High-performance assays of small molecules: Enhanced sensitivity, rapidity, and convenience demonstrated with a noncompetitive immunometric anti-immune complex assay system for digoxin

Abstract

We report the first clinical application of a noncompetitive immunometric assay system that provides advantages for the rapid and robust assay of small molecules similar to those realized for larger molecules with two-site immunometric assays. This anti-immune complex assay is based on the interaction of a receptor such as a primary antibody with its ligand, such that new binding sites, recognizable by a secondary antibody, are formed. In this report the system is applied to the measurement of digoxin in serum. Utilizing an anti-complex antibody that recognizes a digoxin-bound primary antibody with affinity >2000-fold over its binding to the primary antibody alone, we show that this anti-complex assay system provides a high- performance assay for serum samples, being conveniently simple (immobilized primary antibody binds digoxin and then labeled secondary antibody so that when excess unbound label is washed away the immunometric read-out reflects the digoxin concentration), rapid (incubation time 1-10 min), sensitive (detection limit 30 ng/L), precise (3-4% within-run CV, 1-8% total CV), and free from interference from digoxin-like immunoreactive factors.