Construction and clinical validation of a sensitive and specific assay for serum mature calcitonin using monoclonal anti-peptide antibodies.

Using calcitonin (CT) as an hapten, we have generated a library of monoclonal antibodies. Six monoclonal antibodies were developed and analyzed with respect to affinity and specificity for epitopes on CT by enzyme linked immunosorbent assay and radioimmunoassay. These antibodies were used in the construction and the optimization of a two-site monoclonal immunoradiometric assay (m-IRMA) for CT. We used a combination of two monoclonal antibodies to develop an assay which is rapid (overnight incubation), sensitive (10 pg/ml) and strictly specific for the mature form of calcitonin, ie the 32 amino acid-long polypeptide bearing a prolineamide as the C-terminal residue. This assay was utilized to demonstrate that mature calcitonin circulates as heterogeneous molecular species resulting from polymerization of calcitonin by disulphide linkage and/or by aggregation on irrelevant proteins. The clinical relevance of this assay was studied on a series of patients with medullary carcinoma of the thyroid (MCT) and the characteristics of the assay was compared with those of a conventional polyclonal radioimmunoassay. The m-IRMA for CT proved to be useful for both the diagnosis and the follow-up of MCT.

Utilization of synthetic peptides for the study of calcitonin and biosynthetic precursors for calcitonin.

By using synthetic peptides and a library of monoclonal anti-peptide antibodies, we have developed a panel of techniques that allow the dissection of circulating immunoreactive calcitonin in the serum. C Cells of the thyroid were found to release both mature calcitonin and biosynthetic intermediates in the circulation. Finally, these products were found to circulate as heterogenous molecular species. A methodology for the standardization of the measurement of calcitonin is proposed in the form of a two-site immunoradiometric assay specific for mature calcitonin.

Radioiodination of human calcitonin using the Iodogen reagent.

A method is described for the radioiodination of human calcitonin using the Iodogen reagent. The tracer was purified by 2 successive gel filtrations. The first, using Sephadex G-10, resulted in the complete removal of free 125iodide as assessed by ascending paper chromatography. The second, involving Sephadex G-75, permitted the separation of the tracer from high molecular weight aggregates. Analytical RP-HPLC showed a slight proportion (13%) of a contaminating product, probably (sulfoxyl-methionine9)calcitonin. The final yield, after radioiodination, was found to be 42.7 +/- 5.5% and the tracer displayed a specific activity of 850 +/- 110 Ci/mmol. The effects of long-term storage of the frozen tracer were studied.