Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-09-22
2003-01-14
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S014000, C435S019000, C435S283100, C435S286500, C435S028000, C422S068100
Reexamination Certificate
active
06506575
ABSTRACT:
BACKGROUND AND SUMMARY OF THE INVENTION
The application concerns an analytical element for determining the amount of an analyte in a liquid containing a sample application zone and a detection zone which are in liquid-transferring contact, the latter containing at least one enzyme and an indicator substance, the enzyme catalysing a reaction in which the analyte or a substance derived from the analyte participates and the indicator substance forming a signal when the analyte is present which signal correlates with the amount of analyte, and containing a liquid-permeable interference-reducing layer in direct contact with the detection zone arranged such that liquid does not reach the detection zone until it has passed through the interference-reducing layer, the interference-reducing layer containing at least one enzyme which participates in a reaction of the analyte to be determined or of a substance derived from the analyte. The application additionally concerns a method for the determination of an analyte in a liquid by means of such a multilayer analytical element.
Results for analytes to be detected that are too high i.e. false positive often occur with analytical elements of the dry chemistry type which are often called test strips on which undosed sample volumes and hence volumes which vary widely are applied. In addition it is often not possible to recognize when the detection reaction is completed. The detection reaction often slowly declines over a long time period (reaction creep).
The Japanese laid-open specification No. Hei 5-23199 (publication date Feb. 2, 1993) describes an analytical element and a corresponding analytical method for analysing neutral fats. The object of the invention described in the patent application is to remove glycerol that was originally present in the samples to be examined before carrying out an enzymatic analysis of neutral fats via glycerol as an intermediate step. This is achieved by providing analytical elements for the analysis of neutral fats which have at least one reagent layer on a transparent support and a reaction layer thereon. The reaction layer which is the first layer to come into contact with the sample to be examined contains glycerol dehydrogenase and NAD
+
. The reagent layer also contains glycerol dehydrogenase but no co-enzyme. Consequently glycerol is converted into dihydroxyacetone and NADH in the reaction layer which acts as an interference-reducing layer and is thereby removed. Apparently the NAD
+
which diffuses together with the sample from the reaction layer into the reagent layer, activates the glycerol dehydrogenase that is present there such that glycerol formed in the course of the analytical reaction of the neutral fats is also converted in the reagent layer into dihydroxy-acetone and NADH. The NADH that is formed reacts with a chromogen in the reagent layer to form a coloured detection product. It is to be expected that NADH which diffuses from the reaction layer into the reagent layer during the course of the analysis would influence the colour reaction in a false-positive manner.
In view of this prior art it was regarded as an object to provide analytical elements which also give correct results with undosed samples i.e. results which agree with those obtained with the respective reference methods. In addition the result should be available after a short time. It should be possible to clearly recognize the end of the detection reaction by the fact that there is no further substantial change in a signal that is used to determine an analyte.
This object is achieved according to the invention by the subject matter which is characterized in more detail in the patent claims.
The invention concerns in particular an analytical element for determining the amount of an analyte in a liquid containing a sample application zone and a detection zone which are in liquid-transferring contact, the latter containing at least one enzyme and an indicator substance, the enzyme catalysing a reaction in which the analyte or a substance derived from the analyte participates and the indicator substance forming a signal when the analyte is present which signal correlates with the amount of analyte, and containing a liquid-permeable interference-reducing layer in direct contact with the detection zone arranged such that liquid does not reach the detection zone until it has passed through the interference-reducing layer, the interference-reducing layer containing at least one enzyme which participates in a reaction of the analyte to be determined or of a substance derived from the analyte, characterised in that the analyte or the substance derived from the analyte is converted enzymatically in the interference-reducing layer into end products which cannot contribute to the signal formation by the indicator substance.
The invention also concerns a method for the determination of an analyte in a liquid by means of an analytical element described above characterised in that liquid is applied to the sample application zone in an undosed manner, liquid passes through the interference-reducing layer into the detection zone and there the amount of analyte in the liquid is determined in this process the detection zone is filled with liquid and excess liquid remains in the interference-reducing layer and optionally in the sample application zone.
A subject matter of the invention is especially the use of a layer in a multilayer analytical element which converts an analyte to be determined or a substance derived therefrom into products which do not contribute to the signal formation in this multilayer analytical element for the determination of this analyte, to prevent rediffusion of analyte from other zones into the zone of the analytical element in which it is intended to detect the analyte when the detection zone is filled with liquid.
An analytical element according to the invention contains a matrix material which contains a sample application zone and a detection zone. Several matrix materials may also be present one of which carries a sample application zone and the other carries the detection zone. All absorbent or swellable materials which can imbibe a liquid can basically be used as matrix materials. These can be fibrous materials such as fleeces, fabrics or knitted fabrics or non-fibrous materials such as porous or non-porous films or membranes.
In an analytical element according to the invention the sample application zone and the detection zone are in a liquid-transfer-enabling contact. The sample application zone can touch the detection zone. However, both zones can also be separate provided that liquid which is applied to the sample application zone can pass into the detection zone in the analytical element.
In order to improve the handling of the analytical element, the sample application zone and the detection zone can be disposed on an inert stiff support material. All inert, adequately stiff materials such as glass, hydrophobised cardboard or polymer materials are potentially suitable for such a support material. Stiff polymer foils which are for example composed of methacrylate/acrylate, polystyrene or polycarbonate are preferably used. In addition the support material can be transparent or impermeable to light.
In an analytical element according to the invention the sample application zone and detection zone can be arranged next to one another or above one another.
The sample application zone refers to the area of the analytical element according to the invention on which the liquid sample is applied. The detection zone of the analytical element according to the invention is understood as the area in which, in the presence of the analyte, a signal is produced which correlates with the amount of the analyte.
The reagents required to determine the amount of an analyte can be distributed over several layers in the detection zone. This has proven to be especially advantageous when certain reagents are not compatible with one another and thus cannot be accommodated within one layer. The detection zone contains a
Gaa Otto
Knappe Wolfgang Reinhold
Pachl Rudolf
Knauer Richard T.
Leary Louise N.
Roche Diagnostics GmbH
Woodburn Jill L.
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