Development of sequence specific radioimmunoassay of human parathyroid hormone and its use in the diagnosis of hyperparathyroidism

Abstract

Two antisera which were raised against bovine parathyroid hormone (bPTH), and which cross-reacted with the human hormone, have been characterized. The antisera which originated from rooster and guinea-pig, were found to contain several populations of antibodies directed against both N-terminal and C-terminal sequences of the hormone. However, at proper dilutions the rooster antiserum did not bind the N-terminal fragment nor could this fragment displace the [¹²⁵] bPTH (1-84 amino acid residue) from binding to the antiserum. Furthermore, preincubation experiments with excess N-terminal fragment showed only a negligible reduction in maximal binding of the iodinated intact hormone using the rooster antiserum. In contrast, the guinea-pig antiserum reacted equally well with the N-terminal fragment and the intact hormone, and preincubation with this fragment reduced the binding of the [¹²⁵]bPTH (1-84 amino acid residues) by 75%. Gel filtration of hyperparathyroid serum on Bio-Gel P-60 showed immuno-reactive material which was measured with both antisera, eluting at a position similar to the intact hormone. However, in the C-terminal specific, but not in the N-terminal specific radioimmunoassay the major component eluted together with or somewhat earlier than the N-terminal bPTH fragment (1-34 amino acid residue), and this peak represented more than 90% of total immunoreactive PTH (iPTH) in serum. This major iPTH component must therefore represent fragment(s) with intact carboxy-terminal sequences. The N-terminal specific radioimmunoassay was unable to measure iPTH in about 80–90% of healthy individuals while the C-terminal specific assay detected iPTH in about 88% of these sera (equal to or above 0.1 μg/1). Similarly, the N-terminal specific antiserum measured consistently lower serum iPTH concentrations in patients with primary hyperparathyroidism. In thirty-four out of forty-one patients with surgically verified primary hyperparathyroidism, serum iPTH concentrations equal to or above 0.60 μg/1 were demonstrated using the C-terminal, specific radioimmunoassay.

Key Words:

• hyperparathyroidism
• parathyroid hormone
• radioimmunoassay
• sequence specific