Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-06-29
2003-09-09
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S968000, C435S975000
Reexamination Certificate
active
06617125
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to enzymatic assays, and more particularly to enhancers for use in catalyzed reporter deposition.
BACKGROUND OF THE INVENTION
Peroxidase, because of its high turnover rate, good stability, and availability is widely used in enzyme-based analytical methods. For example, horseradish peroxidase (HRP) (EC 1.11.1.7) catalyzes the oxidation of a large variety of hydrogen-donating substrates with hydrogen peroxide. HRP is also one of the preferred enzymes for use in catalyzed reporter deposition.
Catalyzed reporter deposition (CARD) is a novel method of signal amplification which constitutes the subject matter of U.S. Pat. Nos. 5,863,748; 5,688,966; 5,767,287; 5,731,158; 5,583,001 and 5,196,306. It is also discussed in Bobrow et al.,
Journal of Immunological Methods,
125: 279-285 (1989) and in Bobrow et al.,
Journal of Immunological Methods,
137: 103-112(1991).
The method utilizes an analyte-dependent enzyme activation system (“ADEAS”) to catalyze the deposition of a detectable label onto the solid phase of an assay platform. These enzymatically deposited labels may be detected directly or indirectly and results in signal amplification and improved detection limits. In a preferred embodiment, HRP is the enzyme.
HRP reacts with a conjugate consisting of a detectably labeled substrate specific for the ADEAS. When the ADEAS and the conjugate react, an activated conjugate is formed which deposits covalently wherever receptor site for the activated conjugate is immobilized.
For analytical use, substrate oxidation by HRP has been used to generate products which become colored, fluorescent or chemiluminescent. These products either remain soluble or become insoluble and precipitate on the solid phase. The CARD method differs in this respect as the products of the detectably labeled phenol substrate become covalently bound to the solid phase.
To improve detection limits in analytical methods, it is desirous to increase or enhance the substrate to product conversion by enzymes. Although a substance which enhances HRP catalysis regardless of the substrate used has not been discovered, several enhancers specific for HRP substrates which form soluble products have been described. One enhancer specific for the substrate diaminobenzidine, which forms an insoluble product has been described. Enhancers for substrates which, by the catalytic activity of HRP, form covalently depositable products have not been described.
J. R. Whitaker and A. L. Tappel,
Biochimica et Biophysica Acta
, pages 310-317, Vol. 62, 1962 show that KCl, NaCl, Na
2
SO
4
and to a lesser extent, LiCl enhance the oxidation of guaiacol.
U.S. Pat. No. 4,598,044 issued to Kricka et al. on Jul. 1, 1986 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,3-dihydro-1,4-phthalazinedione, which forms a soluble chemiluminescent product, by various phenolic compounds.
U.S. Pat. No. 4,729,950 issued to Kricka et al. on Mar. 8, 1988 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,3-dihydro-1,4-phthalazinedione, by various aromatic amine compounds. Tables 1 and 2 summarize various substrate/enhancer combinations. The Tables and the discussion (column 3 line 67 to column 4 line 34) lead to the conclusion that whether an HRP catalyzed oxidation of a substrate will be enhanced by a given compound is not predictable.
U.S. Pat. No. 5,629,168 issued to Kricka on May 13, 1997 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,3-dihydro-1,4-phthalazinedione, by aromatic organoboron compounds.
U.S. Pat. No. 4,521,511 issued to Stout on Jun. 4, 1985 describes the enhancement of the HRP catalyzed oxidation of the substrate, 2,2′-azino-di(3-ethyl-benzothiazolone-6-sulfonic acid), by various phenolic compounds.
W. Straus,
Journal of Histochemistry and Cytochemistry
, Vol. 30, pages 491-493, 1982, shows that imidazole enhances the HRP catalyzed oxidation of diaminobenzidine which forms in insoluble product.
A. S. H. de Jong et al.,
Histochemical Journal
, Vol. 17, pages 1119-1130, 1985 also show that imidazole enhances the oxidation of diaminobenzidine by approximately four fold, a substrate combination of p-phenylenediamine-pyrocatechol by two fold and has no effect on the substrate 4-chloro-1-naphthol, all of which form insoluble products.
The aforementioned enhancers, with the exception of imidazole, only enhance the conversion of soluble substrates to soluble products. In addition, the enhancers are substrate specific. The KCl, NaCl, Na
2
SO
4
and LiCl enhancement of the oxidation of guaiacol is specific for guaiacol. These salts do not enhance the oxidation of substrates which form insoluble products nor do they enhance the oxidation of commonly used substrates that form soluble products, such as orthophenylediamine or tetramethylbenzidine. The enhancers for 2,3-dihydro-1,4-phthalazinedione also do not enhance the oxidation of substrates which form insoluble products nor do they enhance the oxidation of commonly used substrates that form soluble products, such as orthophenylediamine or tetramethylbenzidine. Imidazole, which has been demonstrated to enhance the oxidization of diaminobenzidine, has a marginal effect on p-phenylenediamine-pyrocatechol, no effect on 4-chloro-1-naphthol, and no effect on substrates which form covalently depositable products. Whether the oxidation of a given substrate by HRP will be enhanced by a given compound cannot be predicted.
Accordingly, it would be advantageous and desirable to have reagents for enhancing the catalysis of HRP and to have an enhancement effect greater than would be expected based on previous technology.
SUMMARY OF THE INVENTION
The present invention concerns enhancing a Catalyzed Reporter Deposition (CARD) method by reacting a conjugate comprising a detectably labeled phenol-containing molecule with a peroxidase enzyme, wherein the reaction is carried out in the presence of an enhancing reagent including, an organic enhancing compound or synergistic mixtures of an inorganic salt and the organic enhancing compound. The organic enhancing reagent has the structure:
wherein each R is independently selected from the group consisting of: hydrogen and a C
1-12
substituent; where V, W, Y and Z are each independently selected from the group consisting of: H, halogen, the C
1-12
substituent, NR
2
, OR and SR; and where X is selected from the group consisting of: H, Br, Cl, F, the C
1-12
substituent, NR
2
, OR and SR; or synergistic mixtures of an inorganic salt and the organic compound. The C
1-12
substituent is linear, branched or cyclic. The C
1-12
substituent is alkyl, alkenyl, alkynyl, heteroatom substituted alkyl, heteroatom substituted alkenyl, heteroatom substituted alkynyl, aryl, arylalkyl, arylalkenyl or arylalkynyl. Further, the heteroatom is N, O, S or halogen. Any C, N, O or S in the C
1-12
substituent optionally has a pendant moiety which is carbonyl, hydroxyl, carboxyl, amine, thiol, thioester, thioether, phosphate, alkoxy, aryl, arylalkyl, sulfonamide or alkyl halide.
A kit is provided containing an enhancing reagent for enhancing the detection of an enzyme reaction as described herein, together with instructions for use.
REFERENCES:
patent: 4521511 (1985-06-01), Stout
patent: 4598044 (1986-07-01), Kricka et al.
patent: 4729950 (1988-03-01), Kricka et al.
patent: 5196306 (1993-03-01), Bobrow et al.
patent: 5356893 (1994-10-01), Bradshaw et al.
patent: 5467287 (1995-11-01), Wenner et al.
patent: 5583001 (1996-12-01), Bobrow et al.
patent: 5629168 (1997-05-01), Kricka
patent: 5688966 (1997-11-01), Bobrow et al.
patent: 5731158 (1998-03-01), Bobrow et al.
patent: 5835101 (1998-11-01), Otsuka
patent: 5863748 (1999-01-01), Bobrow
patent: 6372937 (2002-04-01), Bobrow et al.
Hark R. Novel Approaches Toward Ninhydrin Analogs. Tetrahedron Letters 35(42)7719-7722, 1994.*
Whitaker, et al., Modification of Enzymic Activity, II. Effects of Salts on A-Amylase, Alcohol Dehydrogenase, Peroxide and Hematin Catalysis, Nov. 15, 1961, pp. 310-317.
The Journal of Histoc
Gifford, Krass, Groh Sprinkle, Anderson & Citkowski, P.C.
Gitomer Ralph
PerkinElmer Life Sciences, Inc.
LandOfFree
Compositions for enhanced catalyzed reporter deposition does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Compositions for enhanced catalyzed reporter deposition, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compositions for enhanced catalyzed reporter deposition will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3040633