Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1996-07-09
1998-10-27
Hollinden, Gary E.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 4, C12Q 102
Patent
active
058276785
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a cell reagent, an assay method for measuring an analyte and a kit therefor.
2. Description of Related Art
British patent specification No. 2,005,018 describes a method of detecting a toxic substance by forming a suspension of a luminous micro-organism in an aqueous medium, contacting the suspension with the toxic substance, and sensing a decrease in the light output of the luminous micro-organisms caused by the toxic substance. An as-say kit embodying this principle is marketed by Microbics Inc. under the Trade Mark Microtox, and is in routine use in a number of environmental laboratories.
Although the Microtox kit is believed to use a naturally occurring bacterium, there is interest in the use of genetically modified luminous micro-organisms, as they potentially have a wider area of application. For example, different micro-organisms can be made with sensitivities to different ranges of toxic substances or even individual toxic substances. The use of genetically modified organisms for this purpose is described in U.S. Pat. No. 4,581,335.
There is an increasing need to perform these assays at the site of potential pollution, and this may well involve tests outside normal laboratory facilities. But such cells are often pathogenic or subject to controlled use. One potential problem when using genetically modified organisms is the possibility that they might escape into the environment and cause harm by growing in an uncontrolled manner. Legal restrictions on use of genetically modified organisms in the field are, or may be, applied in various countries.
It is an object of this invention to avoid the above problem by using genetically modified organisms which have been killed. But as is explained below, the invention is not limited to genetically modified organisms, nor to organisms that emit light, nor to organisms for use in the stated assay for toxic substances.
When micro-organisms are subjected to increasing doses of ionizing radiation, the organisms viability progressively decreases. Thus, a dose of 26 kGy is recommended for sterilizing micro-organisms, and is amply sufficient to reduce their viability to zero.
Non-recombinant luminescent organisms have been subjected to ionising radiation, and the luminescent property used to monitor the radiation dose (Phys. Med. Biol. 28, 599-602, 1983). But the radiation doses were not sufficient to kill all the organisms present.
SUMMARY OF THE INVENTION
In one aspect, this invention provides an assay reagent comprising bacterial cells which have been killed but which retain a functional metabolic activity, the reagent not containing corresponding bacterial cells which have not been killed.
Cells which have been killed have 0% viability. They are unable to reproduce. Cells preferably retain a detectable functional metabolic activity and structural function including one or more of the following properties: cell-wall integrity; membrane/energy function; co-factor provision; metabolic requirement; gene expression; protein synthesis; cytoplasmic enzyme activity; vegetative metabolic processes involving energy usage and transfers.
Preferably the detectable functional metabolic activity is bioluminescence. It will be appreciated that continued bioluminescence may involve continued protein synthesis. It is surprising that cells which have been killed nevertheless retain a functional metabolic activity, such as bioluminescence, albeit at reduced intensity compared to the non-irradiated cells. More surprising is the fact that the functional metabolic activity in these killed cells is altered in a dose-responsive way by substances which alter the functional metabolic activity in a dose-responsive way in the corresponding living cells. Thus, for example, luminescence of killed bioluminescent organisms is changed in a dose-responsive way in the presence of a substance which is a toxicant for the corresponding living organism.
Preferably the cells used in this invention are micro-organisms whic
REFERENCES:
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Dox et al (The Harper Collins Illustrated Medical Dictionary, 1993, p. 501).
Mantel et al., "The Effect of Radiation on Bioluminescent Bacteria: Possible Use of Luminescent Bacteria as a Biological dosemeter," Phys. Med. Biology, 1983, 28, 599-602.
Kaplan, "Macromolecular Basis of Radiation-Induced loss of Viability in cells and Viruses," Actions Chimiques ert Biologiques des Radiations: The Chemical and Biological Action of Radiations, Chapt. II, Haissinsky, ed., 1968, 12, 71-94.
Stults et al., "Use of Recombinant Biotinylated Aequorin in Microtiter and Membrane-Based Assays: Purification of Recombinant Apoaequorin from Escherichia coli," Biochemistry, 1992, 31, 1433-1442, Jan. 1992.
Journal of Pharmacy and Pharmacology, vol. 18, No. Supp, 1966, London GB, pp. 33S-38S, N.D. Harris and M. Whitefield "The effect of the addition of manganese dioxide to media on the viability of bacteria damaged by x-rays, phenol and radiomimetic agents".
Actions Chimiques Biologiques Et Radiations, vol. 12, 1968, Paris, FR, pp. 71-94, Henry S. Kaplan "Macromolecular basis of radiation-induced loss of viability in cells and viruses".
Physics in Medicine and Biology, vol. 28, No. 5, 1983, London GB, pp. 599-602, J. Mantel et al., "The effect of radiation on bioluminescent bacteria as biological dosemeter".
Mutation Research, vol. 267, No. 1, May 1992, Amsterdam NL, pp. 19-30, C. Seymour et al., "All colonies of CHO-K1 cells surviving gamma-irradiation contain non-viable cells".
Hesslewood Ian Philip
Stewart Gordon Sydney Anderson Birnie
Hollinden Gary E.
Jones Dameron
Merck Patent GmbH
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