AJP - Endocrinology and Metabolism, Vol 261, Issue 2 E262-E268, Copyright © 1991 by American Physiological Society
Homologous amino-terminal radioimmunoassay for rat parathyroid hormone
M. S. Calvo, C. M. Gundberg, H. Heath 3rd and J. Fox
Division of Endocrinology and Metabolism, Mayo Clinic, Rochester, Minnesota 55905.
Existing radioimmunoassays for parathyroid hormone (PTH) in rat plasma are
based on cross-reactivity of rat PTH (rPTH) with heterologous antisera. We
used the synthetic NH2-terminal fragment of rPTH [rPTH-(1-34)] to develop a
homologous radioimmunoassay for circulating PTH. An antiserum to
rPTH-(1-34) was raised in a goat (G-813), and the same peptide was used as
radioligand (125I) and standard. Purification of the label by
high-performance liquid chromatography (HPLC) increased specific binding
greater than twofold and sensitivity by 50-100%. With a final antiserum
dilution of 1:70,000, maximum specific binding of 30-33%, nonspecific
binding of 1-5%, and 50-microliters sample additions, the assay detection
limit was 1.8-2.5 pmol/l. A midregional fragment of human PTH did not
displace 125I-labeled rPTH-(1-34). HPLC of extracts of rat parathyroid
glands and hyperparathyroid plasma showed only a single peak of
immunoreactivity that eluted 2 min after rPTH-(1-34). Dose dilution curves
for rat parathyroid gland extracts, rPTH-(1-34) added to rat plasma, and
endogenous rat plasma PTH all paralleled the standard curve. Immunoreactive
PTH (irPTH) was detectable in greater than 90% of fasting normal rat plasma
and changed appropriately in response to hyper- and hypocalcemia induced by
low-calcium and vitamin D-deficient diets, injections of calcium and EDTA,
and after thyroparathyroidectomy. The normal range for rat plasma irPTH was
less than 2.0-12 pmol/l, in general agreement with bioassay results of
others. Thus rPTH-(1-34) is an excellent immunogen for raising antisera to
rPTH, and assays incorporating it may be of great value in studying rat
parathyroid physiology.
