JP2003528623A - Reagent system for detecting the presence of reduced cofactor in a sample and methods of use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a signal generation system, a reagent composition, a test strip and a kit thereof, and a method of using the same in detecting an analyte in a sample. The signal generation system is characterized by having at least a first electron transfer agent and a second electron transfer agent and a redox indicator. In many preferred embodiments, the system includes proteinaceous and non-proteinaceous electron transfer agents, such as phenazine compounds and diaphorase. In many preferred embodiments, the systems and kits further comprise an enzyme cofactor and an enzyme having analyte oxidizing activity, such as an analyte dehydrogenase. The systems, reagent compositions, test strips and kits are used for detecting various analytes in a sample, such as a physiological sample such as blood or a piece of blood.

Description

Detailed Description of the Invention

[0001]Introduction Technical field   The field of this invention is the detection of analytes, and more particularly, reagent systems used for analyte detection.
It is about.

[0002]Conventional technology   The detection of analytes in physiological fluids such as blood or blood extracts has become a reality in today's society.
It is becoming more and more important. Sample detection and measurement methods are clinical laboratory tests, home
Used for various purposes such as testing, these test results are used for diagnosis and treatment of various medical conditions.
Plays an outstanding role in. Samples of interest include alcohol,
Lumaldehyde, glucose, glutamic acid, glycerol, β-hydroxybutene
Includes sylate, L-lactate, leucine, malic acid, pyruvic acid, steroids, etc.
Be done. Depending on this increase in importance of analyte detection, various analyte detection protocols
And analyte detection devices have been developed for both clinical and home use. Ever developed
Many of the protocols and devices used in this study exist in physiological samples such as blood.
A signal generation system for recognizing the sample is used.

One type of signal producing system used to detect a variety of analytes is that dehydrogenase oxidizes the analyte in the signal producing system and concomitantly with N 2.
Some reduce enzymatic cofactors such as AD (P) +. The reductant of a cofactor such as NAD (P) H is subsequently transferred by a cofactor oxidant such as phenazine methosulfate or diaphorase to a redox indicator such as a tetrazolium salt to produce a detectable product. To be detected.

Although various signal generating systems as described above have been developed so far for detecting various analytes, further development of such systems continues to be sought. For example, there may be great interest in signal generation systems that increase reaction rates and reduce costs.

[0005]Related literature   Related patents are US Pat. Nos. 4,629,697 and 5,126,247.
And No. 5,902,731. Also, Histochem.J.
1983) 15: 881-893.

[0006]Summary of the invention   Signal generation systems, reagent compositions, test strips and their kits, and
Methods for their use in detecting analytes in samples are provided. This signal generation system
The system includes at least a first electron transfer agent and a second electron transfer agent and redox instructions.
Characterized by having a drug. In many preferred embodiments, the system is
Is a proteinaceous and nonproteinaceous electron transfer agent such as a phenazine compound and
And diaphorase. In many preferred embodiments, the system and kit
Is an enzyme having an enzyme cofactor and an analyte oxidative activity, such as analyte dehydrate.
Contains a logenase. The system, reagent composition, test strips and kits
Detecting various analytes in a sample, such as a physiological sample such as blood or blood pieces
Used for output.

[0007]BEST MODE FOR CARRYING OUT THE INVENTION   Signal generation systems, reagent compositions, test strips and their kits, and
Methods for their use in detecting analytes in samples are provided. This signal generation system
The system includes at least a first electron transfer agent and a second electron transfer agent and redox instructions.
Characterized by having a drug. In many preferred embodiments, the system is
Is a proteinaceous and nonproteinaceous electron transfer agent such as a phenazine compound and
And diaphorase. In many preferred embodiments, the system and kit
Is an enzyme having an enzyme cofactor and an analyte oxidative activity, such as analyte dehydrate.
Contains a logenase. The system, reagent composition, test strips and kits
Detecting various analytes in a sample, such as a physiological sample such as blood or blood pieces
Used for output.

Before the invention is described in detail, it should be understood that the particular embodiments described below can be modified in various ways, which modifications are still covered by the claims. Is not limited by the particular embodiment described below. Also, it is to be understood that the terminology used is for the purpose of describing the embodiments and is not limiting. Rather, the scope of the invention will be defined by the appended claims.

In this specification and claims, the singular reference includes the plural reference unless specifically stated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Signal Generation System As described above, the present invention provides a signal generation system capable of detecting the presence of reduced enzyme cofactors in a sample. Signal generating system means the collection of two or more compounds or molecules that can act cooperatively when bound to produce a detectable signal that is indicative of the presence and often the amount of a particular analyte in a given sample. To do. The term signal producing system is broadly meant to be a mixture of all reagent components of the signal producing system, with one or more reagent components remaining in the system,
For example, it is used to include both the meaning of a system separated from the components present in the kit.

A characteristic of the signal generating system is that it has two different electron transfer agents.
By electron transfer agent is meant a compound or molecule capable of transferring an electron from the reduced enzyme cofactor in the form of a hydride ion to a redox indicator. In the present signal generation system, the first electron transfer agent is a low molecular weight molecule and the second electron transfer agent is a high molecular weight molecule. As used herein, low molecular weight is a range not exceeding about 2000 Daltons, usually about 1000 Daltons, and in many embodiments about 5 Daltons.
It is 00 Dalton. High molecular weight means a molecular weight of at least about 5000 Daltons, and in many embodiments 10,000 Daltons or 20,000 Daltons or higher. The molecular weight of a high molecular weight electron transfer agent is usually about 100,0.
Will not exceed 00 Daltons. In many embodiments, the high molecular weight electron transfer agent is a proteinaceous compound, while the low molecular weight electron transfer agent is a non-proteinaceous compound. By proteinaceous is meant a polypeptide or pseudo-polymeric form thereof.

There is interest in a variety of low molecular weight nonproteinaceous electron transfer agents. These agents include riboflavin (RBF), alloxazine (ALL) and lumichrome (
LC) flavin, phenazine, phenazine methosulfate (PMS)
, Phenazines such as phenazine ethosulfate, methoxyphenazine methosulfate and safranine, methyl-1,4-naphthol (menadione), P
T and its radical cation PT +, thionine (TH), Azure A (AA)
, Phenothiazines such as Azure B (AB), Azure C (AC), methylene blue (MB), methylene green (MG) and toluidine blue O (TOL), phenoxazine (POA), basic blue 3 (BB3), brilliant clay. Silblue ALD (BCBA), phenoxazine such as benzo-α-phenazoxonium chloride (Medola's blue), indophenol such as 2,6-dichlorophenolindophenol (DCIP), bindschedler's green (Bindschedler's green) ), Indamine and the like such as phenylene blue. Of particular interest in many embodiments are phenazine compounds such as PMS, phenazine ethosulfate, methoxyphenazine methosulfate and safranine. In many embodiments, PMS is used as the low molecular weight non-proteinaceous electron transfer agent.

[0013] In many embodiments, the high molecular weight, proteinaceous electron transfer agent is, for example, NAD.
An enzyme capable of oxidizing a reduced cofactor such as (P) H and concomitantly reducing a redox indicator. In many embodiments, the electron transfer enzyme is a diaphorase such as lipodehydrogenase, ferredoxin-NADP reductase, lipoamide dehydrogenase, NADPH dehydrogenase.
Various diaphorases are available and may be used. Representative commercially available diaphorases that may be present in the signal generating system include Bacillus diaphorase, Clostridium diaphorase, Vibrio diaphorase, Porcine diaphorase and the like.

In the present signal generating system, the ratio of the first electron transfer agent to the second electron transfer agent is selected to provide a faster reaction rate than the comparison standard. A comparison standard is a comparable signal generating system having only one electron transfer agent, such as either PMS or diaphorase. Generally, the ratio of the first electron transfer agent to the second electron transfer agent in the system is about 0.001 (nmole / U) to 10 (nmole / U).
), Usually 0.01 (nmole / U) to 1.0 (nmole / U), and even 0.05 (nm
ole / U) to 0.5 (nmole / U).

In addition to the first electron transfer agent and the second electron transfer agent described above, the signal generating system also includes a redox indicator. By redox indicator is meant a compound that can be reduced by an electron transfer agent to produce a detectable product such as a chromogenic product. When the redox indicator produces a chromogenic product, i.e. when the redox indicator is the primary colorant, a suitable primary colorant is any compound that undergoes a color change upon reduction by one or more electrons. This suitable primary color usually receives electrons from the electron transfer agents described above.

There is interest in various redox indicator compounds. Compounds of interest include oxazine, thiazine and tetrazolium salts. Particularly preferred in many embodiments is a tetrazolium salt, which is capable of receiving the hydride captured from the electron transfer agent and producing a colored formazan product. In many embodiments, these salts have a light yellow color in their oxidised form, but have the advantageous feature of being readily transformed into a bright visible color by electron reduction and converted into a formazone dye. Tetrazolium compounds or salts of particular interest include 2- (2'benzothiazolyl) -5-styryl-3- (4'-phthalhydrazidyl) tetrazolium (BSPT), 2-benzothiazolyl- (2) -3,
5-diphenyltetrazolium (BTDP), 2,3-di (4-nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3- (4-styrylphenyl) tetrazolium (DPSP), distyrylnitroblue tetrazolium (D
S-NBT), 3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl)
-4,4'-Diyl] -bis [2- (4-nitrophenyl) -5-phenyl] (-
2H tetrazolium (NBT), 3- (4,5-dimethyl-2-thiazolyl)-
2,5-Diphenyl-2H tetrazolium (MTT), 2-phenyl-3- (4
-Carboxyphenyl) -5-methyltetrazolium (PCPM), tetrazolium blue (TB), thiocarbamyl nitroblue tetrazolium (TCNBT)
), Tetranitroblue tetrazolium (TNBT), tetrazolium violet (TV), 2-benzothiazothiazolyl-3- (4-carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl). Phenyl] -2H
-Tetrazolium (WST-4), 2,2'-dibenzothiazolyl-5,5'-
Bis [4-di (2-sulfoethyl) carbamoylphenyl] -3,3 '-(3,3
'-Dimethoxy-4,4'-biphenylene) ditetrazolium, disodium salt (WST-5), 2- (p-nitrophenyl) -3- (p-iodophenyl)-
5-phenyltetrazolium chloride (INT) and the like. WST-5 is preferred in many embodiments. This is because WST-5 dissolves rapidly in aqueous media and is most compatible with biological samples. Furthermore, the formazone compounds produced show a strong absorption of the spectrum in the violet-blue region, which reduces the need to correct the background signal due to hemoglobin. Other useful tetrazolium salts are disclosed in U.S. Pat. Nos. 4,490,465 and 4,491,631.
No. 4,598,042, 4,351,899, 4,271,
No. 265, No. 4,247,633, No. 4,223,090, No. 4,2
No. 15,917, No. 4,142,938, No. 4,024,021, No. 3,867,259, No. 3,867,257, No. 3,791,931 and No. 4,254,222, the disclosures of which are incorporated herein by reference.

The signal generating system described above is based on the reduced enzyme aid present in a sample, in particular an aqueous sample, more particularly in a biological sample such as whole blood, whole blood pieces or whole blood extracts. The presence of the factor can be detected. A variety of reduced reductase cofactors may be detected using the present signal generation system, representative reductase cofactors including β-
Nicotinamide adenine dinucleotide (β-NAD), β-phosphate nicotinamide adenine dinucleotide (β-NADP), thionicotinamide adenine dinucleotide, phosphate thionicotinamide adenine dinucleotide, nicotinamide 1, N6-ethenoadenine Included are dinucleotides, nicotinamide 1, N6-phosphate ethenoadenine dinucleotide and reductants of cofactors such as pyrroloquinoline quinone (PQQ). This signal generation system is particularly suitable for NADH or NA.
It is suitable for use in detecting D (P) H.

In many applications where the present signal generating system is used, the reductant of the enzyme cofactor is generated following oxidation of the analyte of interest in the sample. Therefore, in many embodiments, the signal generating system further comprises an enzyme cofactor and an analyte oxidase capable of oxidizing the analyte of interest and concomitantly reducing the enzyme cofactor. Enzyme cofactors of interest include those mentioned above. That is, β-nicotinamide adenine dinucleotide (β-NAD), β-nicotinamide adenine dinucleotide (β-NADP), thionicotinamide adenine dinucleotide, phosphoric acid thionicotinamide adenine dinucleotide, nicotinamide 1, Includes N6-ethenoadenine dinucleotide, nicotinamide 1, N6-phosphate ethenoadenine dinucleotide and pyrroloquinoline quinone (PQQ). Enzyme cofactors of particular interest that may be included in the present signal generation system include NADH or NAD (
P) There is H.

The analyte oxidase present in the signal generating system is always determined by the type of analyte detected by the system. Analyte oxidases of interest include alcohol dehydrogenase for alcohol, formaldehyde dehydrogenase for formaldehyde, glucose dehydrogenase for glucose,
Phosphate glucose-6 dehydrogenase for phosphate glucose-6, glutamate dehydrogenase for glutamate, glycerol dehydrogenase for glycerol, β-hydroxybutyrate dehydrogenase for β-hydroxybutyrate, hydroxysteroid for steroids Included are dehydrogenase, L-lactate dehydrogenase for L-lactate, leucine dehydrogenase for leucine, malate dehydrogenase for malate and pyruvate dehydrogenase for pyruvate. As can be seen from the list above, the analyte oxidase is generally a dehydrogenase.

Reagent Compositions The present invention also provides reagent compositions used to detect at least reduced enzyme cofactor in a sample, and in many embodiments, analytes. The reagent composition may be a liquid, such as water, or may be a dry composition. In many embodiments it is a dry composition. The reagent composition comprises at least a first electron transfer agent and a second electron transfer agent.
It includes an electron transfer agent and a redox indicator, and these components are as described above. Such a reagent composition is suitable for use in detecting reduced enzyme cofactors such as NAD (P) H in a sample. However, in many embodiments, the reagent composition further comprises an enzyme cofactor and a sample oxidase, these components being as described above.

Reagent Test Strips The present invention also provides reagent test strips used to detect the presence of an analyte in a sample. In particular, the invention provides dry strips for measuring specific analytes in whole blood, such as β-hydroxybutyrate, glucose and the like. In the broadest sense, a reagent test strip has a hollow support and a dry reagent composition thereon. This dry reagent composition is made up of all of the reagent compounds necessary to produce a detectable signal in the presence of the analyte of interest. In most embodiments of the invention, the dry reagent composition present on the test strip comprises an analyte oxidase, an enzyme cofactor, a first electron transfer agent and a second electron transfer agent, a redox indicator. Each of these components is as described in detail above.

In many embodiments, the test strips include a thin film test pad secured to a hollow support. The support material may be a plastic sheet such as polystyrene, nylon, polyester, a metal sheet, or any suitable material known in the art. In many embodiments, the test pad preferably comprises an absorbent agent such as filter paper or a polymer film. Coated on the test pad or contained within it is the reagent composition that binds to the test pad. The strip may also be designed in an intricate configuration, for example a test pad may be present between the support and a surface layer where one or more reagents used during sample processing may be present. Further, as known to those of skill in the art, flow paths or pathways may be present on the test strip. Test strip configurations of interest in many embodiments are disclosed in US Pat. No. 5,902,731, the disclosure of which is incorporated herein by reference.

The test strip may be made using any convenient protocol. One convenient protocol is to contact at least the test pad portion of the strip with an aqueous composition that includes all components of the reagent composition that bind to the test pad in the final reagent test strip. Conveniently, the test pad may be soaked in the aqueous composition, maintained for a sufficient period of time, and then dried to produce a test pad of a reagent test strip that binds to the reagent composition. As noted above, the aqueous composition will include various components of the reagent composition to be associated with the test pad of the reagent test strip. The various components are present in an amount sufficient to provide the desired amount of reagent composition produced on the test pad. Therefore, the concentration of the non-proteinaceous electron transfer agent present in the aqueous composition is typically about 1 μM to 1000 μM, or even about 10 μM to 500 μM. In contrast, the concentration of proteinaceous electron transfer agent in the aqueous composition is about 50 U to 3000 U, usually about 100 U to 1000 U. The concentration of redox reagent present in the aqueous composition is about 3 mM to 36 mM, usually about 6 mM to 24 mM. When present, the enzyme cofactor has a concentration of about 1.5 mM to 28 mM, usually about 3.5 mM to 14 mM.
Similarly, when a sample oxidase is present, the concentration of the sample oxidase is about 100 U to 2000 U, usually about 200 U to 1000 U. Other ingredients used to produce the reagent test strip may be present in the aqueous composition, such as sodium chloride, magnesium chloride, Tris, PSSA, Tetronic 1307.
, Crotane-SPA, sucrose, oxamic acid, sodium salt and the like. See the experimental section below for a more detailed description of an exemplary method of producing the present reagent test strips.

Analyte Detection Methods It will be appreciated that the signal generating system, reagent composition and test strips described above are used in methods to detect the presence and often the amount of an analyte in a sample. Various analytes can be detected by this method. Representative specimens include, for example, alcohol, formaldehyde, glucose, glutamic acid, glycerol, β-hydroxybutyrate, L-lactate, leucine, malic acid, pyruvic acid, steroids, etc. described above. In principle, the method can be applied to various biological samples such as urine,
It may be used to measure the presence and concentration of analytes in tears, saliva, etc.
It is particularly suitable for use in measuring the concentration of an analyte in blood or a blood sample such as a blood extract sample, and especially whole blood.

An important feature of the method is that the present signal generating system including a first electron transfer agent and a second electron transfer agent provides a faster reaction rate as compared to a standard system, eg, a system including one electron transfer agent. It is to be. Usually, the reaction rate is accelerated 1 to 3 times based on the ratio of PMS and diaphorase. Furthermore, the reaction rate is 1 to 3 times greater than the theoretical rate expected based on the sum of the rates provided by each electron transfer agent. K / S with reaction rate per second (see experimental section below)
, The K / S per second in the reaction of the signal generating system is typically in the range of about 0.01 to 10, even about 0.05 to 5, and even 0.1 to 2. To be

In the method, the sample and the signal generating system are combined into a reaction mixture. The reaction is allowed to proceed for a sufficient time to produce a signal that is indicative of the presence (and often the amount) of analyte in the sample. The signal produced is detected and correlated with the presence (and often its amount) of analyte in the sample. In the broadest sense, the reaction mixture is produced in any convenient environment, such as a cuvette or other liquid storage means. However, in many embodiments, the above steps are performed on the reagent test strips described above. Therefore, the method is further discussed as a method using a reagent test strip.

In practicing the method, the first step is to apply a quantity of physiological sample to the test strip. The test strip is as described above.
The amount of physiological sample, such as blood, applied to the test strip varies, but is usually about 2 μL to 40 μL, often about 5 μL to 20 μL. Due to the nature of the test strip, the blood sample size applied to the test strip may be relatively small, about 2 μL to 40 μL, usually about 5 μL to 20 μL. When the physiological sample is blood, the hematocrit value is about 20% to 65%, or even 25.
% To 60%, blood samples with different hematocrit values can be measured by the method.

After applying the test strip to the sample, the sample is reacted with the components of the signal generating system to produce a detectable product. This detectable product is produced in an amount proportional to the initial amount of analyte of interest present in the sample. The amount of detectable product, or signal produced by the signal producing system, is then measured and correlated with the concentration of analyte in the initial sample. In one embodiment,
An automated device is used that performs the detection and association steps described above. Regarding the above-mentioned reaction step, detection step, association step and devices for performing these operations, US Pat. Nos. 4,734,360, 4,900,666, and 4,935 are applicable.
, 346, 5,059,394, 5,304,468, 5,
306,623, 5,418,142, 5,426,032, 5,515,170, 5,526,120, 5,563,042.
No. 5,620,863, No. 5,753,429, No. 5,573.
No. 452, No. 5,780,304, No. 5,789,255, No. 5,8
No. 43,691, No. 5,846,486, No. 5,968,836, and No. 5,972,294. These disclosures are incorporated herein by reference. Regarding the concentration of the sample derived in the association step, a certain proportion of the competitive reaction to the actually measured signal is taken into consideration by calibrating the device.

Kits The invention also provides kits for use in carrying out the method. As mentioned above, the kit of the present invention includes at least a signal generation system. The components of this signal generation system may be combined into one reagent composition, or may be separated into separate containers or the like. In certain embodiments, the signal generating system will be present in the kit in the form of reagent test strips as described above. The kit may further comprise means for obtaining a biological sample. For example, if the physiological sample is blood, the kit may further include means for obtaining a blood sample, such as a lance for puncturing a finger, lance actuation means, and the like. Further, the kit may include a standard solution, for example, a sample standard solution containing a sample at a standard concentration. In certain embodiments, the kit also includes an automated method for detecting the amount of product produced on a strip after application of the sample as described above and correlating the detected product with the amount of analyte in the sample. Included equipment. Finally, the kit comprises instructions for using the kit elements in determining the concentration of an analyte in a physiological sample. These instructions may be present on one or more of the packaging, inserted labels, containers present in the kit, and the like. The following examples are given by way of illustration and not limitation.

[0030]Example A. Making a ketone test strip   0.8 μm Nylon Thin from Cuno (Meridian, CT)
The membrane was dipped in the reagents shown in Table 1 and thoroughly saturated.

[Table 1] Excess reagent was squeezed out using a glass rod. This thin film was attached to a drying chamber and dried at 56 ° C. for 10 minutes. Porex (thickness 0.6 mm) was dipped in a 5% nitric acid solution, then attached to a drying chamber and dried at 100 ° C. for 10 hours. Finally, the thin film was prepared from polyester material (ICI America, Wilmington, Del.).
Laminated between 4 mm Melenex® polyester) and nitric acid soaked Porex.

B. Measurements The following protocol was used to exchange 10 μL of an aqueous sample containing 40 mg / dL (D) β-hydroxybutyrate while exchanging strips based on the amount of PMS and / or diaphorase present on the strips described above. Testing was done on strips. A 10 ml aqueous sample was applied onto a freshly prepared test strip. The strip was inserted into a reflectometer and data acquisition was started. The reflectance of the readout strips was measured at 660 nm for ½ second intervals for 2 minutes. Next, the acquired data was uploaded from the reflectometer memory buffer to the personal computer via a modified serial cable. The reaction rate was calculated based on the initial rate of change of K / S in the linear range of reaction characteristics. The results shown in Table 2 are average values of the results of experiments repeated 5 times. The reaction rate was measured (in terms of K / S per second) in individual measurements. (K / S indicates the ratio of reflectance, which is described in US Pat. No. 4,935,3
No. 46, col. 14, disclosure of which is incorporated by reference) and the results are shown in Table 2.

[Table 2]

[0032] C. Measured speed vs. theoretical speed   Table 3 below shows a comparison of the measured and theoretical speeds for the above measurements.

[Table 3]

FIG. 1 is a graph showing measured speed vs. theoretical speed. As can be seen in FIG. 1, the reaction rate of the test strip's signal generation system is accelerated by the presence of both PMS and diaphorase. That is, the magnitude of the measured acceleration is PM
It is unexpectedly greater than the theoretical amount based on the sum of reaction rates for systems with S or diaphorase respectively.

From the above results and discussions, it is possible according to the present invention to significantly and unexpectedly accelerate the reaction rate measured by the analyte detection protocol based on the oxidation of the analyte and the accompanying reduction of the redox indicator. it is obvious. Further, according to the present invention, it is possible to detect an analyte by a more economical method as compared with the conventional method in which only diaphorase is used as the electron transfer agent. Therefore, the present invention makes a significant contribution to the art.

All publications and patent references cited in this specification are hereby incorporated by reference in the same manner as they are specifically and individually indicated to be referenced. Part of the calligraphy. All publications are cited as disclosed before the filing date of the present application and admit that the present invention is not entitled to antecedence to these publications by virtue of prior invention. Not a thing.

While the present invention has been described in detail with the aid of illustrations and examples for a clear understanding of the invention, some changes and modifications may be made without departing from the scope of the claims according to the teaching of the present invention. It is obvious to those skilled in the art that the above can be done.

[Brief description of drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 graphically illustrates the reaction rates actually measured in test strips according to the present invention versus the theoretically predicted reaction rates in test strips containing PMS and diaphorase. It has been clearly shown that the use of both non-protein and protein electron transfer agents such as PMS and diaphorase results in unexpectedly increased reaction rates.

[Procedure amendment]

[Submission date] February 5, 2003 (2003.2.5)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, J P, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, R O, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Oo Young Tianmei             United States, 94539 California             State, Fremont, Via San Gabriel               41945 F term (reference) 2G045 AA25 BB51 CA25 CB03 CB07                       DA04 DA20 DA30 DA31 DA35                       DA36 DA60 DA74 DA77 FB01                       FB11 FB17 GC11 JA01                 2G054 AA06 AA07 AB02 AB05 CA21                       CA23 CA24 CA25 CE02 CE08                       EA05 EB01 GA03 GE06 JA01                       JA06                 4B063 QA18 QA19 QA20 QQ03 QQ24                       QR02 QR64 QR65 QR66 QS02                       QS28 QX02

Claims (35)

[Claims] 1. A signal generating system for detecting the presence of a reduced enzyme cofactor in a sample, the first electron transfer agent and the second electron transfer agent capable of oxidizing the reduced cofactor. , And a signal generation system with a redox indicator. 2. The signal generating system according to claim 1, wherein the first electron transfer agent is a low molecular weight electron transfer agent. 3. The signal generation system according to claim 2, wherein the low molecular weight electron transfer agent is a phenazine compound. 4. The signal generating system according to claim 1, wherein the second electron transfer agent is a high molecular weight electron transfer agent. 5. The signal generation system according to claim 4, wherein the high molecular weight electron transfer agent is a protein compound. 6. The signal generating system according to claim 5, wherein the proteinaceous compound is an enzyme. 7. The signal generating system according to claim 1, wherein the redox indicator is a tetrazolium compound. 8. The signal generating system according to claim 1, wherein the signal generating system further comprises an enzyme cofactor. 9. The signal generation system according to claim 1, wherein the system further comprises a sample oxidase. 10. The signal generating system of claim 1, wherein the signal generating system is present as a reagent composition. 11. A reagent composition used for detecting the presence of an analyte in a sample, comprising: an analyte oxidase, an enzyme cofactor, a non-protein electron transfer agent, and a protein electron transfer agent. A reagent composition comprising a redox indicator. 12. The reagent composition according to claim 11, wherein the enzyme cofactor is NAD (P) +. 13. The reagent composition according to claim 11, wherein the non-proteinaceous electron transfer agent is a phenazine compound. 14. The reagent composition according to claim 11, wherein the proteinaceous electron transfer agent is diaphorase. 15. The reagent composition according to claim 11, wherein the redox indicator is a tetrazolium compound. 16. The reagent composition of claim 11, wherein the composition is a dry reagent composition. 17. A reagent composition used for detecting the presence of an analyte in a sample, comprising a sample dehydrogenase, NAD (P) +, a phenazine compound, a diaphorase, and a tetrazolium compound. Composition. 18. The reagent composition, wherein the phenazine compound is phenazine methosulfate. 19. The reagent composition according to claim 17, wherein the composition is a dry reagent composition. 20. The reagent composition of claim 19, wherein the dry reagent composition is on a test strip. 21. (a) a support member, (b) (i) a sample oxidase, (ii) an enzyme cofactor, (iii) a non-protein electron transfer agent, and (iv) a protein electron transfer agent. And a dry reagent composition comprising (v) a redox indicator, a reagent test strip. 22. The reagent test strip of claim 21, wherein the first enzyme is dehydrogenase. 23. The reagent test strip of claim 21, wherein the cofactor is NAD (P) +. 24. The reagent test strip of claim 21, wherein the non-proteinaceous electron transfer agent is a phenazine compound. 25. The reagent test strip of claim 24, wherein the phenazine compound is phenazine methosulfate. 26. The reagent test strip of claim 21, wherein the proteinaceous electron transfer agent is diaphorase. 27. The reagent test strip of claim 21, wherein the redox indicator is a tetrazolium compound. 28. A method for detecting the presence of an analyte in a sample, comprising:
A method with the improvement of using a signal generation system according to claim 1. 29. The method of claim 28, wherein the signal generating system is present as a reagent composition according to claim 11. 30. The method of claim 29, wherein the reagent composition is present on a reagent test strip according to claim 21. 31. A kit for use in detecting the presence of an analyte in a sample, the kit comprising a signal generating system according to claim 1. 32. The kit of claim 31, wherein the signal generating system is present as a reagent composition according to claim 11. 33. The kit of claim 32, wherein the reagent composition is present on a reagent test strip according to claim 21. 34. The kit of claim 31, wherein the kit further comprises means for obtaining a physiological sample. 35. The kit of claim 34, wherein the means for obtaining a physiological sample is a lance.