A new sensitive homologous radioimmunoassay for amino-terminal parathyroid hormone in the rat

• Published in: Journal of bone and mineral research Vol. 7; no. 2; p. 229
• Main Authors: Finch, J L, Rapp, N, Martin, K J, Slatopolsky, E
• Format: Journal Article
• Language: English
• Published: United States 01.02.1992
• Subjects: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Iodine - metabolism; Molecular Sequence Data; Parathyroid Hormone - blood; Parathyroid Hormone - secretion; Radioimmunoassay; Rats; Sensitivity and Specificity; Sequence Homology, Nucleic Acid
• ISSN: 0884-0431
• DOI: 10.1002/jbmr.5650070215

The determination of circulating biologically active PTH in the rat has been difficult due at least in part to the inability to develop an antibody suitable for RIA of rat PTH. However, since the amino acid sequence of the rat PTH molecule has been deduced by molecular techniques, corresponding synthetic peptides have made it possible to produce such an antibody. A total of 12 roosters were immunized with synthetic rat PTH-(1-34), and one animal, RD1, developed a sensitive antibody against this amino-terminal region of the rat PTH molecule. To further increase the sensitivity of the RIA, we utilized an analog of rat PTH, Tyr1 rat PTH-(2-34), as the radioligand, which can be iodinated to high specific activity (450 microCi/micrograms). The iodinated peptide was purified by HPLC using a C18 Nova Pak HPLC column and a 20-60% acetonitrile gradient in 0.1% TFA. Synthetic rat PTH-(1-34) was used as the standard. To validate the RIA, we measured PTH under a variety of metabolic conditions. Normal values for PTH were 55.6 +/- 3.9 pg/ml (n = 26). Levels in parathyroidectomized (PTX) rats (n = 9) were undetectable, but renal insufficiency and vitamin D deficiency increased PTH to 587.4 +/- 141.3 pg/ml (n = 73) and 1662.0 +/- 137.8 (n = 27), respectively. Intraperitoneal (IP) administration of ethylenediaminetetraacetic acid (EDTA), 200 mg/kg, was used to decrease ionized calcium (ICa) from 4.75 +/- 0.07 to 3.55 +/- 0.10 mg/dl, which increased PTH from 51.3 +/- 5.9 to 109.3 +/- 13.4 pg/ml (n = 12).