Immunological, biochemical, and enzymic validation of radioimmunoassays specific to the amino and carboxy terminal of human calcitonin.

We characterized rabbit antisera to synthetic human calcitonin that specifically recognize the aminoand carboxy-terminal regions of calcitonin (antisera 1-506 and HMS-385, respectively), by both classical (parallelism of dose-response curves) and (owing to the unavailability of a variety of analog fragments of human calcitonin) biochemical plus enzymic alteration of synthetic human calcitonin. We used nonequilibrium double-antibody assay conditions and incubation times of 48 + 72 + 24 h. Final concentrations in the assay tube (1 mL) for 1-506 and HMS-385 were 1:100 000 and 1:200 000, respectively. Midpoint of the calcitonin dose-response curve was 95 and 425 pg in the 1-506 and HMS-385 assays, respectively. The intraand inter-assay CV was <8 and 17%, respectively, for each assay. The HMS-385 antiserum was shown to recognize the products of chymotryptic digestion plus oxidation and reduction of the amino-terminal disulfide bond of calcitonin in addition to complete immunological recognition of a synthetic carboxy-terminal hexapeptide fragment. The 1-506 antiserum failed to recognize the calcitonin fragments after chymotrypsin digestion and the carboxy-terminal hexapeptide fragment. Immunoreactivity in the 1-506 assay was also reduced by about 90% after oxidation and reduction of the amino-terminal disulfide bond. HMS-385 cross reacted completely with rat and human calcitonin; 1-506 was completely specific only to human calcitonin. Neither antisera bound salmon, porcine, or bovine calcitonins. The immunological determinants for the HMS-385 and 1-506 antisera evidently reside in the amino acid sequence of the extreme carboxy-terminal region and the conformation plus amino acid sequence of the amino terminal, respectively. In normal human sera, immunoreactive calcitonin was undetectable (<10-20 ng/L) in 87 and 53% of samples with the carboxyand amino-terminal antisera, respectively. In 97% of all normal sera analyzed with both antisera, immunoreactive calcitonin was <200 ng/L. In contrast, higher concentrations of carboxythan amino-terminal immunoreactivity were noted in extracts of normal thyroids and sera from cancer patients.