An immunoassay for small analytes with theoretical detection limits

Anew-based immunoassay that uses microspheres as the solid phase accomplish ed the theoretical limit of detect ability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was b riefly exposed to a bead pack containing immobilized estriol in a flow cell . A small portion of free antibody was separated rapidly from the mixture b y binding it to immobilized hormone, but the antibody-hormone complex was k inetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labe ling the separated antibodies on the beads with a Cy5-conjugated anti-speci es secondary antibody. By labeling after the separation step, perturbing th e liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumul ating free antibody onto the beads prior to quantification and, thus, offer ed ideal conditions to achieve theoretical limits of detectability. The opt imum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the K-d Of the antibody-estriol interaction, when the concentration of the antibody was below the K-d, the smallest detectable estriol concentration a pproached the theoretical limit of detectability achievable with this antib ody.