Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-03-17
1994-10-18
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 4, 435 77, 435 772, 435 14, 435 25, 435 26, 435174, 435176, 435288, C12Q 100, G01N 3353
Patent
active
053567803
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an assay technique. In particular, it relates to a competitive binding assay technique for substances which can serve as substrates for coenzyme-requiring enzymes.
A large number of substrates of coenzyme-requiring enzymes are small molecules having no antigenic determinant and consequently cannot be assayed by conventional immunoassay techniques. Commonly, such substrates are assayed by monitoring enzymic substrate conversion. Thus, for example, glucose biosensors are known which depend upon the sample to be assayed for glucose being contacted with glucose oxidase (a coenzyme-requiring oxidoreductase enzyme), enzymic conversion of any sample glucose being determined by indirect detection of H.sub.2 O.sub.2 production or by monitoring electron transfer from the redox centre of the enzyme to a working electrode via an electron transfer mediator. Glucose sensing systems of the latter type are disclosed, for example, in EP-A-076836 and one such biosensor employing disposable electrode strips carrying immobilised glucose oxidase together with an electron transfer mediator is currently made available commercially by MediSense Inc.
Enzyme electrodes for assaying of enzyme substrates are generally considered favourable from the point of view of convenience of use and a low degree of interference, but their lower limit of sensitivity is significantly higher than for known immunosensors for antigenic species owing to the kinetics of enzymic substrate conversion. Thus, whereas immunosensors commonly cover a concentration range of 1 nM to 0.1 mM, enzyme electrodes are typically only of use in assaying substrate concentrations in the range 10 .mu.M to 1M.
Fibre-optic biosensors for glucose are also known which depend upon competitive binding of sample glucose and fluorescently-labelled dextran to the lectin concanavalin A [Schultz et al, Diabetes Care 5, 245-253 (1982); Anal. Chem. 58, 766A-770A (1986)]. Analogous fibre-optic biosensors for non-haptenic small molecules other than lectin-binding sugars are, however, not known.
The present invention now provides a competitive binding assay technique which is applicable not only to glucose, but also inter alia to any other substrate of a coenzyme-requiring enzyme for which an apoenzyme binding protein with no functional coenzyme component can be obtained.
Thus, according to one aspect of the invention, there is provided a method of assaying within a liquid sample a substance capable of serving as a substrate for a coenzyme-requiring enzyme which method comprises contacting the said sample with an assay system including (i) a substrate analogue and (ii) a moiety capable of binding specifically to the substrate without catalysing the conversion of the substrate (the said substrate analogue being a substance capable of binding to the said moiety competitively with any of the said substrate present in the sample), and determining the presence or amount of the said substrate by monitoring the extent to which there are formed or remain complexes between the said moiety and substrate analogue and/or substrate respectively.
The moiety capable of binding specifically to the substrate without catalysing its conversion will hereinafter be referred to, for brevity's sake, as a specific binding partner. It is to be understood that, at commencement of an assay according to the above method, the specific binding partner and the substrate analogue may be either separate components (the standard format of a competitive assay) or already mutually bound (the format of a displacement competitive assay).
In a preferred embodiment of the invention, the specific binding partner is an appropriate apoenzyme in the absence of its functional coenzyme required for catalytic activity. Accordingly, the invention will be described hereinafter with particular reference to this preferred embodiment.
Other appropriate specific binding partners for use in the method of the invention include partly denatured enzymes (i.e. enzymes which have been appropriately deactivated by
REFERENCES:
patent: 4810635 (1989-03-01), Ledden et al.
patent: 4978503 (1990-12-01), Shanks et al.
Hurrell John G.
Robinson Grenville A.
Applied Research Systems Holding N.V.
Scheiner Toni R.
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