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Amylin is a 37-amino-acid polypeptide synthesized in and secreted from pancreatic beta cells along with insulin. Its biological actions include the slowing and reduction of postmeal increases in plasma glucose concentrations. Studies of the basic amylin biology in humans have been hampered by the lack of a rapid, sensitive assay capable of measuring physiological concentrations of amylin in small volumes of plasma. We report here two sandwich-type immunoassays that use pairs of monoclonal antibodies, the fluorescent substrate 4-methylumbelliferyl phosphate, and the enzyme alkaline phosphatase. The minimum detectable concentration of amylin in 50 microL of plasma was 0.5 to 2 pmol/L, and the dynamic range was 2 to 100 pmol/L. The assays had average intraassay CVs of <10%, average interassay CVs of <15%, and good linearity on dilution and recovery of added amylin. The two assays use the same detection antibody, which binds to the carboxyl terminus of the molecule, but different capture antibodies. One of the assays measures only human amylin; the other also detects amylin-like peptides. Examples of measurements in human plasma are provided in subjects with impaired glucose tolerance and in nondiabetic controls.

Highly selective monoclonal antibodies to the peptide hormone human amylin have been produced and characterized. These antibodies are produced by hybridomas resulting from the fusions of BALB/c-derived myelomas and splenocytes from either inbred or outbred mouse strains. Certain of these antibodies recognize epitopes at the amino-terminus or the amidated carboxy-terminus, as well as conformational epitopes within the central region of the 37 amino acid peptide. Several of these antibodies show less than 0.1% cross-reactivity with related peptide hormones such as calcitonin and calcitonin gene-related peptide (CGRP) and have apparent affinities in the low nanomolar range. Antibody pairs were selected for use in two-site assays for the direct measurement of endogenous amylin and the synthetic human amylin analogue, pramlintide (25, 28, 29 tripro-human amylin), which is presently under clinical investigation for improving glucose control in patients with both Type I and Type II diabetes treated with insulin.