Homologous amino-terminal radioimmunoassay for rat parathyroid hormone.
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.
Calvo
M S
MS
Division of Endocrinology and Metabolism, Mayo Clinic, Rochester, Minnesota 55905.
Gundberg
C M
CM
Heath
H
H
Fox
J
J
D000818:Animals; D002118:Calcium / Q000008:administration & dosage / Q000097:blood; D004032:Diet; D005069:Evaluation Studies as Topic; D006041:Goats; D008297:Male; D010281:Parathyroid Hormone / Q000097:blood* / Q000737:chemistry; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats / Q000097:blood*; D011919:Rats, Inbred Strains; D013045:Species Specificity
D010281:Parathyroid Hormone; D002118:Calcium