NEW NONCOMPETITIVE IMMUNOASSAYS OF SMALL ANALYTES

We developed a novel noncompetitive immunoassay format for monoepitopi c analytes and describe here a model assay for triiodothyronine (T-3), performed on Ciba Coming's ACS:180 analyzer. Acridinium eater (AE)-la beled bivalent anti-T-3, was incubated with the sample, producing AE-a nti-T-3/T-3 complexes and unreacted AE-anti-T-3. Controlled-pore glass particles (CPG) with immobilized diiodothyronine (T-2) were then adde d in excess, to bind AE-anti-T-3 possessing two unoccupied binding sit es but not AE-anti-T-3 bound to one or two T-3 molecules. Paramagnetic particles (PMP) with immobilized anti-AE were then added to the same cuvette to capture AE-anti-T3T3 complexes; AE-anti-T-3 bound to the su rface 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 chemilumines cence associated with the PMP remaining in the cuvette was then measur ed. This noncompetitive T-3 assay exhibited a 10-fold lower detection limit than the equivalent competitive T-3 assay, i.e., 0.3 vs 3 pg/tes t. 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.