Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1989-03-23
1995-01-10
Beisner, William H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 4, 435968, 435975, 435 71, 436 63, 436172, 436800, 436809, 546184, 546112, 546208, 546226, 546245, 546273, 548181, 548204, 5483121, 5483147, 5483647, 548451, 548455, 544129, C12M 128, C07C 1300, C07C20505, C07D47100
Patent
active
053806506
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an enhanced luminescent assay, particularly immunoassay, and to a diagnostic kit for use in the assay. The luminescent assays with which the present invention is concerned are those depending on a chemiluminescent reaction (a chemical reaction that results in the emission of light). The luminescent emission is generally of sufficient duration to enable the light emitted to be detected or measured, and thereby to allow the detection or quantification of an analyte. The chemiluminescent reaction with which this invention is concerned is that between a 2,3-dihydro-1,4-phthalazinedione (DPD), especially luminol or isoluminol, with an oxidant, especially hydrogen peroxide, and a catalyst, especially peroxidase enzyme, which catalyses the oxidation of the DPD by the oxidant. The oxidation is accompanied by emission of light.
2. Description of the Prior Art
Luminescent assays making use of the above-mentioned peroxidase catalysed oxidation of a DPD can be of several major types, the commonest of which are those wherein horseradish peroxidase is conjugated to a ligand in order to label it and a luminescent reaction is used to detect or quantitate the label. The present invention relates exclusively to assays of a different kind, namely those wherein a chemiluminescent compound is used directly to label ligands such as proteins, hormones, haptens, steroids, nucleic acids, metabolites, antigens and/or antibodies. The chemiluminescent DPD such as luminol or isoluminol is normally conjugated to a ligand. Chemiluminescence can be detected by adding peroxidase and an oxidant to the reacted conjugate.
A review of luminescent assays has been published by T. P. Whitehead et. al., Clinical Chemistry 25, 1531-1546 (1979).
The sensitivity of the peroxidase-catalysed chemiluminescent oxidation of DPDs can be enhanced by including in the reagents an enhancer, namely a 6-hydroxybenzothiazole (European Patent 87959B of NRDC), a phenol selected from a narrowly defined class (European Patent 116454B or U.S. Pat. No. 4,598,044 assigned to NRDC) or an aromatic amine selected from a narrowly defined class (UK Patent Application 2162946A of NRDC or European Patent Publication 219352A (Minnesota Mining & Mfg. Co.).
In European Patent Application Publication 210449A (Molecular Diagnostics Inc.) it is alleged that ammonia and water-soluble organic amines are enhancers of chemiluminescent reactions. The amines specifically mentioned are aliphatic polyamines such as spermire, spermidine and butylenediamine, tertiary alkylamines, pyridine, azoles, thiazines, aryl amines and benzylamine. However, little evidence of enhancement is provided in that patent specification.
Despite these prior disclosures, none of which exemplifies enhancement of an assay using direct labelling of a ligand by a DPD, it has been a problem to find amines which enhance such an assay to a high level of light intensity and provide a significantly greater light intensity when the DPD conjugate is present than when it is absent.
SUMMARY OF THE INVENTION
It has now been found that certain saturated heterocyclic amines enhance the chemiluminescent oxidation of a DPD. These amines have the characteristic that they lose an electron easily to form positive ions. They are either tertiary amines or secondary amines in which the hydrogen atom attached to the nitrogen is well shielded or sterically hindered. More precisely, the amines of interest are saturated bicyclic compounds having a nitrogen atom at one or both bridgehead positions and piperidine ring compounds having four lower (C.sub.1-4) alkyl groups at the 2- and 6- positions (the carbon atoms adjacent to the nitrogen). The invention provides a luminescent or luminometric assay which comprises carrying out a chemiluminescent reaction between a catalyst having an accessible heme group, preferably a peroxidase enzyme, most preferably microperoxidase, an oxidant, preferably hydrogen peroxide, and a chemiluminescent DPD conjugated to a
REFERENCES:
patent: 4842997 (1989-06-01), Carter
Johnson J Immunological Methods 55 (1982) pp. 231-242.
T. P. Whitehead et al., Clinical Chemistry 25, 1531-1546 (1979).
G. J. Barnard et al. in "Alternative Immunoassays" ed. W. P. Collins, John Wiley & sons Chichester, U.K. 1984, pp. 123-152.
W. Klingler et al., Trends in Analytical Chemistry 2, 132-136 (1983).
P. Bischof et al., Tetrahedron Letters 46, 4025-4028 (1969).
N. Duran et al., Cellulose Chemistry & Technology 18, 411-419 (1985).
K. Honda et al., Analytica Chimica Acta 177, 111-120 (1985).
A. Lundin & L. Hallander Poster displayed at the 4th International Symposium on Biolumiescence and Chemiluminescence held at Freiburg, W. Germany, 8-10 Sep. 1986.
Barnard Geoffrey J. R.
Davidson Robert S.
Goodwin Dean
Beisner William H.
British Technology Group Ltd.
Williams Jane
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