Capture asays

Details Capture asays Capture asays

1997-01-06

1998-08-25

Woodward, Michael P.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435 732, 435 15, 435 19, 436518, G01N 33573, G01N 33569

Patent

active

057982147

DESCRIPTION:

BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 USC 371 of PCT/GB95/01643 filed Jul. 12, 1995.
The present invention relates to a method for detecting and assaying microorganisms, to agents for use in such a method, and to test kits comprising essential reagents for carrying out the method.


BACKGROUND OF THE INVENTION

European Patent Application Publication No. 217,583 describes an assay for a ligand extracted from cells, in which the ligand is simultaneously extracted from the cell and reacted with two specific anti-ligands to yield a detectable product.
All living organisms utilise adenosine triphosphate (ATP) as a source of chemical energy and it is known to assay this using the ATP driven luciferase/luciferin reaction. Light generated by this enzymic reaction can be measured using a luminometer and related to the amount of ATP present. The usefulness of ATP as an index of microbial numbers has been known since the mid 1960's (see ATP Luminescence Rapid Methods in Microbiology (1989) editor Stanley et al.; Blackwell Scientific Publications, London, see pages 1-10); its main advantage being speed and sensitivity. Utilising this assay format simple samples can be analysed in a matter of minutes while complex ones samples can be analysed in a matter of minutes while complex ones routinely take only half an hour with a detection capability provided down to 10.sup.-12 mol/l ATP. There is however a need for methods which provide still further sensitivity when detecting microorganisms or their contents while retaining speed and ease of performance.
The present inventor has determined that the speed and sensitivity of ATP based method can be enhanced significantly by shifting the target of the assay from ATP to an enzyme which generates it, particularly to adenylate kinase. Adenylate kinase is an enzyme used by all organisms for the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). The targeting of this enzyme in preference to ATP, by using the preferred method, reagents and kits of the invention, allows the detection of down to at least 10.sup.-20 moles of intracellular marker adenylate kinase.
It is known to assay adenylate kinase using the luciferase/luciferin system (see Brolin et al Journal of Biochemical and Biophysical Methods 1 (1979) 163-169 and Shutenko et al. Biotekhnologiya, No 4, PA (1988) 542-547) for the purpose of determining its activity and this has been applied to study of certain mammalian and plant tissues (e.g. see Rodionova et al Fiziologiya Rastenii (1978) 25, 4, P731-734). The use of such assay system for the detection and assay of microorganisms however has not been suggested and the advantages of doing such, i.e. enhanced sensitivity so provided, have not been relevant to those studying the enzyme itself.
Although adenylate kinase is present in smaller quantities than ADP or ATP, its use as a biological marker for microorganisms provides enhanced sensitivity with a typical amplification available of 400,000 by measuring its presence through the ATP it produces; that is for every mole of enzyme present 400,000 moles of ADP are converted to ATP in a 10 minute incubation. Thus estimation of the enzyme by measuring the substrate or product of the reaction it catalyses provides for detection down to as low as 10.sup.-20 moles.
The applicant's copending PCT applications PCT/GB94/00118, now U.S. Pat. No. 5,648,232, and PCT/GB94/00766, relate to methods of estimation of microorganisms in a sample from its ability to convert ADP to ATP, and relating that to the presence of microorganisms or their intracellular materials. These applications exemplify methods where magnesium ions, necessary for the reaction of two molecules of ADP with each adenylate kinase active site, are either added as a reagent or provided by any bacteria cells present and as an impurity in the other reagents. The number of cells detected in the examples using such technique proved to be about 10.sup.2, with results of a more statistically valid nature being obtained at 10.sup.3

REFERENCES:
patent: 3933592 (1976-01-01), Clendenning
patent: 4806415 (1989-02-01), Fossati
patent: 5648232 (1997-07-01), Squirrell
Chemical Abstracts, vol. 93, No. 5, issued 1980, Aug. 4, (Columbus, Ohio U.S.A.), S.E. Brolin et al.: "Firefly luciferase assay of adenylate kinase in insulin and glucagon-producing cells" p. 370, No. 40 098j; & Proc.-Int. Symp. Anal. Appl. Biolumin. Chemilumin. 1978 (Pub. 1979), 458-66.

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