Chemiluminescent acridinium esters (AEs) permit the development of high sensitivity ligand binding assays due to a combination of high intensity light emission and very low backgrounds. Here these advantages are exploited for use in homogeneous nucleic acid hybridisation assays using quenched chemiluminescence. AE chemiluminescence is conventionally initiated at highly alkaline pH. Novel “active” AEs were designed that permit initiation under conditions compatible with maintenance of nucleic acid hybrids (i.e. pH less than 9). Methyl red was found to be a dark quencher species capable of functioning at this pH. Practical application of the chemiluminescence quenching assay system has been demonstrated using two model nucleic acid hybridisation assays based on intra- and intermolecular emitter/quencher pairs.
