New patented approach allowing the development of immunometric assays for haptens

Since the seventies, it has been largely demonstrated that immunometric assays (mainly two-site immunometric assays) allow much more sensitive measurements than conventional competitive immunoassays. Immunometric assays have not been currently applied to haptenic molecules because it is well known that haptens are to small to allow simultaneous binding of two different antibodies. Consequently, the detection limit for the compounds remains far above those observed with high molecular weight antigens.

We have developed a new procedure that is based on the use of a single monoclonal anti-body using the same epitope for capture and tracer binding. This method named SPIE-IA (Solid-Phase Immobilised Epitope-Immuno-Assay) is suitable for establishing immunometric assays for haptens whatever their size. It is based on the following principles illustrated in the cartoons.

All in all, after specific immuno-reaction with a solid-phase immobilised monoclonal antibody (step 1), the hapten is covalently linked to the antibody by means of a bifunctional reagent (step 2). After washing the plate, a dissociating and/or denaturing treatment is applied so that the immobilised hapten is released from the antibody binding site (step 3). In a final step, the released epitope, covalently bound to the solid-phase, is reacted with the same monoclonal antibody labelled with acetylcholinesterase.

This procedure leads to linear standard curve with a signal proportional to the concentration of hapten introduced in the assay. A significant improvement in sensitivity is observed in comparison to the corresponding competitive assay (a 10 to 300 fold increase). This has been successfully applied to the determination of Angiotensin II. The Angiotensin II EIA kit is introduced hereafter.

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