Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1991-10-11
1994-04-19
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 791, 435 795, 422 8205, 422 85, 422 681, 422 91, 356318, 356445, C12Q 100, G01N 122, G01N 2155, G01J 330
Patent
active
053044657
DESCRIPTION:
BRIEF SUMMARY
This invention concerns methods of performing enzyme assays using the technique of surface plasmon resonance spectrometry (SPSR).
The phenomenon of SPR is well known and will not be described in detail (see EPA 305109 for example). Briefly, the intensity of monochromatic plane-polarised light (conveniently obtained from a laser) reflected from the interface between an optically transparent material, e.g. glass, and metal depends on the refractive index of material on the downstream side of the metal. Accordingly, by measuring changes in intensity of reflected light an indication can be obtained of changes in refractive index of material at a particular point on the downstream surface of the metal. The intensity of reflected light also varies with the angle of incidence, and reflectively drops sharply to a minimum at a particular angle which is characteristic of the equipment.
International Patent Application PCT/GB90/00432 filed Mar. 21, 1990 makes use of this phenomenon in an enzyme assay. The solid surface carries thereon an immobilized enzyme which catalyses the reaction between two or more reactants resulting in the production of a reaction product, or a substrate for the enzyme. The method involves bringing into contact with the solid surface a fluid medium containing the remaining reactants and if not already present the enzyme, under conditions to cause the reaction product to be deposited on the solid surface. One molecule of enzyme can catalyze the deposition of many molecules of reaction product on the solid surface. The change in refractive index that results from deposition of the reaction product is monitored by SPRS. That method provides a satisfactory assay for the enzyme, but one which requires the addition of one or more reagents to the fluid medium.
This invention is a development of that method, and has the advantage that no additional reagents are required, apart from a pretreated solid surface and a liquid sample of the analyte.
a method of performing an assay for an enzyme according to the invention involves the use of a solid surface carrying an immobilized reagent which is capable of being released by the enzyme. A fluid sample is brought into contact with the immobilised reagent under conditions such that the enzyme if present in the sample catalyzes release of the reagent from the surface. The solid surface is provided by a metallic layer applied to a block of material transparent to electromagnetic radiation. Release of the reagent from the surface of the metallic layer is assayed by SPRS.
The immobilized reagent may be a substrate for the enzyme. Examples of suitable pairs of enzyme and substrate are DNAse/DNA; RNAse/RNA; amylase/starch; various glycosidases/their polysaccharide substrates; peptidases/polypeptides. In addition to these natural substrates, synthetic substrates can be made combining a molecule or particle (of high or alternatively low refractive index with respect to the bulk phase) with the metal surface via an enzymatically sensitive linker that is cleavable by the enzyme under study. Release of the molecule or particle is then easily detected as a change in the SPRS signal.
As noted above, a major advantage of this technique is that it requires no reagent other than the enzyme substrate pre-coated on the solid surface of the SPR device. A further advantage is its directness. The technique also meets a need--the classes and types of enzymes suited for this approach are very difficult to assay directly and at high sensitivity by any other means.
It is not necessary that the enzyme be the primary analyte under study. There are very many assays in existence for a variety of analytes which result in the production of an enzyme in solution at a concentration related to the concentration of primary analyte in a sample. This invention provides a convenient and rapid technique for observing the presence of concentration in a fluid of an enzyme from any source.
EXAMPLE
A glass slide coated with 50 nm thick silver-metal was placed on a fan beam SPR instrument. The silv
REFERENCES:
patent: 4877747 (1989-10-01), Stewart
patent: 5064619 (1991-11-01), Finlan
Sigma Chemical Co Catalog 1992 St. Louis, Mo. p. 254.
Garland Peter B.
Malan Philip G.
Amersham International plc
Itomer Ralph G.
Wityshyn Michael G.
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