Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Patent
1997-04-07
1998-09-01
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
435 4, 435968, 435975, 435 21, 435 18, 536 2626, C12Q 166, C12Q 100, C12Q 134, C12Q 142
Patent
active
058010076
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to method for calibrating chemical assays and more particularly but not exclusively to a method for calibrating assays for analytes of importance in analytical microbiology such as an assay for adenosine 5'-triphosphate (ATP).
Accurate detection and quantification of particular substances or microorganisms for analytical purposes are essential laboratory tools. Detection and quantification of contaminant substances or contaminant microorganisms for the purposes of product or process control, or for food safety reasons, are of paramount importance to the food and beverage industries. Microbial biomass estimation is also important in a number of other applications, including control of waste processing, monitoring or sterilization processes and monitoring of air quality. In many cases, it is advantageous to be able to estimate the degree of microbial or chemical contamination within the shortest possible period of time.
Numerous assay techniques are available for detection and quantification of analytes of interest, and examples include enzymic assays, enzyme linked immunosorbent assays (ELISA), spectrophotometric assays, luminescence assays, fluorescence assays, chromatographic assays, assays based on measurement of changes in optical rotation of test sample, ion selective assays (including titrimetric assays) and colorimetric assays. The particular assay method used mainly depends upon the analyte to be detected and quantified.
It is essential that the particular assay method chosen be as accurate and as reproducible as possible. In order to maximize the accuracy of such assays, standardization or calibration of the assay is important. A variety of factors can affect the assay reaction and may give rise to sources of error and thus the effects of these factors must be minimized or taken into account.
Standardization techniques are known which use radioactive isotopes for the purpose of standardizing the instrumentation rather than the assay per se. An example of such radioactive standardization is embodied in the Biolink Light Standard (Leaback, D H, Easy-to-use light standards as aids to luminometry, Szalay, A. A. et al (Eds) pp 33-37--Bioluminescence and Chemiluminescence, Status Report. Proceedings of VII International Symposium of Bioluminescence and Chemiluminescence John Wiles & Sons, Chichester 1993). Such standards are based upon gaseous-tritium-activated phosphors which emit light of a defined spectral range and exhibit predictable decay on long term storage. Such devices serve only to assist in calibration of the luminometers themselves.
It is equally important that the assay per se is also calibrated or standardized so that variations in sample and reagent compositions can be taken into account.
For example, adenosine 5'-triphosphate (ATP) is an important analyte in microbiological assays. ATP is found in live cells but not in dead cells and its presence in a sample can be indicative of microbial contamination.
One widely used technique for assaying ATP is an ATP bioluminescence technique.
In the presence of a purified enzyme (luciferase) from the American firefly, Photinus pyralis, a substrate, D-luciferin, and sufficient magnesium ions and dissolved oxygen, the following reaction takes place:
______________________________________ Mg-ATP + O.sub.2 + D-luciferin
.dwnarw. firefly luciferase
Oxyluciferin + AMP + PP.sub.1 + CO.sub.2 + light
______________________________________
AMP=adenosine 5'-monophosphate; PP.sub.i =inorganic phosphate; CO.sub.2
=carbon dioxide).
Under appropriate conditions, the amount of light produced by the reaction is directly proportional to the ATP concentration and can be detected using a sensitive light detector. This is the basis of the ATP-bioluminescence assay.
Assays for ATP using the firefly luciferase reaction can be calibrated in two ways (Jago, P. H., Stanfield, G. Simpson, W. J. & Hammond, J. R. M. 1989. In ATP Luminescence: Rapid Methods in Microbiology, Society of Applied Bacteriology Technical Series, Vol. 26, St
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Little et al. (1985) J. Food Protection 48:1022-4.
Denburg et al. (1970) Arch. Biochem. Biophys. 141:668-75.
Jago et al. (1989) In "ATP luminescence: Rapid Methods in Microbiology", Soc. Appl. Bacteriol. Tech. Series, vol. 26, Stanley P.E. et al. (eds), pp. 53-61.
Simpson et al. (1991) J. Chemilumin. Biolumin. 6:97-106.
Pye Julian Mark
Simpson Willaim John
BRF International
Leary Louise N.
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