New noncompetitive immunoassays of small analytes

We developed a novel noncompetitive immunoassay format for monoepitopic analytes and describe here a model assay for triiodothyronine (T3), performed on Ciba Corning's ACS:180 analyzer. Acridinium ester (AE)-labeled bivalent anti-T3 was incubated with the sample, producing AE-anti-T3/T3 complexes and unreacted AE-anti-T3. Controlled-pore glass particles (CPG) with immobilized diiodothyronine (T2) were then added in excess, to bind AE-anti-T3 possessing two unoccupied binding sites but not AE-anti-T3 bound to one or two T3 molecules. Paramagnetic particles (PMP) with immobilized anti-AE were then added to the same cuvette to capture AE-anti-T3/T3 complexes; AE-anti-T3 bound to the surface of CPG, however, was not captured, because of steric hindrance. After the incubation, the PMP was magnetically separated to remove the liquid phase and the suspended CPG from the cuvette. The chemiluminescence associate with the PMP remaining in the cuvette was then measured. This noncompetitive T3 assay exhibited a 10-fold lower detection limit than the equivalent competitive T3 assay, i.e., 0.3 vs pg/test. Imprecision (CV) in the clinically significant range was 6% or less. The assay also displayed two- to sevenfold lower cross-reactivities and a wider dynamic range.