A new rapid and reproducible homologous immunoradiometric assay for amino-terminal parathyroid hormone in the rat

Abstract

Measurement of parathyroid hormone (PTH) in the rat is most often performed with competitive ligand radioimmunoassays (RIA) utilizing heterologous antibodies. We report here the validation of a newly developed homologous immunoradiometric assay (IRMA) for rat PTH. Two different goat antibodies to the amino-terminal sequence of rat PTH are utilized; one is immobilized onto plastic beads to capture the PTH molecules and the other is radiolabeled for detection. To test this new IRMA, 30 Sprague-Dawley rats were randomized into three treatment groups to receive by intraperitoneal injection: (1) saline 1 ml/kg (control); (2) calcium chloride 40 mg/kg (hypercalcemic); and (3) EDTA 300 mg/kg (hypocalcemic). Blood samples were taken at 0, 30, 60, 180, and 300 minutes after administration of the assigned treatment for measurement of ionized calcium (Ca²⁺) and serum PTH. Most of the variance in PTH levels was found to be due to changes in Ca²⁺ (r²=0.780, P<0.0001). There was also a close temporal relationship between the two, with the highest levels of PTH occurring at the same measured time points as the lowest Ca²⁺, and vice versa. The measured detection limit of the IRMA was 3 pg/ml with intra-and interassay coefficients of variation of 1.74% and 3.07%, respectively. Serial dilutions with pooled rat serum, synthetic rat PTH-(1–34), and synthetic human PTH-(1–34) showed good parallelism with increased specificity for the pooled and synthetic PTH, despite a degree of crossreactivity with hPTH. The assay is able to quantitate rapid changes in PTH, providing all the advantages of IRMA methodology including technical simplicity and speed of performance, and is likely to become a useful tool in investigations of bone, mineral, and renal homeostasis using the rat.