Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1992-04-01
1994-04-26
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 18, 435 21, 435188, 435 28, 436172, G01N 2176, G01N 3353, C12Q 128, C12N 996
Patent
active
053066219
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to diagnostics, more specifically to the assay of enzyme activity, using a chemiluminescent reaction (a chemical reaction that results in the emission of light). The particular chemiluminescent reaction is that between a 2,3-dihydro-1,4-phthalazinedione (DPD), especially luminol or isoluminol, with an oxidant, especially hydrogen peroxide, and a peroxidase enzyme which catalyses the oxidation of the DPD by the oxidant. The oxidation is accompanied by the emission of light, thus generating a signal by which the enzyme activity can be assayed (detected or measured).
2. Description of the Prior Art
Enzymes are extensively used as labels in ligand-binder assays (e.g., immunoassay, DNA hybridization assay) and the signal amplification inherent in an enzyme catalyzed reaction confers great sensitivity on the assay. Chemiluminescent reactions are very sensitive and have been successfully used to measure enzyme activity with improvement in the detection limit for the enzyme over conventional enzyme assays L. J. Kricka, and T. P. Whitehead, Journal of Pharmaceutical and Biomedical Analysis, 5, 829-833 (1987).
Alkaline phosphatase can be measured chemiluminescently using the phosphate substrates disclosed in European Patent Application Publication No. 254051A and by Bronstein et al., Journal of Bioluminescence and Chemiluminescence, 4, 99-111, (1989). The enzyme cleaves the phosphate from the adamantly 1,2-dioxetane phosphate to produce a dephosphorylated intermediate which decomposes with the emission of light. A bioluminescent assay for this enzyme is also possible using firefly luciferin-O-phosphate (PCT Application Publication WO 87/02667). The enzyme cleaves the phosphate group and liberates firefly luciferin, which, unlike the luciferin phosphate, is a substrate in the bioluminescent reaction catalyzed by firefly luciferase: ##STR1##
Peroxidase activity can be measured chemiluminescently using luminol as a substrate and the analytical utility of this reaction is significantly improved by the addition of certain enhancers, especially certain phenols, U.S. Pat. No. 4,598,044 and British Patent Application 2205945A or amines (U.S. Pat. No. 4,729,950), all owned by National Research Development Corporation.
It has been a problem that such enhanced chemiluminescent assays require the use of a peroxidase enzyme and are therefore primarily of use in assays in which the ligand or its binding partner are peroxidase-labelled. It would be desirable to have a chemiluminescent assay in which the ligand or binding partner can be labelled with a different enzyme, especially alkaline phosphatase.
Further prior art is mentioned after the "Summary of the invention", without which its context would not be clear.
SUMMARY OF THE INVENTION
The present invention is based on idea of using the phenolic enhancer of the above-mentioned existing peroxidase-based chemiluminescent assay to make the assay sensitive to the presence of a second enzyme. Pursuant to this idea, it has been found certain enzymes will catalyse reactions of certain non-enhancing or poorly-enhancing compounds, generating a good enhancer and thus allowing an enhanced peroxidase-catalyzed chemiluminescent reaction to proceed and, furthermore, that the light emission from this reaction is dependent on the enzyme used in the enhancer generation reaction. It has further been found that such assays can be performed as single tube assays in which the non-enhancing or poorly-enhancing compound is reacted with the enzyme in the presence of the reagents which give the chemiluminescent reaction.
The non-enhancing or poorly-enhancing compound is referred to herein as a "pro-enhancer". It will normally be a derivative of the enhancer and be cleavable by the action of the enzyme to yield the enhancer. Preferably it is a phosphate and the enzyme used to cleave it is an alkaline phosphatase.
It has also been found that certain "anti-enhancers" inhibit the effect of the enhancer on the chemilumin
REFERENCES:
patent: 4598044 (1986-07-01), Kricka et al.
patent: 4729950 (1988-03-01), Kricka et al.
P. D. Mize et al., Anal. Biochem. 179, 229-235 (1989).
British Technology Group Limited
Kepplinger Esther L.
Parsons Nancy J.
LandOfFree
Enhanced chemiluminescent assay does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Enhanced chemiluminescent assay, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enhanced chemiluminescent assay will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1711111