A method is provided for determining in a medium the presence or amount of an
analyte which is capable of binding to a ligand partner to form a ligand complex, which method comprises: (a) contacting the medium with either: (i) a ligand partner conjugated with an
enzyme being capable of catalysing a reaction, or one or more reactions in a
cascade thereof, to produce
hydrogen peroxide, or (ii) a ligand partner and either a competing
analyte or an analog of said
analyte, a competing analyte or analyte analog being capable of forming a ligand complex with the ligand partner and the competing analyte or analyte analog being conjugated with an
enzyme capable of catalysing a reaction, or one or more reactions in a
cascade thereof, to produce
hydrogen peroxide, (b) optionally separating complexed and uncomplexed
enzyme conjugates, (c) causing or allowing the reaction or
cascade of reactions to occur to produce
hydrogen peroxide by contacting the complexed or uncomplexed enzyme conjugate with a corresponding enzyme substrate; (d) contacting
hydrogen peroxide with a substance capable of exhibiting
chemiluminescence in the presence of
hydrogen peroxide selected from the group consisting of acridinium compounds and analogs thereof having a conjugate base with a pKa less then 9 to generate a chemiluminescent reaction, and (e) detecting the occurrence of said chemiluminescent reaction to determine the presence or amount of the analyte. Kits for use in connection with the method are also disclosed.