JPH0353919B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an amplified and highly sensitive measurement method for various substances.

Various high-sensitivity measurement methods have been developed, such as fluorescence measurement and luminescence measurement, but when the sample is body fluid, the blind value may increase due to substances contained in the body fluid, and errors may occur due to quenching. easy.

The coenzyme cycling method is a well-known method [Biochemistry Experiment Course 5, Enzyme Research Methods,
1, 121-135, edited by Nippon Biokagaku, Tokyo Kagaku Doujin].
However, in the conventional method, an indicator reaction must be further performed after the cycling reaction, which has problems in operability, and the indicator reaction itself is often influenced by substances other than the analyte. Moreover, it requires a special reaction container, such as an oil-based one, and has been carried out using a very small amount of reaction liquid. Furthermore, in conventional methods, when decomposing unreacted coenzymes, adding acid or alkali, heat treatment, and neutralizing, the amount of alkali or acid for neutralization must be added very precisely. However, if accurate neutralization is not possible and variations in pH occur, this greatly affects the cycling rate and causes errors in measurement values.

In order to solve the various drawbacks mentioned above, the present inventors
As a result of various studies aimed at improving the highly sensitive amplification measurement method using the coenzyme cycling method, we found that the cycling reaction itself can be used as the indicator reaction, which eliminates the need for operations and reagents for the indicator reaction, and allows only the end point method to be used. We have discovered that this is a very simple method, which has the feature that a late method is also possible, and also does not require a special reaction vessel, allowing the reaction to be carried out in a general thermostat. Furthermore, in order to eliminate the influence of PH variation on the cycling rate in the neutralization reaction after destroying unreacted coenzymes,
By adding an excess amount of formic acid lower alkyl ester
We have found a way to easily set the PH.

The present invention was completed based on the above-mentioned various findings, and in quantifying a substance to be measured, NAD ⁺ or
A main reaction system in which an oxidoreductase that uses the analyte as a substrate acts in the presence of NADH, NADP ⁺ or NADPH to convert a coenzyme according to the amount of the analyte. A decomposition reaction system that decomposes the enzyme and neutralizes it by adding formic acid lower alkyl ester, and a coenzyme and the coenzyme in the presence of an excess of the second substrate and a coenzyme converted in accordance with the amount of the substance to be measured. A reaction consisting of a cycling reaction system in which a second redox reaction system acting on the second substrate and a reaction system in which a tetrazolium salt is converted to formazan in the presence of diaphorase or an electron transfer substance are amplified by coenzyme cycling in combination. , is a highly sensitive measuring method for a substance to be measured, which is characterized by colorimetrically quantifying the produced formazan, and preferably includes an improvement measure by adding an alkali formate ester.

The objects of the present invention are as follows: (1) To provide a measurement method in which the cycling reaction itself is an indicator reaction, does not require any operation or reagent for the indicator reaction, and is not affected by substances other than the analyte. (2) The conventional method was only an end point method, but the present invention provides a measurement method that is capable of both an end point method and a late method. (3) A special reaction vessel is used. (4) To provide a very simple measurement method that does not require a very small amount of reaction liquid and can be carried out in a general thermostat. In the neutralization reaction after destroying the coenzyme, if the amount of acid or alkali is not carried out very precisely, variations in pH will occur.
This greatly affected the cycling rate and caused errors in measurement values, but in the present invention, by adding an excessive amount of formic acid lower alkyl ester, the pH
(5) Since the substrate of the analyte is amplified to the extent that it does not interfere with the measurement of other coexisting substances, the measurement method does not require purification and concentration of the analyte. It is about providing law.

An example of the principle of the reaction in the present invention is as shown in the following formula.

Main reaction system AH ₂ +NAD (P) ⁺ A + NAD (P) H + H ⁺ oxidoreductase AH ₂ or A; analyte NAD (P) ⁺ ; Indicates NAD ⁺ or NADP ⁺ .

NAD(P)H: Indicates reduced form of NAD ⁺ or NADP ⁺ .

Decomposition reaction system Neutralization reaction Neutralization of alkali; MOH + HCOR → HCOOM + ROH M: Alkali metal, R: Lower alkyl Cycling reaction In the present invention, the oxidoreductase used in the main reaction uses the analyte as a substrate, and NAD(P) ⁺ or
The oxidoreductase may be of any origin as long as it uses NAD(P)H as a coenzyme. These enzymes may be known or new. Examples of known enzymes and their substrates include:
It is described in "Enzyme Handbook" (published by Asakura Shoten) supervised by Shiro Akahori.

For the above main reaction, the reaction solution volume can range from 10 μm to 3 ml. Coenzyme is 0.1
It may be present in concentrations ranging from molar to 10 mmol. The minimum amount of the analyte that can be quantified is about 10 ^-8 to 10 ^-15 mol, and the amount of coenzyme is present in excess of the molar ratio at which the analyte acts in the reaction.
The above reaction is usually carried out within the optimum pH and temperature range of the oxidoreductase.

Next, when the main reaction stops, unreacted coenzymes are destroyed. In this decomposition reaction system, if the unreacted coenzymes are oxidized NAD ⁺ or NADP ⁺ , alkali By adding and heating, only the reduced NADH or NADPH produced by the main reaction remains. Also,
If the unreacted coenzyme is NADH or NADPH, add acid and heat to
Only NAD ⁺ or NADP ⁺ remains.

As the alkali, an aqueous solution of alkali hydroxide, such as sodium hydroxide or potassium hydroxide, is usually used. Furthermore, an aqueous solution of lithium hydroxide, sodium carbonate, potassium carbonate, or the like may be used. The concentration of alkali is the concentration of its aqueous solution.
A concentration that exhibits alkalinity with a pH of 12 or higher is preferably used. For example, a concentration of 0.001M to 0.5M is desirable. Heating conditions include alkali concentration,
Although it varies depending on the heating temperature and heating time, NAD ⁺ ,
NADP ⁺ is 10 at 100℃ in 0.1N−NaOH aqueous solution.
Completely destroyed within minutes.

As the above acid, an acid whose aqueous solution has an acidic pH of 2 or less is used. Hydrochloric acid, sulfuric acid, etc. are usually used. The acid concentration is preferably from 0.001M to 0.5M. Heating conditions include acid concentration,
Although it varies depending on the heating temperature and heating time, NADH,
NADPH is destroyed in 0.1N hydrochloric acid at 50°C in about 3 minutes.

In the neutralization reaction after the decomposition reaction of unreacted coenzymes, if NAD ⁺ or NADP ⁺ is destroyed with alkali, it is neutralized with formic acid lower alkyl ester. Examples of the formic acid lower alkyl esters include methyl formate, ethyl formate, propyl formate, butyl formate, and the like. In the conventional method, if the amount of acid was not adjusted very precisely, variations in pH occurred, which greatly affected the cycling rate and caused errors in measurement values. However, with the above method, neutralization Even if the amount of formic acid lower alkyl ester used is excessive, for example, 10 times the amount required for neutralization, only the amount of formic acid lower alkyl ester corresponding to the amount of alkali is reacted, so neutralization can be performed accurately. , the pH for the decomposition reaction can be easily set.

Even when NADH or NADPH is destroyed by acid, it can be accurately neutralized by first neutralizing with a slight excess of alkali, and then neutralizing with formic acid lower alkyl ester in the same manner as above. As the alkali mentioned above, an aqueous alkali hydroxide solution is usually used.

Next, an amplification reaction is carried out by coenzyme cycling, and in the present invention, the coenzyme is reacted in the presence of the remaining coenzyme converted in accordance with the amount of the analyte and an excess of the second substrate. A cycling reaction is performed by a combination of a second oxidoreductase reaction system that acts on a second substrate, and a reaction system that converts a tetrazolium salt into formazan using diaphorase or an electron transfer substance.

The oxidoreductase in the second oxidoreductase reaction system is not particularly limited, and uses NAD(P) ⁺ as a coenzyme and acts on a specific second substrate used in excess to produce NAD(P). The dehydrogenase that produces H may be an enzyme of any origin. Examples of these enzymes and second substrates are described in the "Enzyme Handbook" as above. For example, when NAD is used as a coenzyme, the second substrate and enzyme of the second oxidoreductase reaction system include alcohols and alcohol dehydrogenase (EC1.1.1.1), glycerol and glycerol dehydrogenase (EC1.1.1.6). ), glycerol-3-
Phosphate and glycerol-3-phosphate dehydrogenase (EC1.1.1.8), xylitol and xylitol dehydrogenase (EC1.1.1.9), lactate and lactate dehydrogenase (EC1.1.1.27), malate and malate dehydrogenase (EC1) .1.1.37),
Examples include glucose and glucose dehydrogenase (EC1.1.1.47), galactose and galactose dehydrogenase (EC1.1.1.48), 3α-hydroxysteroids and 3α-hydroxysteroid dehydrogenase (EC1.1.1.50), and
The second substrate and enzyme of the second oxidoreductase reaction system when NADP is used as a coenzyme include alcohols and alcohol dehydrogenase (EC1.1.1.2), gulonic acid and glucuronate reductase (EC1.1.1). .19), aldoses and aldose reductase (EC1.1.1.21), glucose and glucose dehydrogenase (EC1.1.1.47), glucose-6-phosphate and glucose-6-phosphate dehydrogenase (EC1.1.1.49) , 3α-hydroxysteroids and 3α-hydroxysteroid dehydrogenase (EC1.1.1.50), glycerol and glycerol dehydrogenase (NADP ⁺ )
(EC1.1.1.72), glutamic acid and glutamate dehydrogenase (glutamate dehydrogenase (NADP ⁺ ) (EC1.4.1.4), etc., and these are examples and are not limited in any way.
The amount of enzyme used depends on the enzyme titer, type of substrate and coenzyme cycling rate. The amount of the second substrate used is an incomparably larger molar amount than that of the cycling coenzyme, but it may be at least at a concentration that maximizes the reaction rate of the redox enzyme. Typically it may be present in a concentration range of 0.1mM to 10mM. Diaphorase is NADH
Or it may be highly specific to NADPH,
It may be something that acts on both. The concentration used may generally range from 0.1 to 50 U per ml of reaction solution.

As an electron carrier, NAD(P)H is NAD
It is a substance that has the ability to oxidize to (P) ⁺ and does not adversely affect the coenzyme cycling reaction, such as phenacine metal salephate, Meldora Blue, and pyrocyanine. The concentration to be used can be set according to the cycling reaction rate, but it is usually 1 μg or more per ml of reaction solution.
It may be present in a concentration range of 1 ml.

The tetrazolium salt is 3,3'-(3,
3'-Dimethoxy-4,4'-diphenylene)bis[2-(P-nitrophenyl)-5-phenyl-tetrazolim chloride] (NTB), 2-(P-nitrophenyl)-3-(P-iodophenyl)- 5-phenyltetrazolim chloride (1NT), 2-
(4',5'-dimethyl-2'-thiazolyl)-3,5-
Diphenyltetrazolimbrolide (4,5-
MTT), etc. The concentration used is rather limited by the aqueous solubility of both the tetrazolium salt and the ultimately formed formazan, but typically may be present in a concentration range of 3 μg to 100 μg per ml of reagent.

The above cycling reaction is usually carried out at room temperature to 37°C.
It is carried out at a temperature in the vicinity range, preferably between 30 and 37°C. Rapid termination of the reaction at the end of the predetermined time is achieved by adding an acid, such as hydrochloric acid, phosphoric acid, etc.

In the above reaction, it is advantageous to add a surfactant to help prevent formazan precipitation. As a surfactant, Triton X-100,
Examples include nonionic surfactants such as Adekatol SO-145. The concentration used is 0.01 to 3 for the reagent.
% concentration range. By adding this surfactant, it is possible to increase the sensitivity of measurement values and stabilize the formazan dye.

Colorimetric determination of the formed formazan dye can be performed by measuring the absorbance (OD) at the specific absorption wavelength of formazan. For example, the absorbance can be measured at a wavelength of 500 to 550 nm to quantify the analyte. can.

According to the present invention, not only the end point method but also the late method is possible.

Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 Quantification of NAD ⁺ [Reagent] (1) 0.2M phosphate buffer (PH8.0) (2) Add 100ml of 2% Adecatol-SO-145 aqueous solution
INT-adecatol solution containing 100mg of INT (3) Diaphorase or electron carrier diaphorase (NADH) solution (100U/ml) Phenazine metasulfate (PMS) aqueous solution (100mg/20ml) Pyrocyanine aqueous solution (10mg/20ml) Meldora・Blue aqueous solution (10mg/20ml) (4) Ethanol (5) Alcohol dehydrogenase (250U/ml) (6) 10μMNNAD ⁺ (NAD ⁺ is generated in the main reaction system, and unreacted NADH is neutralized with alkali after acidic heating. (7) 0.1N hydrochloric acid [Procedure] In separate test tubes, add 0.5ml of phosphate buffer, 0.2ml of INT-adecatol solution, 20μ of ethanol, and 30μ of water.
and 0.1 ml of alcohol dehydrogenase,
After adding 50μ of diaphorase (NADH) or electron mediator and incubating at 37°C for 3 minutes, 10μMNAD ⁺ solution was added to each of 0, 10, 20,
Add 40, 60, 80 and 100μ and make the total volume 1.0 with water.
ml and incubated at 37°C for exactly 10 minutes. To stop the reaction, 2 ml of 0.1N hydrochloric acid was added to each, and the absorbance (OD) at 500 nm was measured. For blind testing, use 0.1ml of water instead of NAD solution.
was used.

〔result〕

As shown in Figure 1, an extremely good direct relationship was obtained between the amount of NAD ⁺ and absorbance when using either diaphorase or various electron carriers, indicating that the cycling reaction was carried out very accurately. The results showed that the sensitivity was extremely high.

Example 2 Effect of oxidoreductase used in cycling reaction [Reagents] (1) 0.2M phosphate buffer (PH8.0) (2) INT-adecatol solution (same as Example 1) (3) Diaphorase (NADH) Solution (100U/ml) (4) Substrate solution 100mM methyl alcohol 100mM glycerol-3-phosphate aqueous solution 100mM lactic acid 100mM malic acid (5) Redox enzyme Alcohol dehydrogenase (ADH) solution
(25000U/ml) Glycerol-3-phosphate dehydrogenase (G-3-PDH) solution (600U/ml) Lactate dehydrogenase (LDH) solution
(1500U/ml) Malate dehydrogenase (MDH) solution
(4600U/ml) 10μM NAD ⁺ solution 0.1N hydrochloric acid [Procedure] In separate test tubes, add 0.5ml of phosphate buffer, 0.2ml of INT-adecatol solution, and diaphorase (NADH) solution.
Add 50μ of substrate solution, 5μ of oxidoreductase solution that acts on the substrate, and 95μ of water, incubate at 37℃ for 3 minutes, and then add each enzyme separately.
10μMNAD ⁺ solution at 0, 10, 20, 40, 60, 80 and
Add 100μ, adjust the total volume to 10ml with water, and heat at 37℃.
Incubate for exactly 10 minutes. To stop the reaction, add 2 ml of 0.1N hydrochloric acid to each, and then
The optical density (OD) was measured at as a blind test
100μ of water was used instead of NAD ⁺ solution.

〔result〕

As shown in FIG. 2, very good linearity was obtained with each oxidoreductase, indicating that the cycling reaction was carried out accurately, and the sensitivity was very high.

Example 3 Quantification of NADP ⁺ [Reagent] (1) 0.2M phosphate buffer (PH8.0) (2) Add to 100ml of 2% Adecatol SO-145 aqueous solution
INT-adecatol solution containing INTmg (3) NADPH-diaphorase (NADPH) solution
(100U/ml) (4) Substrate solution 100mM glucose-6-phosphate
(G-6-P) 100mM glutamic acid (GA) (5) Redox enzyme solution Glucose-6-phosphate dehydrogenase (G-6-PDH) solution (1000U/ml) Glutamate dehydrogenase (GlDH)
Solution (1000U/ml) (6) 10 μM NADP ⁺ solution (7) 0.1N hydrochloric acid [Procedure] In separate test tubes, add 0.5 ml of phosphate buffer solution, 0.1 ml of INT-adecatol solution, and diaphorase (NADPH)
100μ, substrate solution 50μ, enzyme solution 50μ and water
After adding 30 μM NADP + and incubating for 3 minutes at 37°C, add 10 μM NADP ⁺ to each of 0, 10, 20, 40, 60,
80 and 100μ were added, the total volume was adjusted to 1.0ml with water, and incubated at 37°C for exactly 10 minutes. To stop the reaction, 2 ml of 0.1N hydrochloric acid was added to each,
The optical density (OD) at 500 nm was measured. As a blind test, 100μ of water was used instead of the NADP solution.

〔result〕

As shown in FIG. 3, it was found that NADP ⁺ was measured with high sensitivity, showing an extremely good linear relationship with glucose-6-phosphate dehydrogenase and glumate dehydrogenase.

Example 4 Quantification of malic acid [test solution] Malic acid was measured at 1, 2, 4, 6, 8, and
Aqueous solution containing 10μM 20μ [Reagents] (1) Reagent I; 0.2M phosphate buffer (PH8.0) 0.2ml,
Mixed solution of 0.1ml of 10mMNAD ⁺ solution and 10μ of malate dehydrogenase (120U/ml) solution (2) 0.2N-NaOH aqueous solution (3) Ethyl formate (4) Reagent: 0.5M phosphate buffer (PH8.0) 0.1ml Ethyl alcohol 10μ, alcohol dehydrogenase (250U/ml) 50μ, diaphorase (NADH) (100U/ml) 100μ, 1% NTB solution 20μ and 10% Triton X-100 solution 20μ
Mixed solution (5) 0.1N hydrochloric acid [Procedure] The test solution was placed in separate test tubes, 10.2 ml of reagent was added to each tube, and the tubes were incubated at 37° C. for 15 minutes. Add 0.1ml of 0.2N-NaOH to the reaction solution and incubate at 100℃.
After heat treatment for 10 minutes, it was immediately cooled and neutralized with 20μ of ethyl formate. After incubating at 37°C for 5 minutes, 0.3 ml of reagent was added and incubated at 37°C for exactly 10 minutes. To stop the reaction, 2.5 ml of 0.1N hydrochloric acid was immediately added to each, and the absorbance (OD) at 550 nm was measured. For blind testing, 100μ of water was used instead of NAD ⁺ solution.

[Results] As shown in Figure 4, the amount of malic acid can be spotted on a straight line in proportion to the OD value, and the measurement error is very small, indicating that malic acid can be measured with high sensitivity.

Example 5 Quantification of γ-aminobutyric acid [Test solution] 50μ of human serum to which γ-aminobutyric acid was added at concentrations of 0, 10, 20, 30, 40, and 50μM [Reagents] (1) Reagent; 0.1M pyrophosphate Buffer (PH8.3) 1.0
ml, 10mM NADP ⁺ aqueous solution 0.2ml, 0.1M α-ketoglutaric acid aqueous solution 1.0ml and commercially available enzyme GABASE derived from Pseudomonas fluorescens
(2U/ml) 0.4ml mixed solution (2) 0.5M-NaOH aqueous solution (3) Ethyl formate (4) Reagent: 2% Adecatol SO-145 aqueous solution
1.0ml of INT-adecatol solution with 100mg of INT dissolved in 100ml, 1.0ml of diaphorase (NADPH) solution (100U/ml), 0.2ml of 0.1M glucose-6-phosphate aqueous solution and glucose-6
-Phosphate dehydrogenase solution (100U/
ml) 20μ mixed solution [Procedure] Add 0.35ml of reagent to the test solution, incubate at 37℃ for 30 minutes, then add 0.1ml of 0.5N-NaOH,
Heat treatment was performed at 100°C for 10 minutes. After cooling, 20μ of ethyl formate was added to neutralize, and the mixture was incubated at 37°C for 5 minutes. After that, 0.2ml of reagent was added and incubated at 37°C for exactly 10 minutes. Due to reaction termination,
Immediately after adding 2 ml of 0.1N hydrochloric acid, the absorbance (OD) at 550 nm was measured, and the OD in the case of a test solution containing no γ-aminobutyric acid was corrected as 0.

〔result〕

As shown in Figure 5, the amount of γ-aminobutyric acid in human serum can be detected linearly in proportion to the OD value, and can be detected with high sensitivity and without the need for deproteinization. This shows that it is possible to quantify.

[Brief explanation of drawings]

Figure 1 shows the NAD ⁺ standard curve when diaphorase and various electron transfer substances are used in the indicator reaction system, and Figure 2 shows the influence of the oxidoreductase used in the cycling reaction using the NAD ⁺ standard curve. , Figure 3 shows the influence of the oxidoreductase used in the cycling reaction using the NADP ⁺ standard curve, Figure 4 shows the calibration curve for quantifying malic acid according to the present invention, and Figure 5 shows the influence of the oxidoreductase used in the cycling reaction. γ- added to serum
A calibration curve for quantifying aminobutyric acid is shown.

Claims (1)

[Claims] 1. When quantifying a substance to be measured, NAD ⁺ or NADH or
The main reaction system converts coenzymes in proportion to the amount of the analyte by acting with an oxidoreductase that uses the analyte as a substrate in the presence of NADP ⁺ or NADPH.The main reaction system is stopped and unreacted coenzymes are removed. A decomposition reaction system that decomposes and neutralizes by adding formic acid lower alkyl ester, and a coenzyme and a second substrate in the presence of a coenzyme converted in proportion to the amount of the substance to be measured and an excess of the second substrate. A reaction consisting of a coenzyme cycling reaction system is performed by combining a second redox reaction system that acts on the substrate and a reaction system that converts a tetrazolium salt into formazan in the presence of diaphorase or an electron carrier, and the resulting formazan is measured by colorimetry. A highly sensitive measurement method for a substance to be measured, which is characterized by quantitative determination. 2. The measurement method according to claim 1, wherein the amount of coenzyme in the main reaction system is in excess of the molar ratio of the substance to be measured. 3. Unreacted coenzyme in the decomposition reaction system becomes NAD ⁺
Alternatively, in the case of NADP ⁺ , the measuring method according to claim 1, wherein unreacted coenzyme is decomposed and neutralized by addition of an aqueous alkali hydroxide solution and heat treatment. 4. The measuring method according to claim 3, wherein the aqueous alkali hydroxide solution has a concentration such that it exhibits alkalinity of PH12 or higher. 5. The measuring method according to claim 4, wherein the alkali hydroxide is sodium hydroxide or potassium hydroxide. 6. The measuring method according to claim 3, wherein the heat treatment is performed until NAD ⁺ or NADP ⁺ is completely destroyed. 7 Unreacted coenzyme in the decomposition reaction system
In the case of NADH or NADPH, the measuring method according to claim 3, wherein unreacted coenzyme is decomposed and neutralized by addition of an acid aqueous solution and heat treatment. 8. The measuring method according to claim 8, wherein the acid aqueous solution has a concentration such that it exhibits acidity of PH2 or less. 9. The measuring method according to claim 8, wherein the acid is an acidic substance having a pH of 2 or less in an aqueous solution. 10. The measuring method according to claim 9, wherein the acid is hydrochloric acid or sulfuric acid. 11. The measuring method according to claim 7, wherein the heat treatment is performed until NADH or NADH is completely destroyed. 12. The measuring method according to claim 11, wherein neutralization is carried out by adding a slightly excess aqueous alkali hydroxide solution and immediately adding lower alkyl formic acid ester. 13. The measuring method according to claim 12, wherein the alkali hydroxide is sodium hydroxide or potassium hydroxide. 14 Formic acid lower alkyl ester is methyl formate,
The measuring method according to claim 1 or 12, wherein the measuring method is ethyl formate, propyl formate, or butyl formate. 15. The measuring method according to claim 1, wherein the electron carrier in the cycling reaction system is phenazine metasulfate, meldorable, or pyronisyan. 16. The measuring method according to claim 16, which comprises adding a surfactant in the cycling reaction system.