In an assay method in which a member of a specific binding pair is detected by
means of an optically detectable reaction, the improvement wherein the optically
detectable reaction includes the reaction, with an enzyme, of a dioxetane having
the formula
##STR1##
where T is a cycloalkyl or polycycloalkyl group bonded to the 4-membered ring
portion of the dioxetane by a spiro linkage; Y is a fluorescent chromophore; X
is H, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl,
or enzyme-cleavable group; and Z is H or an enzyme-cleavable group, provided that
at least one of X or Z must be an enzyme-cleavable group, so that the enzyme cleaves
the enzyme-cleavable group from the dioxetane to form a negatively charged substituent
bonded to the dioxetane, the negatively charged substituent causing the dioxetane
to decompose to form a luminescent substance that includes group Y of said dioxetane.
