906:Antibodies / Q000032:analysis; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019382:Human Growth Hormone / Q000032:analysis* / Q000097:blood / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000032:analysis* / Q000097:blood / Q000276:immunology; D010902:Pituitary Gland / Q000737:chemistry
D000906:Antibodies; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D019382:Human Growth Hormone