Immunometric Approach to Peptide Hormone Analysis

Abstract

Theoretically, reagent-excess assays employing labelled Ab’s* (immunometric assays) offer several advantages over limited-reagent labelled antigen assays (e.g. RIA) for peptide hormones, notably in respect of detection limits, specificity, working range, incubation time and ruggedness — as now explored. For ACTE we have developed and selected compatible mouse MAb’s directed towards the near-N-terminal (residues 14–18) and C-terminal (residues 24–39) sequences. A two-step protocol is employed: incubation of standard or test sample with ¹²⁵I-labelled N-terminal Ab, then addition of excess solid-phase (Sephacryl S300) C-terminal Ab. There was marked benefit, vs. RIA using the same Ab’s, to detection limit (2 ng/l), working range and assay time (<24 h). The assay detected intact 1–39 ACTE, large mol. wt. precursors of the proopiomelanocortin family but not ACTE fragments.

For TSE we have compared 6 commercial two-site assays employing MAb’s and either radioisotopic or non-isotopic labels. All had detection limits <0.2 (some 0.05) ml]/l, wide working ranges and overall incubation times <24 h. All were more sensitive with wider working ranges than current RIA’s, and consistently allowed thyrotoxic and euthyroid patients to be distinguished by TSE levels. We conclude that the IMA approach to peptide hormone analysis is an important technical advance with major clinical applications.