Chemiluminescent reagent and assay using a substituted acetanilide for light generation

An aqueous composition for providing a chemiluminescent signal comprising:

• a) an oxidant in an amount of from about 1 to about 10 mmolar,
• b) a low molecular weight cationic surfactant present at from about 0.05 to about 2% above its critical micelle concentration, or a cationic polymer present at from about 0.01 to about 2% by weight,
• c) a buffer to maintain the pH of said composition at from about 6 to about 8.5, and
• d) a substituted acetanilide which is present in a total amount of from about 0.05 to about 10 mmolar as the sole chemiluminescent signal generating reagent which provides a signal in response to the catalytic activity of peroxidase, said substituted acetanilide having the structure (I):
• ##STR4## wherein R.sup.1 is hydrogen or alkyl of 1 to 4 carbon atoms,
• R.sup.2 is hydrogen, alkyl of 1 to 4 carbon atoms, alkoxyalkyl of 1 to 4 carbon atoms, hydroxyalkyl of 1 to 4 carbon atoms, aminoalkyl of 1 to 4 carbon atoms, haloalkyl of 1 to 4 carbon atoms, or alkenyl of 2 to 5 carbon atoms,
• R.sup.3 is hydrogen or alkyl of 1 to 4 carbon atoms,
• at least one of R.sup.4 and R.sup.5 is an electron withdrawing group having a Hammet sigma value of at least about 0.01, wherein any R.sup.4 or R.sup.5 which is not said electron withdrawing group is hydrogen, and R.sup.6 and R.sup.7 are independently hydrogen, halo, cyano or methyl,
• provided that said composition is substantially free of any other chemiluminescent agent.