Generation, characterization and utilization of anti-human growth hormone 1–43, (hGH 1–43), monoclonal antibodies in an ELISA

Abstract

The major isoform of hGH is a polypeptide of 191 amino acids. Human GH 1–43 is an amino terminal segment of hGH 1–191 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 hGH 1–43 an assay to measure its levels in biological fluids and extracts is needed. This communication describes the development of anti-hGH 1–43 monoclonal antibodies and their use in the development of an indirect competitive ELISA for the quantification of hGH 1–43. Hybridomas were produced by the fusion of FOX–NY myeloma cells with spleen cells taken from a mouse immunized with hGH 1–43. The hybridomas were screened for production of antibodies to hGH 1–43 by antibody capture ELISA. Hybridomas which produced antibodies reactive to hGH 1–43 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 hGH 1–43 but minimally reactive towards hGH 1–191. The isotypes of the mAbs secreted by CCL-1, CCL-2 and CCL-3 were all IgG1 κ 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 hGH 1–191, hPRL, and hPL indicated minimal cross-reactivity of mAbs with these hormones and conversely, a high degree of specificity for hGH 1–43. 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 hGH 1–43 will help us answer questions regarding the biosynthesis, regulation of secretion, and role of hGH 1–43 in the control of glucose homeostasis.