Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-09-05
2003-02-04
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S004000, C435S014000, C435S023000, C436S066000, C548S250000
Reexamination Certificate
active
06514720
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of measuring an analyte in a sample using a redox reaction.
BACKGROUND OF THE INVENTION
Traditionally, measurement of the quantity of an analyte in a sample using a redox reaction has been utilized in a wide range of applications. For example, such a measurement has been utilized for measuring glycated proteins in applications such as biochemincal analyses, clinical tests, and the like.
For instance, glycated proteins in blood, particularly glycated hemoglobin (HbAlc) in erythrocytes, are significant indicators in the diagnosis and therapy of diabetes, because they reflect the patient's past history of blood sugar value. Glycated proteins in erythrocytes are measured using a redox reaction, for example, as follows:
First, erythrocytes are hemolyzed to prepare a sample. The hemolyzed sample is treated with a suitable protease or the like, and then treated with fructosyl amino acid oxidase (hereinafter referred to as FAOD) so as to form hydrogen peroxide. The quantity of the hydrogen peroxide formed corresponds to the quantity of glyated proteins in erythrocytes. Then, a peroxidase (hereinafter referred to as POD) and a reducing agent are added to the sample, so that a redox reaction occurs between the hydrogen peroxide and the reducing agent with the POD as a catalyst. At this time, when a reducing agent that develops color when it is oxidized is used, the quantity of the hydrogen peroxide can be determined by measuring the color. As a result, the quantity of the glycated proteins in erythrocytes can be determined.
However, various kinds of reducing substances, such as L-ascorbic acid (AsA) and bilirubin, are usually present in blood. Moreover, various types of reducing substances such as glutathione (GSH) and the like are present in erythrocytes. These reducing substances may reduce the hydrogen peroxide, or may inhibit the redox reaction, or may reduce the reducing agent after it develops color, so as to cause degradation of the color. Therefore, there has been a problem that it is difficult to determine the quantity of the glycated proteins in erythrocytes accurately.
There has been also another problem, that precision of the measurement may deteriorate because the concentrations of the reducing substances contained in samples are not constant.
In order to avoid these problems, for example, various types of oxidizing agents have been added to samples. For example, Publication of Unexamined Japanese Patent Application No. Sho 56-151358 discloses a method of using halogen oxides, such as iodic acid or periodic acid, as oxidizing agents. Publications of Unexamined Japanese Patent Applications No. Sho 57-13357, No. Sho 57-161650, No. Sho 59-193354, No. Sho 62-169053, and No. Hei 3-30697 also disclose methods of using complexes of metals such as cobalt, iron, cerium, etc. as oxidizing agents.
However, the effect of the reducing substances on the measurements can not be avoided sufficiently even with the use of these oxidizing agents. In particular, these oxidizing agents performed poorly when the analyte was a component in erythrocytes.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a highly reliable method of measuring an analyte in a sample using a redox reaction.
In order to accomplish this object, the present invention provides a method of measuring an analyte in a sample using a redox reaction, comprising: adding a tetrazolium compound prior to the redox reaction to a sample so as to eliminate the influence of any reducing substance contained in the sample; then forming a reducing substance or an oxidizing substance derived from the analyte; measuring the quantity of the formed substance derived from the analyte by the redox reaction; and determining the quantity of the analyte from the quantity of the formed substance. The tetrazolium compound herein refers to a compound having a tetrazole ring.
As a result of extensive studies. the inventors found that the cause of the problems in the conventional methods was not that the influence of the low molecular weight reducing substances such as the above-mentioned GSH and AsA were not eliminated, but that the influence of high molecular weight reducing substances such as proteins or the like were not eliminated. The inventors also found that, not only the influence of the low molecular weight reducing substances, but also influence of other reducing substances can be eliminated by the use of the tetrazolium compound, and thus have reached the method of the present invention. According to the method of the present invention, the quantity of the analyte can be determined with greater reliability. Thus, it is used suitably for various kinds of tests. e.g. in clinical medicine.
In the method of the present invention, it is preferable that the tetrazolium compound has cyclic substituted groups in at least two positions of its tetrazole ring. More preferably, it has cyclic substituted groups at three positions thereof.
When the tetrazolium compound has cyclic substituted groups in at least two positions of its tetrazole ring as mentioned above, it is preferable that the substituted groups are at positions 2 and 3 thereof. Moreover, when the tetrazolium compound has cyclic substituted groups at three positions of its tetrazole ring, it is preferable that the substituted groups are at positions 2, 3, and 5 thereof.
In the method of the present invention, it is preferable that at least two of the cyclic substituted groups have benzene rings. Moreover, cyclic substituted groups other than those having benzene rings include, for example, substituted groups containing S or O in their ring skeletons and having resonance structures, such as thienyl and thiazolyl groups, and the like.
In the method of the present invention, it is preferable that the tetrazolium compound has cyclic substituted groups in at least three positions of its tetrazole ring, and that at least two of the cyclic substituted groups have benzene rings.
In the method of the present invention, it is preferable that at least one of the cyclic substituted groups has functional groups. It is more preferable that the number of the functional groups is large.
Preferable examples of the functional groups are electron attractive functional groups, e.g. halogen atoms or ether, ester, carboxyl, acyl, nitroso, nitro, hydroxyl or sulfo groups, and the like. Examples other than these functional groups are groups containing oxygen, such as hydroperoxy, oxy, epoxy, epidioxy, and oxo groups, and groups containing sulfur, such as mercapto, alkylthio, methylthiomethyl, thioxo, sulfino, benzenesulfonyl, phenylsulfonyl, p-toluenesulfonyl, p-tolylsulfonyl, tosyl, sulfamoyl, and isothiocyanato groups. Among the electron attractive functional groups, preferable are halogen atoms and nitro, sulfo, carboxyl, hydroxyl, methoxy, and ethoxy groups. Furthermore, examples other than the above-mentioned electron attractive functional groups include unsaturated hydrocarbon groups, such as phenyl group (C
6
H
5
—), styryl group (C
6
H
5
CH═CH—), and the like. Moreover, the functional groups may be ionized by dissociation.
In the method of the present invention, it is preferable that the tetrazolium compound has benzene rings at positions 2 and 3 of its tetrazole ring, and that at least one of the benzene rings has at least one functional group selected from the group consisting of halogen atoms and carboxyl, nitro, hydroxyl, sulfo, methoxy, and ethoxy groups. Moreover, both of the benzene rings may have such functional groups. The benzene ring may have the functional groups at any position (i.e. ortho-, meta-, or para-). Furthermore, the number of the functional groups is not particularly limited, and the benzene ring may have either the same or different functional groups.
In the method of the present invention, examples of the tetrazolium compound include those having substituted groups with benzene rings at positions 2, 3 and 5 of their tetrazole rings, e.g. 2-(4-iodophenyl)-3-(4-nitrophe
Komori Tsuguki
Yonehara Satoshi
Kyoto Dai-ichi Kagaku Co. Ltd.
Leary Louise N.
Merchant & Gould P.C.
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