JPH0673473B2 - Analytical element - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an analytical element, particularly an analytical element for analyzing a specific component in a fluid sample, and more specifically, a specific component in a biological fluid sample in a reduced form. The present invention relates to a dry analytical element for analysis via coenzyme.

[Conventional technology]

Various types of dry analytical elements are known for analyzing a specific component in a biological fluid sample. Among them, an analytical element utilizing a reaction system for forming a dye by transmitting a reduced coenzyme generated under the participation of a dehydrogenase and an oxidized coenzyme to a chromogenic precursor through an electron transfer agent, For example, it is described in detail in JP-A-59-88097, JP-A-59-91896 and Japanese Patent Application No. 60-104776.

[Problems to be solved by the invention]

However, the analytical element described in each of these specifications is a biological element except for the analysis of lactate dehydrogenase and the analysis of specific components by the use of oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) as an oxidized coenzyme. Of lactate dehydrogenase (LDH) and lactate-derived oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) in biological fluid samples due to non-specific reduction There is.

An object of the present invention is to provide an analytical element utilizing a reaction system in which a reduced coenzyme produced under the participation of a dehydrogenase and an oxidized coenzyme is transferred to a pigment forming precursor through an electron transfer agent to form a pigment. In order to eliminate the error caused by LDH and lactic acid in the biological fluid sample, it is to provide an analytical element with improved analytical accuracy.

[Means for solving problems]

Briefly describing the present invention, the present invention relates to an analytical element, comprising: a light-transmissive liquid-impermeable support;
Reagent layer, a second reagent layer and a porous spreading layer above the same, and an electron transfer agent, a dye-forming precursor, an oxidized coenzyme,
An analysis element for analyzing a specific component in the fluid sample, which comprises a buffer and a reagent capable of converting the oxidized coenzyme into a reduced coenzyme through the specific component in the fluid sample, It is characterized in that a coenzyme is contained in the porous developing layer and an LDH inhibitor is contained only in the porous developing layer or in the second reagent layer and the porous developing layer.

Hereinafter, the present invention will be specifically described.

The electron transfer agent according to the present invention is present in the presence of a reduced coenzyme produced by the reaction of at least one of the reagents according to the present invention and an oxidized coenzyme, with the presence of a specific component in a biological fluid sample. The electron transfer agent, which has been reduced by the above method, further reduces the dye-forming precursor to form a dye having absorption in the visible region.

Specific components in the fluid sample that can be measured in the present invention include, for example, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), amylase (AMY), creatine phosphokinase (CPK) and triglyceride (TG). Can be mentioned.

The reagent according to the present invention contains an enzyme and optionally a substrate.

These reagents are appropriately selected depending on the specific components in the biological fluid sample to be measured, for example, aspartic acid, α-ketoglutarate and glutamate dehydrogenase (GlDH) when measuring GOT, and when GPT. Is alanine, α-ketoglutarate and glutamate dehydrogenase (GlDH), AMY
In the case of, altopentose, orthophosphate, β-phosglucomutase (β-PGM), glucose oxidase (G
OD) and maltose phosphorylase (MP), creatine in the case of CPK, adenosine triphosphate (ATP), hexonase (HK) and glucose-6-phosphate dehydrogenase (G
-6-PDH) and TG, lipoprotein lipase (L
PL), glucerokinase (GK), glucerophosphate dehydrogenase (GPDH) and adenosine triphosphate (ATP) or L
PL and glycerol dehydrogenase (GDH).

The oxidized coenzyme according to the present invention refers to NAD ^+, NADP ^{+ and the} like. The reduced coenzyme is a reduced form of the oxidized coenzyme. The reduced form of NAD ⁺ is NADH and the reduced form of NADP ⁺ is NADPH. In the present invention, NAD ⁺ is particularly preferably used, and
Converted to NADH. By incorporating NAD ⁺ into the porous spreading layer according to the present invention, the analytical sensitivity of the specific component can be improved.

Below is shown a reaction formula in which NADH is produced by the reaction of at least one of the reagents according to the present invention with NAD ⁺ through the intervening specific component in the biological fluid sample according to the present invention.

GOT GPT AMY CPK TG Examples of the electron transfer agent used in the present invention include N-methylphenazine methosulfates (for example, N-methylphenazine methosulfate, 1-methoxy-N-methylphenazine methosulfate, etc.), Meldola blue, methylene blue and diaphorase. Preferred electron transfer agents that can be used include N-methylphenazine methosulfates and diaphorase.

On the other hand, tetrazolium salts are usually used as the dye-forming precursor according to the present invention. Most of the tetrazolium salts used in the present invention become hardly soluble or insoluble in water after dye formation, and although it is usually difficult to use by the wet chemistry method, the dye formed is diffusion resistant and Prevent desired ringing,
It can be preferably used because it improves the quantitativeness of measurement.

Examples of the tetrazolium salt useful in the present invention include, for example, 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis [2- (p-nitrophenyl) -5.
-Phenyl tetrazolium chloride], 3,3 '-(3,3'
-Dimethoxy-4,4'-biphenylene) -bis [2,5-diphenyltetrazolium chloride], 3- (4'5'-
Dimethyl-2-thiazolyl) -2,4-diphenyltetrazolium bromide, 3- (p-iodophenyl) -2-
(P-Nitrophenyl) -5-phenyl-tetrazolium chloride, 2,2 ', 5,5'-tetra- (p-nitrophenyl) -3,3'-(3,3'-dimethoxy-4,4'-biphenylene ) -Ditetrazolium chloride, 2,3,5-triphenyltetrazolium chloride, 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis- [2,5-bis (p-nitrophenyl) ) Tetrazolium chloride] and
3,3 '-(4,4'-biphenylene) -bis [2,5-diphenyltetrazolium chloride] and the like can be mentioned.

Among the above tetrazolium salts, those preferably used include 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis [2- (p-nitrophenyl) -5-phenyltetrazolium chloride. ] And 3,3 '-(4,4'-
Biphenylene) -bis [2,5-diphenyltetrazolium chloride] can be mentioned, and it is preferable to include it in the first reagent layer according to the present invention.

The buffer used in the present invention is appropriately selected depending on the optimum pH in the above reaction. For example, tris buffer (known as a combination of trishydroxymethylaminomethane and trishydroxymethylaminomethane hydrochloride), known as a good's buffer, carbonate buffer and the like can be preferably used.

The buffering agent is preferably contained in a layer different from the above-mentioned dye-forming precursor. It goes without saying that these are laminated in a state where they are not mixed at the time of production and application of the sample. For this reason, it is preferable that the buffer agent is dispersed in the binder, and it is preferable that the buffer agent is contained in the second reagent layer according to the present invention.

The LDH inhibitor according to the present invention refers to a substance that inhibits the activity of LDH, and as the LDH inhibitor, oxalic acid and its salt, pyruvic acid and its salt, malonic acid and its salt, oxamic acid and its salt. , Tartronic acid and its salts, ethylenediaminetetraacetic acid and its salts, iodoacetamide, 2,
4-dinitrofluorobenzene, mercuric p-chlorobenzoate, iodide, silver salt and mercuric acid salt can be used. Preferred lactic acid dehydrogenase inhibitors include oxalic acid and its salt, pyruvin. Mention may be made of acids and salts thereof, oxamic acid and salts thereof, tartronic acid and salts thereof and iodoacetamide.

The effect of the present invention becomes particularly large by including the LDH inhibitor only in the second reagent layer and the porous spreading layer or the porous spreading layer according to the present invention.

The characteristics of the binder that constitutes the first reagent layer and the second reagent layer according to the present invention are important. The binder of the second reagent layer is preferably laminated with a solvent that is insoluble in the binder of the first reagent layer. That is, the second
A combination of binders in which the solvent of the binder of the reagent layer does not dissolve the binder of the first reagent layer is preferable. For example, the binder of the first reagent layer is preferably a water-soluble polymer, and the binder of the second reagent layer is preferably a combination of hydrophilic and organic solvent-soluble polymers.

As the binder for forming the first reagent layer according to the present invention, gelatin, gelatin derivatives such as phthalated gelatin, water-soluble cellulose derivatives such as hydroxyethyl cellulose, carboxymethyl cellulose sodium salt, polyvinyl alcohol, polyacrylamide, polymethacrylamide and the like. , Poly (mono- or dialkyl-substituted) acrylamide, poly (mono- or dialkyl-substituted) methacrylamide, and water-soluble copolymers thereof. Gelatin and its derivatives are preferably used.

Examples of the binder for forming the second reagent layer of the present invention include poly (N-vinylpyrroline), poly (N-vinylimidazole), poly (N-vinyltriazole) and their derivatives or their copolymers. , Cellulose derivatives such as ethyl cellulose and methyl cellulose, Japanese Patent Application No. 60
The copolymers and the like described in the specification of -104776 can be mentioned. These polymers are mainly polymeric substances which are soluble in alcohols such as ethanol, propanol and butanol and are hydrophilic. The copolymer described in Japanese Patent Application No. 60-104776 is preferably used.

The electron transfer agent and various other reagents according to the present invention may be contained in any of the first reagent layer, the second reagent layer and the porous spreading layer.

The amount of the electron transfer agent used in the present invention contained in the analytical element of the present invention varies depending on the amount of the specific component, but in the case of an electron transfer agent other than diaphorase, it is usually 1 mg / m ^{2 to} 1 g.
/ m ² , preferably 10 to 500 mg / m ² .

Furthermore, when diaphorase is used as an electron transfer agent, it varies depending not only on the amount of the specific component but also on the origin of diaphorase and the method for measuring the activity value. Usually 100U
/ m ^{2 to} 100,000 U / m ² , preferably 500 to 50,000 U / m ² .

The amount of the dye-forming precursor according to the present invention contained in the analytical element of the present invention is usually 10 mg / m ^{2 to} 10 g / m ² , and preferably 50 mg / m ^{2 to 3} g / m ² . Further, the amount of the oxidized coenzyme according to the present invention contained in the analytical element of the present invention is usually 10
mg / m ^{2 to} 50 g / m ² , preferably 50 mg / m ^{2 to} 10 g / m ² .

The amount of the LDH inhibitor according to the present invention contained in the analytical element of the present invention is usually 5 mg / m ^{2 to} 50 g / m ² , preferably 30 mg.
/ m ^{2 to} 10 g / m ² .

The liquid-impermeable light-transmitting support according to the analytical element of the present invention (hereinafter, abbreviated as the support according to the present invention) may be any type as long as it is liquid-impermeable and light-transmitting. However, various polymeric materials such as cellulose acetate, polyethylene terephthalate, polycarbonate or polystyrene are suitable for this purpose. Furthermore, not only the above-mentioned polymer materials but also inorganic materials such as glass can be used as well. The thickness of the support according to the present invention is arbitrary, but is preferably 50 to 250 μm. Further, one side surface of the support according to the present invention on the observation side can be optionally processed according to its purpose. Further, a light-transmitting undercoat layer may be used on the side of the support on which the reagent layer is to be laminated to improve the adhesion between the reagent and the support.

The porous spreading layer according to the present invention has (1) a function of uniformly distributing a fixed volume of fluid sample to the reagent layer per unit area. Furthermore, the performance described in JP-B-53-21677, that is, (2) a function of removing a substance or a factor that inhibits an analysis reaction in a fluid sample and / or (3) a spectrophotometric analysis Further, it is preferable that it has a function of performing a back ground function of reflecting the measurement light transmitted through the support. Therefore, the porous spreading layer according to the present invention is a layer having only the function of the above (1),
In addition to (1), it may be any of the layers having the functions of (2) and / or (3), or (1)
It is also possible to appropriately separate a plurality of functions including, and to use a different layer for each function. Furthermore, a layer having two functions among the functions (1), (2) and (3). And a layer having the remaining one function can be used in combination. For example, a development layer of a non-fibrous porous medium called a brush polymer composed of titanium dioxide and cellulose diacetate, which is described in JP-B-53-21677, described in JP-A-55-164356. Development layer of hydrophilically treated woven fabric, JP-A-57-94658, 57-125847, 57
-197466 and 58-70161, and the like, the fiber structure developing layer described in JP-A-58-90167 and the particle structure developing layer described in JP-A-58-90167. In particular, the above-mentioned fibrous structure-developed layer and particle-conjugated structure-developed layer are particularly useful as a material capable of rapidly transferring a blood cell portion. The film thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, but is preferably about 10
The thickness is 0 to 500 μm, more preferably about 150 to 350 μm. Also, the porosity is preferably about 20-85%.

As other additional additives, various additives such as preservatives and surfactants can be added as desired.

In particular, the surfactant can be effectively used for controlling the permeation rate when the fluid sample is applied to the device of the present invention.

As the usable surfactant, ionic (anionic or cationic) and nonionic surfactants can be used, but nonionic surfactants are effective. Examples of nonionic surfactants include, for example, 2,5-di-t.
Examples include polyalkylene glycol derivatives of alkyl-substituted phenols such as butyl phenoxy polyethylene glycol, p-octyl phenoxy polyethylene glycol, p-isononyl phenoxy polyethylene glycol, and polyalkylene glycol esters of higher fatty acids. These surfactants have the effects of controlling the permeation rate of the fluid sample into the reagent layer and, at the same time, suppressing the undesirable occurrence of the "chromatographic phenomenon".

The surfactant may be used in a widely selected amount, but may be used in an amount of 25% by weight to 0.005% by weight, preferably 15 to 0.05% by weight, based on the weight of the coating solution.

The analysis element of the present invention may be used, for example, as described in U.S. Pat.
No. 992,158, reflective layer, undercoat layer, U.S. Pat.
Radiation blocking layer described in 4,042,335, barrier layer described in U.S. Pat.No. 4,066,403, migration blocking layer described in U.S. Pat.No. 4,166,093,
The scavenger layer described in JP-A-55-90859 and the destructible pot-shaped member described in U.S. Pat.No. 4,110,079 can be arbitrarily combined to form an arbitrary structure for the purpose of the present invention. .

Various layers of these analytical elements are used by appropriately selecting the slide hopper coating method, the extrusion coating method, the dip coating method, etc., which are conventionally known in the photographic industry, according to the desired configuration on the support according to the present invention, and are sequentially used. By stacking, a layer having an arbitrary thickness can be applied.

Using the analytical element of the present invention, the amount of a specific component in a fluid sample can be measured from the support side according to the present invention by reflection spectrophotometry according to the initial velocity method or the reaction end point method. The amount of the specific component can be determined by applying the measured value thus obtained to a calibration curve prepared in advance.

The amount of the fluid sample applied to the analytical element of the present invention can be arbitrarily determined, but is preferably about 5μ to about 50μ.
And more preferably 5 μ to 20 μ. Normal
It is preferred to apply a 10μ fluid sample.

The analysis element of the present invention can be used for any analysis of whole blood, serum and plasma without any inconvenience. Furthermore, other body fluids such as urine, lymph fluid, and cerebrospinal fluid can be used without any inconvenience. If whole blood is used, the radiation blocking layer or other reflective layer described above may optionally be provided to prevent the radiation for detection from being interfered by blood cells.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Analytical element for GOT) A first reagent layer having the following composition was provided on a transparent undercoated polyethylene terephthalate support having a film thickness of 180 μm.

First reagent layer (R-1-1) Gelatin 21.0 g / m ² glutamate dehydrogenase 42,000 U / m ² diaphorase 2,100 U / m ² 3,3 ′-(4,4′-biphenylene) -bis (2 , 5-Diphenyltetrazolium chloride) 1.0g / m ² Triton X-100 [Rohm & Has
s) Company] 2.1 g / m ² 1,2-bis (vinylsulfonyl) ethane 0.15 g / m ^{2 A} second reagent layer and a spreading layer shown in the following table are sequentially provided on the first reagent layer, -1 analysis elements 1 to 3 of the present invention
And Comparative Analysis Element-1.

Second reagent layer (R-2) Deployment layer (S) For the analytical elements 1 to 3 and the comparative analytical element-1 of the present invention, dialyzed pooled serum (GOT: 25K-U, LDH: 250W
-U), GOT [Sigma, Porcine Heart (Porcine
Heart)] added pool serum (GOT: 90K-U, LDH: 250W
-U) and pool sera obtained by adding LDH (Rabbit Muscle, Sigma) to the pool sera (GOT: 25K-U, LDH: 730W-U and GOT: 90K-U, LDH:
730W-U) and 0, 10, 20,
After adding 30 mg / dl of each pooled serum to the 10μ development layer, incubate at 37 ° C and measure the reflection density 7 minutes and 11 minutes after addition through a 546 nm filter with a reflection spectrophotometer. The difference between the reflection densities is measured and the result is shown in the table.
Two results were obtained.

As is clear from the results in Table-2, in Comparative Analytical Element-1 which does not contain a lactate dehydrogenase inhibitor, LDH and lactic acid have a large effect, whereas LDH inhibitors (oxalic acid or its salt, oxamine It can be seen that the analytical elements-1 to 3 of the present invention containing an acid salt are hardly affected by the influence and the accuracy of analysis is improved.

Example-2 (analytical element for GPT) A first reagent layer having the following composition was provided on a transparent undercoated polyethylene terephthalate support having a film thickness of 180 μm.

First reagent layer (R-1-2) Gelatin 21.0 g / m ² glutamate dehydrogenase 21,000 U / m ² diaphorase 2,100 U / m ² 3,3 ′-(4,4′-biphenylene) -bis (2 , 5-diphenyltetrazolium chloride) 1.0 g / m ² Triton X-100 2.1 g / m ² 1,2-bis (vinylsulfonyl) ethane 0.15 g / m ^{2 On the} above first reagent layer A second reagent layer and a spreading layer are sequentially provided, and analytical elements 4 to 6 of the present invention shown in Table 3 are shown.
And Comparative Analysis Element-2.

Second reagent layer (R-2) Deployment layer (S) The above-mentioned analytical elements 4 to 6 of the present invention and comparative analytical element-2 were dialyzed with pooled serum (GPT: 20K-U, LDH: 250W).
-U), pool serum (GPT: 80K-U, LDH: 250W-U) to which GPT (Sigma, Poshin Heart) was added, and pool serum to which LDH (Sigma, Rabbit Muskle) was added to each pool serum described above. (GPT: 20K-U, LDH: 730W-U
And GPT: 80K-U, LDH: 730W-U), and each pooled serum added with 0, 10, 20, 30 mg / dl of lithium lactate was added to 1
After dripping on the 0μ development layer, incubating at 37 ° C, and measuring the reflection density after 7 minutes and 11 minutes after the addition using a reflection spectrophotometer through a 546 nm filter, and obtaining the difference in reflection density. The results shown in Table 4 were obtained.

As is clear from the results in Table 4, in Comparative Analytical Element-2 which does not contain a lactate dehydrogenase inhibitor, LDH and lactic acid have a large effect, whereas a lactate dehydrogenase inhibitor (oxalic acid or its Salt, oxamate) -containing analytical elements -4 to 6 of the present invention are hardly affected by the influence,
It can be seen that the accuracy of the analysis has improved.

Example-3 (Analytical element for TG) A first reagent layer having the following composition was provided on a transparent subbed polyethylene terephthalate support having a film thickness of 180 µm.

First reagent layer (R-1) On the first reagent layer, a second reagent layer and a spreading layer shown in the table below are sequentially provided, and the analysis element of the present invention-7 to
9 and Comparative Analysis Element-3 were prepared.

Second reagent layer (R-2) Deployment layer (S) The above-mentioned analytical elements-7 to 9 and comparative analytical element-3 of the present invention
5, dialyzed pooled serum (TG: 125 mg / dl, LD
H: 250W-U), LDH (Sigma), and Rabitto Muskle) added pooled serum (TG: 125 mg / dl, LDH: 730W-
U) and 0, 10, 20, 30 mg / d lithium lactate, respectively.
l Add each pooled serum added to the 10μ layer and add 37
The reflection density after incubating at ℃ for 7 minutes was measured with a reflection spectrophotometer through a 546 nm filter, and
6 results were obtained.

As is clear from the results of Table-6, in Comparative Analytical Element-3 containing no lactate dehydrogenase inhibitor and Comparative Analytical Elements-4 and -5 containing the inhibitor only in the first reagent layer, LD
While the influences of H and lactic acid are large, the analysis elements-7 to 9 of the present invention containing the LDH inhibitor (lithium pyruvate, oxalic acid) are hardly affected by the influence and the accuracy of analysis is high. You can see that it is improving.

In addition, Lipid Salem-I and -II [Eiken Chemical Co., Ltd.
100 mg, 200 mg, and 300 mg / dl of triglyceride standard serum prepared by the above method were dropped on the 10 μ-developed layer in the same manner as described above with respect to the analytical elements-7 to 9 of the present invention and the comparative molecular element-3,
The reflection density after incubating at 37 ° C. for 7 minutes was measured by a reflection spectrophotometer through a filter of 546 nm, and no difference in discriminating ability was observed between these analytical elements.

〔The invention's effect〕

As described in detail above, the analytical element of the present invention is
Also, it has a remarkable effect in that it enables accurate analysis without the influence of lactic acid.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Isao Haga No. 1 Sakuramachi, Hino-shi, Tokyo Photo Konishi Roku Photo Industry Co., Ltd. (72) Inventor Hiroshi Iwadate No. 1 Sakura-cho, Hino City, Tokyo Photo Roku Konishi Photo In-house (56) References JP 59-91896 (JP, A) JP 60-91998 (JP, A) "Biochemistry Data Book ▲ II ▼" edited by the Japanese Biochemical Society Tokyo Kagaku Dojin (1982-10- 10) P. 170-171

Claims (4)

[Claims] 1. A light-transmissive and liquid-impermeable support,
A first reagent layer, a second reagent layer, and a porous spreading layer above the first reagent layer, which have the above-mentioned components through an electron transfer agent, a dye-forming precursor, an oxidized coenzyme, a buffer, and a specific component in a fluid sample. Contains a reagent capable of converting an oxidized coenzyme to a reduced coenzyme,
In an analytical element for analyzing a specific component in the fluid sample, the oxidized coenzyme is contained in the porous developing layer, and a lactate dehydrogenase inhibitor is contained only in the porous developing layer, or An analytical element containing the second reagent layer and the porous spreading layer. 2. The analytical element according to claim 1, wherein the dye-forming precursor is contained in a first reagent layer and the buffer is contained in a second reagent layer. 3. The analytical element according to claim 1 or 2, wherein the oxidized coenzyme is an oxidized nicotinamide adenine dinucleotide. 4. The lactate dehydrogenase inhibitor is oxalic acid and salts thereof, pyruvic acid and salts thereof, malonic acid and salts thereof,
From oxamic acid and its salts, tartronic acid and its salts, ethylenediaminetetraacetic acid and its salts, iodoacetamide, 2,4-dinitrofluorobenzene, mercuric P-chlorobenzoate, iodides, silver salts and mercuric salts. Claim 1 which is at least one selected from the group consisting of
The analytical element according to any one of items 1 to 3.