Development and performance of a highly sensitive and specific two-site immunometric assay of calcitonin gene-related...

Calcitonin gene-related peptide (CGRP), a potent endogenous circulating vasodilator, is produced by the alternative splicing of the calcitonin/CGRP gene and is expressed mainly in neural and cardiovascular tissues. We recently reported a highly sensitive radioimmunoassay of CGRP, based on an antiserum recognizing the C-terminus of the molecule. We have also found that circulating immunoreactive CGRP is heterogeneous; thus we are unable to measure selectively the intact molecule with our one-site competitive approach. We therefore attempted to construct a two-site immunometric assay involving two antibodies, one that detects the C-terminus and another that recognizes the midregion of the molecule. To enhance assay sensitivity, we applied a colorimetric amplification system to this assay. This rapid, robust, and reproducible assay provides more nearly accurate estimates of circulating CGRP and offers a sensitive and more specific alternative to the radioimmunoassay, with advantages in speed, simplicity, and convenience.