Peroxidase indicator system for basic media

Claim 2

The assay of Claim 1 wherein the aqueous medium has a pH of from 10 to 14.

Claim 3

The assay of any of claims 1 and 2 wherein the peroxidatively active substance is hemoglobin.

Claim 4

The assay of any of claims 1 to 3 wherein the phenylenediamine is characterized by the formula: wherein ----NR₂ is in position 2 or 4, wherein the ----NR₂ groups are in positions 4,4'; 4,2'; 2,2' or 5,5' or wherein the ----(NR₂) groups are in positions 1,2; 1,4; 1,5; 1,7; 2,3; 2,6 or 2,8 where the R groups are independently H, CH₃, CH₂,CH₃, CH₂ CH₂OH, C₆H₅ or CH₂CH₂CH₃ wherein the aromatic rings are substituted with 0 to 5 X groups wherein X is independently H, Cl, Br, I, Fl, CN, CH₃, CH₂CH₃, CH₂CH₂CH₃, OCH₃, OCH₂CH₃ or C₆H₅ and the naphthol has the formula: wherein y is CX, S, O or N and is either ortho or meta to the carbon atoms bridging the aromatic rings and wherein the aromatic rings are independently substituted with 0 to 7 X groups where X is H, Cl, Bn, I, Fl, CN, CH₃, CH₂CH₃, CH₂CH₂CH₃, OCH₃, OCH₂CH₃, SO₃H, CO₂H, PO₃H or C₆H₅.

Claim 5

The assay of any of claims 1 to 4 wherein the phenylenediamine is Structure I: bis-hydroxyethyl-1,4-phenylenediamine; 1,2-phenylenediamine, 1,4-phenylenediamine hydrochloride; 3-nitro-1,2-phenylenediamine; 4-nitro-1,2-phenylenediamine; 4-methoxy-1,2-phenylenediamine; 2-chloro-1,4-phenylenediamine sulfate; 2-ethoxy-1,4-phenylenediamine; N,N-dimethyl-1,4-phenylenediamine; 2,3-dimethyl-1,4-phenylenediamine; 4,5-dimethyl-1,2-phenylenediamine; N,N-diethyl-1,4-phenylenediamine dihydrochloride; N-phenyl-1,4-phenylenediamine; N,N,N',N'-tetramethyl-1,4-phenylenediamine; N-methyl-N'-hydroxyethyl-1,4-phenylenediamine; or 2,3,5,6-tetramethyl-1,4-phenylenediamine; Structure II: N,N-dimethyl-1,4-naphthyldiamine; 1,4-naphthyl-diamine; 1,5-diaminonaphthalene; 1,8-diamino naphthalene; 2,3-diaminonaphthalene; 5-chloro-1,4-napthyldiamine or 1-ethoxy-2,3-diaminonaphthalene; Structure III: 3,3',5,5'-tetramethyl-benzidine; 2,2'-dimethyl-biphenyl-4-4'-diamine; 3,3'-diaminobenzidine-tetrahydrochloride; or N,N,N',N'-tetramethyl-biphenyl-4,4'-diamine; and the naphthol is 2-methoxy-1-naphthol; 2-methyl-1-naphthol; 1-naphthol, 2-naphthol, 4-chloro-1-naphthol; 1-bromo-2-naphthol; 6-bromo-2-naphthol; 1,6-dibromo-2-naphthol; 2,4-dichloro-1-naphthol; 6-methyl-2-naphthol; 1-methoxy-2-naphthol; 2-phenyl-1-naphthol; 1-hydroxy quinoline, 5-hydroxy quinoline; 3-hydroxy-1-methyl-quinoline; 6-hydroxy quinoline; 2-hydroxy quinoline, 8-hydroxyquinoline-5-sulfonic acid; 1-naphthol-4-carboxylic acid; 8-hydroxyl-5-nitroquinoline; 5-chloro-8-hydroxyquinoline or 4-ethyl-1-naphthol.

Claim 6

The assay of any of claims 1 to 5 wherein the color forming combination comprises bis-hydroxyethyl-1,4-phenylenediamine and 2-methyl-1-naphthol.

Claim 7

An assay for the detection of hemoglobin in an aqueous test sample having a pH of from 10 to 14 which comprises the steps of adding to an aqueous medium suspected of containing hemoglobin, a peroxide together with a phenylenediamine and a naphthol which, under the conditions of the assay, couple to form a chromogen when hemoglobin is present thereby providing a detectable color change in the assay system.

Claim 8

The assay of Claim 7 wherein the color forming combination comprises bis-hydroxyethyl-1,4-phenylenediamine and 2-methyl-1-naphthol.

Claim 9

An assay device for determining the presence of a peroxidatively active substance in an aqueous test sample which comprises an adsorbant matrix material having adsorbed therein a buffer capable of maintaining the pH of the system at a level of from 10 to 14 and a peroxide together with a phenylenediamine and a naphthol which are capable of coupling in the presence of free oxygen.

Claim 10

The device of Claim 9 wherein the phenylenediame is one or more of the compounds of claims 4 and 5.