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AssigneeNational Research Development Corporation
InventorsLarry J. Kricka, Angela M. O'Toole, Gary H. G. H. Thorpe, Thomas P. Whitehead
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It has been a problem that the chemiluminescent emission produced by the peroxidase-catalyzed oxidation of a 2,3-dihydro-1,4-phthalazinedione is often weak or gives a poor signal-background ratio and therefore does not give the high degree of sensitivity required for assay purposes, particularly for assay of peroxidase used to label a reagent for example in ELISA. The present invention solves the problem by the finding the certain narrowly defined aromatic amines are enhancers of the reaction and accordingly provides for their use in an assay and kit. The amines are of general formula (I) ##STR1## wherein the R symbols (that is, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6) have any of the meanings (a) to (j) given below, all other R symbols in each meaning are hydrogen atoms and the fused rings are to be read in the same configurational sense as formula I: (a) R=R.sup.1 =CH.sub.3 ; R.sup.4 = ##STR2## (b) R.sup.4 = ##STR3## cyclohexyl, or alkyl or alkoxy having 1 to 4 carbon atoms (c) R.sup.2, R.sup.3 and R.sup.4 together represent the polycyclic fused ring system: ##STR4## (d) R.sup.3 and R.sup.4 together represent the fused ring or ring system ##STR5## (e) R.sup.3 and R.sup.4 together represent the fused ring system ##STR6## and R.sup.5 and R.sup.6 together represent the fused ring ##STR7## (f) R.sup.2 =NH.sub.2 and R.sup.4 = ##STR8## (g) R.sup.2 =R.sup.6 =CH.sub.3 and R.sup.4 = ##STR9## (h) R.sup.3 =C.sub.2 H.sub.5 (i) R.sup.2 =R.sup.4 =CH.sub.3 and (j) R.sup.3 =R.sup.4 =CH.sub.3.
In a luminescent or luminometric assay which comprises carrying out a chemiluminescent reaction between a peroxidase, an oxidant, and a chemiluminescent 2,3-dihydro-1,4-phthalazinedione and measuring or detecting the chemiluminescence thereby produced, the improvement wherein the reaction is carried out in the presence of an aromatic amine of the general formula ##STR20## wherein the R symbols, namely R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6, have any of the meanings (a) to (j) given below, all other R symbols in each meaning are hydrogen atoms and the fused rings are to be read in the same configurational sense as formula 1, whereby sides "a", "b" and "d" in formula (1) are fused to sides "a", "b" and "d" respectively in the formulae below:
A kit for use in a luminescent or luminometric assay comprising the following components in separate containers:
Dependencies are inferred from the claim text.