JP5500985B2 - Ultrasensitive measurement method of protein and nucleic acid - Google Patents

Description

Cross-reference of related applications

  This application claims the priority of Japanese Patent Application No. 2007-85550 filed on Mar. 28, 2007, the entire description of which is specifically incorporated herein by reference.

  The present invention relates to a highly sensitive assay method for proteins and nucleic acids using enzyme immunoassay and nucleic acid probe assay. In particular, the present invention applies the thio-NAD cycling method to measure the activity of an enzyme labeled with an antibody or a nucleic acid probe, and enables ultrasensitive measurement of target proteins and nucleic acids even with a widely used colorimetric microplate reader or visually. Regarding the method.

The radioimmunoassay (RIA) method has been technically established as a high-sensitivity measurement method for proteins and nucleic acids. In addition to increasing the sensitivity of detection devices, at present, higher sensitivity of about 10 ^-16 mole or higher is technically required. Impossible. Moreover, in the RIA method, the field of measurement is not limited to the isotope experimental facility, but if a short-lived nuclide is used, the expiration date of the reagent is extremely shortened and the measurement sensitivity is drastically lowered. In addition, there is a problem of radioactive waste unique to radioactivity measurement methods. The problem of disposal is particularly great when long-lived nuclides are used. Therefore, in recent years, research on the development of high-sensitivity measurement methods for proteins and nucleic acids has been developed with “non-radioactive” as a keyword. Therefore, there is almost no research and development or technical improvement on the RIA method.

As a high-sensitivity measurement method replacing the RIA method, an enzyme immunoassay (ELISA method) [FIG. 1] is used for protein measurement, and a PCR method is used for nucleic acid measurement. Enzyme immunoassays have been developed with higher sensitivity (10 ^-15 mole) from the original colorimetric method (10 ^-13 mole) to the fluorescence method and luminescence method, and the development and improvement of dedicated measuring devices are also progressing. However, the sensitivity has reached its limit just by making the measurement operation simple. (Non-Patent Document 1)

In addition, in the PCR method as a highly sensitive method for measuring nucleic acids, nucleic acid quantification is strictly difficult considering the problem of target-specific signal detection, the problem of amplification efficiency, the conditions under which the PCR product reaches a plateau, and the like.
"Ultrasensitive enzyme immunoassay" (Academic Publishing Center, 1993)

Therefore, an object of the present invention is to provide a measurement method using a colorimetric method with the simplest detection method and increasing the sensitivity in a geometric series. Specifically, it is to provide a measurement method with sensitivity increased to 10 ^-18 moles or more.

  In order to measure a protein or nucleic acid with higher sensitivity using an enzyme immunoassay, it is conceivable to amplify a signal substance produced by a label enzyme of the enzyme immunoassay. In addition, several enzyme cycling methods are known as methods for amplifying signal substances. Therefore, the present inventors have made various studies in order to provide a highly sensitive measurement method by colorimetric measurement of protein or nucleic acid using enzyme immunoassay. As a result, the enzyme immunoassay is combined with the enzyme cycling method using the product of the labeling enzyme used in the enzyme immunoassay as a substrate, and thioNAD (P) H is geometrically amplified as a signal substance. The present invention has been completed by finding that a protein or nucleic acid can be quantified or visually detected with high sensitivity by a color method.

The first aspect of the present invention for solving the above problems is an enzyme measurement method using an antibody-enzyme complex,
Enzymatic reaction product quantification of antibody-enzyme complex uses NADH and / or NADPH, thioNAD and / or thioNADP, and dehydrogenase (DH) to produce thioNADH and / or thioNADPH by enzyme cycling reaction The method is carried out by measuring the amount of thio-NADH and / or thio-NADPH produced, or measuring the color change caused by the produced thio-NADH and / or thio-NADPH.
In the above method, the antibody-enzyme complex comprises an antibody specific for the target protein antigen and an enzyme labeled with the antibody, and can be used for measurement of the target protein.

A second aspect of the present invention for solving the above problem is a method for measuring a nucleic acid probe using an enzyme-labeled nucleic acid probe,
Enzymatic reaction product quantification of enzyme-labeled nucleic acid probes uses NADH and / or NADPH, thioNAD and / or thioNADP, and dehydrogenase (DH) to produce thioNADH and / or thioNADPH by an enzyme cycling reaction The method is carried out by measuring the amount of thio-NADH and / or thio-NADPH produced, or measuring the color change caused by the produced thio-NADH and / or thio-NADPH.
In the above method, the enzyme-labeled nucleic acid probe comprises a nucleic acid probe that specifically binds to the target nucleic acid and an enzyme labeled with the nucleic acid probe, and can be used for measurement of the target nucleic acid.

In the first aspect and the second aspect, the following aspects are more preferable. (1) The amount of thio-NADH and / or thio-NADPH produced is measured by measuring the absorbance at the absorption wavelength of thio-NADH and / or thio-NADPH.
(2) The color change due to the generated thio-NADH and / or thio-NADPH is measured visually.
(3) The enzyme (labeled enzyme) of the antibody-enzyme complex or enzyme-labeled nucleic acid probe is a transferase group represented by EC number 2.-, a hydrolase group represented by EC number 3.-, EC number 4 It is at least one enzyme selected from the group consisting of a removed additional enzyme group represented by .- and an isomerase group represented by EC number 5.-.

The third aspect of the present invention for solving the above-mentioned problem is an enzyme immunoassay kit containing the following reagents.
An enzyme labeled with an antibody specific for a target protein antigen Substrate dehydrogenase (DH) of the above enzyme
NADH and / or NADPH,
Thio NAD and / or Thio NADP

A fourth aspect of the present invention for solving the above problems is a kit for measuring a nucleic acid probe comprising the following reagents.
Enzyme labeled with a nucleic acid probe that specifically binds to the target nucleic acid Substrate dehydrogenase (DH) of the above enzyme
NADH and / or NADPH,
Thio NAD and / or Thio NADP

  In the above kit, the enzyme (labeled enzyme) of the antibody-enzyme complex or the enzyme-labeled nucleic acid probe is a transferase group represented by EC number 2.-, a hydrolase group represented by EC number 3.-, an EC number It may be at least one enzyme selected from the group consisting of a removed additional enzyme group represented by 4.- and an isomerase group represented by EC number 5.-.

ADVANTAGE OF THE INVENTION According to this invention, the enzyme immunoassay which improved the measurement sensitivity to 10 < ^-18 > mols or more can be provided, and the highly sensitive measurement of target protein or target nucleic acid is possible.

[Enzyme cycling method]
The present invention is an enzyme measurement method using an antibody-enzyme complex,
Enzymatic reaction product quantification of antibody-enzyme complex uses NADH and / or NADPH, thioNAD and / or thioNADP, and dehydrogenase (DH) to produce thioNADH and / or thioNADPH by enzyme cycling reaction And measuring the amount of thio-NADH and / or thio-NADPH produced, or measuring the color change caused by thio-NADH and / or thio-NADPH.

  The antibody-enzyme complex comprises an antibody specific for a target protein antigen and an enzyme labeled with the antibody, and is used for measurement of the target protein.

Furthermore, the present invention provides a nucleic acid probe measurement method using an enzyme-labeled nucleic acid probe,
Enzyme quantification of enzyme-labeled nucleic acid probes to produce thio-NADH and / or thio-NADPH by enzyme cycling reaction using NADH and / or NADPH, thio-NAD and / or thio-NADP, and dehydrogenase (DH) And measuring the amount of thio-NADH and / or thio-NADPH produced, or measuring the color change caused by thio-NADH and / or thio-NADPH.

  The enzyme-labeled nucleic acid probe comprises a nucleic acid probe that specifically binds to a target nucleic acid and an enzyme labeled with the nucleic acid probe, and is used for measuring the target nucleic acid.

  The enzyme (labeled enzyme) of the antibody-enzyme complex or enzyme-labeled nucleic acid probe used in the above-described method of the present invention is a transferase group represented by EC number 2.-, a hydrolase represented by EC number 3.-. It may be at least one enzyme selected from the group consisting of a decomposing enzyme group, a removed and added enzyme group represented by EC number 4.- and an isomerase group represented by EC number 5.-.

As the transferase group represented by EC number 2.-, for example, any of those classified into the following EC numbers can be used.
Examples of enzymes included in the transferase group represented by EC number 2.- include alanine aminotransferase (ALT) and the like.

As the hydrolase group represented by EC number 3.-, for example, any of those classified into the following EC numbers can be used.
Examples of enzymes included in the transferase group represented by EC number 3.- include, for example, alkaline phosphatase (ALP), inulinase (INL), β-galactosidase (β-Gal), α-glucosidase (α-Glu ), Lipoprotein lipase (LPL) and the like.

As the removal additional enzyme group represented by EC number 4.-, for example, any of those classified into the following EC numbers can be used.
Examples of enzymes included in the transferase group represented by EC number 4.- include, for example:
Examples thereof include fumarase (FUM), phosphoenolpyruvate carboxylase (PEPC), oxaloacetate decarboxylase (OAC), malate synthetase (MLS) and the like.

As the isomerase group represented by EC number 5.-, for example, any of those classified into the following EC numbers can be used.
Examples of enzymes included in the transferase group represented by EC number 5.- include, for example, triose phosphate isomerase (TIM), lactate racemase (LRM), 3-ketosteroid Δ4-Δ5 isomerase and the like. Can be mentioned.

  The ultrasensitive measurement method of the present invention can be carried out in the same manner as the usual enzyme immunoassay method and nucleic acid probe method. For example, use a solid phase carrier used for normal measurement, such as a solid phase carrier to which an antibody or nucleic acid probe that specifically binds to the analyte is fixed, such as a microplate, plastic tube, or magnetic bead. Can do.

  Antibodies and nucleic acid probe-enzyme complexes can be prepared by conventional methods.

The antibody constituting the antibody-enzyme complex can be appropriately selected from antibodies that specifically bind to the analyte to be measured by the enzyme immunoassay method of the present invention. For example, the enzyme immunoassay method of the present invention is used for protein analysis. In this case, the antibody of the antibody-enzyme complex is an antibody that specifically binds to the protein as the analyte. In this case, a substrate on which an antibody that specifically binds to the protein as the analyte is fixed to the surface is used. The antibody constituting the antibody-enzyme complex and the antibody to be fixed to the substrate may be antibody fragments. The analyte in the enzyme immunoassay method of the present invention is not limited to a protein, and can be any substance other than the protein to be measured by a normal enzyme immunoassay method.

  Similarly, a probe complementary to the nucleic acid to be measured can be selected as appropriate for the nucleic acid probe enzyme complex.

  In the method of the present invention, the quantification of the reaction product by the enzyme of the antibody-enzyme complex or the enzyme-labeled nucleic acid probe is performed using NADH and / or NADPH, thioNAD and / or thioNADP, and dehydrogenase (DH). By generating thio-NADH and / or thio-NADPH by an enzyme cycling reaction and measuring the amount of thio-NADH and / or thio-NADPH generated, or measuring the color change due to the generated thio-NADH and / or thio-NADPH Do.

In the method of the present invention, the concentration of each component can be in the following range.
(1) Concentration range of antibody-enzyme complex or enzyme-labeled nucleic acid probe
0.01 μg / ml to 1 mg / ml
(2) Substrate concentration range of the labeling enzyme
1 μM to 500 mM
(3) NADH and / or NADPH concentration range
0.01 mM to 50 mM
(4) Thio NAD and / or thio NADP concentration range
0.01 mM to 100 mM
(5) Dehydrogenase (DH) concentration range
0.01 u / ml to 5000 u / ml

  The reaction conditions can be appropriately determined in consideration of the optimum temperature range of the labeling enzyme and dehydrogenase (DH). For example, the reaction temperature is preferably room temperature since the operation is simple. However, it can be carried out at a temperature higher or lower than room temperature in consideration of the optimum temperature range of the labeling enzyme and dehydrogenase (DH).

  The reaction time accumulates thio-NADH and / or thio-NADPH to such an extent that the amount of thio-NADH and / or thio-NADPH produced can be measured or the color change due to the produced thio-NADH and / or thio-NADPH can be measured Enough time. However, the accumulated amount of thio-NADH and / or thio-NADPH necessary for measurement or measurement varies depending on the measurement or measurement conditions, and can be determined as appropriate according to the conditions.

  The reaction product of the enzyme of the antibody-enzyme complex or the enzyme of the enzyme-labeled nucleic acid probe varies depending on the labeling enzyme and the substance used as the substrate of the labeling enzyme. It can be a monophosphate ester of a compound that can be a substrate for an enzyme. Examples of combinations of labeling enzyme and substrate will be described later.

  Among enzyme cycling systems, cycling systems that utilize thio NAD (P) are unique cycling systems that have appeared relatively recently. This system uses dehydrogenase (DH) that uses NAD (P) / NAD (P) H as a coenzyme. NAD (P) / NAD (P) H and its analog, thioNAD (P) Cycling is performed under the coexistence of / thioNAD (P) H, and amplification and quantification is performed using the substrate of dehydrogenase as thioNAD (P) H (maximum absorption wavelength: 400 nm, molar extinction coefficient = 11,900). The measurement principle of the thio NAD (P) cycling method is as follows.

  The maximum absorption of NADH is 340 nm (molar extinction coefficient = 6,200), whereas thio-NAD (P) H absorbs in the visible range (maximum absorption wavelength: 400 nm, molar extinction coefficient = 11,900). Its advantage is that it can be measured using a type of spectrophotometer or a colorimetric microplate reader.

Cycling systems that use thio-NAD (P) can measure dehydrogenase activity based on increased absorption of NADH, taking advantage of the fact that it can be measured using popular absorption photometers and microplate readers for colorimetry. Some of the conventional methods such as a method for quantifying the substrate have been improved to a method using thio NAD. However, there is still no report that uses this cycling system as a detection system such as an enzyme immunoassay for high sensitivity.
In the present invention, by combining a labeling enzyme and a cycling system, an amplification reaction can be made a geometrical reaction for the first time, and sufficient sensitivity can be increased.

  In the measurement method of the present invention, as shown in the enzyme immunoassay example, the product produced by the enzyme complex and the substrate combined therewith is used as a substrate for the next enzyme cycling reaction, and the thiol produced by the enzyme cycling reaction is used. Colorimetric determination of NAD (P) H absorption. In this reaction, since enzyme cycling is performed with one type of dehydrogenase, the substrate for the enzyme cycling reaction may be a reduced substrate or an oxidized substrate.

It is desirable that the labeling enzyme used as the enzyme complex, its substrate, and the dehydrogenase used in the subsequent cycling reaction have the following properties.
(1) Large turnover number of labeling enzyme (2) Commercially available and widely used labeling enzyme can be used (3) Large number of turnover of enzyme cycling (4) Dehydrogenase used for enzyme cycling reaction No activity similar to enzyme contamination or labeling enzyme (5) The substrate of the labeling enzyme is a commercially available compound or can be easily synthesized and has high stability

Examples of dehydrogenase (DH) include the following dehydrogenases (DH).
More specifically, examples of the dehydrogenase (DH) include malate dehydrogenase, lactate dehydrogenase, glucose dehydrogenase, galactose dehydrogenase, 3α-hydroxysteroid dehydrogenase, galactose dehydrogenase, glucose dehydrogenase, testosterone-17β-dehydrogenase, glycerol-3- Examples thereof include phosphate dehydrogenase.

  Although the following is mentioned as an example of the combination of the typical labeling enzyme which can be used for this invention, a substrate, and the dehydrogenase for enzyme cycling, Of course, it is not necessarily restricted to this combination.

  For example, when 3α-hydroxysteroid dehydrogenase is used as a cycling enzyme in the combination described above, usable substrate candidates include androsterone, 11β-hydroxyandrosterone, 11-oxoandrosterone, 11α-hydroxyandrolone. Examples include sterolone, etiocholanolone, tetrahydrocortisol, pregnanediol, lithocholic acid, deoxycholate, chenodeoxycholic acid and cholic acid.

  As a substrate for cycling, a substrate having a high enzyme reaction rate (high activity against the substrate) and reacting even at a low concentration (small Km value) is preferable. Further, a preferable property as a dehydrogenase is a reaction rate when thioNAD (P) is used as a coenzyme. In many dehydrogenases, the reaction rate when thio-NAD is used as a coenzyme is within several percent of that when NAD is used, whereas in the dehydrogenation reaction of androsterone with 3α-hydroxysteroid dehydrogenase, The reaction rate with NAD is about 59% of the reaction rate with NAD, and it has favorable properties as the cycling enzyme of the present invention. Moreover, it is possible to use a commonly used alkaline phosphatase (ALP) as a labeling enzyme to be combined, and it is also a preferable combination that it is easy to synthesize androsterone 3-phosphate serving as a substrate for alkaline phosphatase.

The enzyme immunoassay method using triosephosphate isomerase (TIM) as the enzyme of the antibody-enzyme complex will be described below as an example.
Triose phosphate isomerase (TIM) is a labeling enzyme with a high turnover number. In this enzyme immunoassay method using TIM, dihydroxyacetone-3-phosphate is generated as a product of TIM. Then, glycerol-3-phosphate dehydrogenase (GAPDH), which is a dehydrogenase using the reaction product of TIM as a substrate, is used for the enzyme cycling reaction.

  Thio NAD cycling allows amplification and quantification of dehydrogenase substrates with an efficiency of several hundred cycles per minute if an appropriate dehydrogenase reaction is selected. Therefore, the activity of an enzyme that gives such a substrate as a reaction product can be measured with extremely high sensitivity by thio-NAD cycling.

[Enzyme cycling kit]
The present invention includes an enzyme cycling method kit comprising an enzyme labeled on a reactive carrier, its substrate, an enzyme for cycling reaction, and its coenzymes thio NAD and NADH.
The reactive carrier represents an antibody, a nucleic acid probe, a lectin or the like having an activity to bind to a measurement object.
The reactive carrier is not particularly limited as long as it is suitable for the measurement target, and the labeling enzyme and its substrate are also suitable.

  More specifically, the present invention includes a kit for an enzyme cycling method comprising a labeling enzyme, its substrate, an enzyme for cycling reaction, and its coenzymes thio NAD and NADH.

The kit of the present invention is an enzyme immunoassay kit containing the following reagents.
An enzyme labeled with an antibody specific for a target protein antigen Substrate dehydrogenase (DH) of the above enzyme
NADH and / or NADPH,
Thio NAD and / or Thio NADP

Furthermore, the present invention is a kit for measuring a nucleic acid probe comprising the following reagents.
Enzyme labeled with a nucleic acid probe that specifically binds to the target nucleic acid Substrate dehydrogenase (DH) of the above enzyme
NADH and / or NADPH,
Thio NAD and / or Thio NADP

  As the labeling enzyme and dehydrogenase (DH), those described in the method of the present invention can be used as they are.

  In the kit of the present invention, a commercially available enzyme-labeled antibody or the like can be used in combination with the constituent reagents of this kit. This kit can be used for an enzyme immunoassay method using an enzyme cycling method.

Hereinafter, the present invention will be described based on examples. However, in the state of the art, such examples can be variously modified. In view of such a technical level, it goes without saying that the present invention should not be limited only to these embodiments.
Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Activity measurement combined with cycling reaction of Fumarase as a labeling enzyme
Reaction reagent
200 mM Tris buffer pH 9.0
9 mM Thio NAD
0.3 mM NADH
300 u / ml MDH
Substrate solution
100 mM Fumaric acid
Measurement sample
1, 5, 10 u / ml Fumarase

Measurement method Dispense 180 μl of the reaction reagent solution into a microplate, add 20 μl of substrate solution, then add 20 μl of the sample solution, and use a 405 nm filter with a microplate reader (Corona MTP-800) at 37 ° C. Measure the change in absorbance.
A highly sensitive reaction curve shown in FIG. 2 was obtained. In particular, since the reaction proceeds exponentially with the passage of time, the sensitivity increases dramatically when the measurement time is increased.

Comparative Example 1 Measurement of activity of Fumarase as a labeling enzyme
Reaction reagent
200 mM Tris buffer pH 9.0
9 mM Thio NAD
300 u / ml MDH
Substrate solution
100 mM Fumaric acid
Measurement sample
1, 5, 10 u / ml Fumarase

Measurement method Dispense 180 μl of the reaction reagent solution into a microplate, add 20 μl of substrate solution, then add 20 μl of the sample solution, and use a 405 nm filter with a microplate reader (Corona MTP-800) at 37 ° C. Measure the change in absorbance.
The reaction curve shown in FIG. 3 was obtained. Since it is not combined with enzyme cycling, the reaction proceeds only linearly, indicating that the sensitivity is low.

Example 2 Activity measurement in combination with cycling reaction of Phosphoenolpyruvate carboxylase as a labeling enzyme
Reaction reagent
100 mM Na ₂ CO ₃ (pH 8.0)
0.3 mM NADH
9 mM Thio NAD
300 u / ml MDH
Substrate solution
50 mM Phosphoenolpyruvate
Measurement sample
0.1 u / ml Phosphoenolpyruvate carboxylase

Measuring method
Dispense 180 μl of the reaction reagent into the microplate, add 20 μl of the substrate solution, and then add 20 μl of the sample solution. Absorbance at 37 ° C. using a 405 nm filter with a microplate reader (Corona MTP-800) Measure changes.
A highly sensitive reaction curve shown in FIG. 4 was obtained. In particular, since the reaction proceeds exponentially with the passage of time, the sensitivity increases dramatically when the measurement time is increased.

Comparative Example 2 Measurement of Phosphoenolpyruvate carboxylase activity as a labeling enzyme
Reaction reagent
100 mM Na ₂ CO ₃ (pH 8.0)
0.4 mM NADH
50 u / ml MDH
Substrate solution
50 mM Phosphoenolpyruvate
Measurement sample
0.1 u / ml Phosphoenolpyruvate carboxylase

Measurement method Dispense 180 μl of the reaction reagent solution into the microplate, add 20 μl of the substrate solution and then add 20 μl of the sample solution, and use a 340 nm filter with a microplate reader (Corona MTP-800) at 37 ° C. Measure the change in absorbance.
The reaction curve shown in FIG. 5 was obtained. It can be seen that the reaction is hardly progressing because it is not combined with enzyme cycling.

Example 3 Activity measurement combined with cycling reaction of Alkaline phosphatase as a labeling enzyme
Reaction reagent
100 mM Tris buffer pH 9.8
1.8 mM Thio NAD
0.9 mM NADH
3.8 mM MgCl ₂
25 units / ml 3α-Hydroxysteroid dehydrogenase

Substrate solution
5 mM Androsterone 3-phosphate
Measurement sample
10, 2.5, 0.625 units / ml Alkaline phosphatase (ALP)

Measurement method Place 800 µl of the reaction reagent in a quartz cell (optical path length 1 cm), add 200 µl of the substrate solution, incubate at 37 ° C for 5 minutes, add 2 µl of the sample to be measured, and measure the change in absorbance at 400 nm.
The reaction curve shown in FIG. 6 was obtained. 0.625 mU / ml can be measured even in a short time.

Comparative Example 3 Measurement of Alkaline phosphatase activity as a labeling enzyme
Reaction reagent
100 mM Tris buffer pH 9.8
1.8 mM Thio NAD
3.8 mM MgCl ₂
25 units / ml 3α-Hydroxysteroid dehydrogenase

Substrate solution
5 mM Androsterone 3-phosphate
Measurement sample
10, 2, 5, 0.625 units / ml Alkaline phosphatase (ALP)

Measurement method Place 800 µl of the reaction reagent in a quartz cell (optical path length 1 cm), add 200 µl of the substrate solution, incubate at 37 ° C for 5 minutes, add 2 µl of the measurement sample, and measure the change in absorbance at 400 nm.
The reaction curve shown in FIG. 7 was obtained. It can be seen that the reaction is hardly progressing because it is not combined with enzyme cycling.

Reference example 1
Preparation of ALP-labeled antibody A mouse-derived monoclonal antibody having specific reactivity with an antigen was dissolved in 1 ml of 100 mM acetate buffer (pH 4.2) at a concentration of 1.0 mg / ml acid 100 mM phosphate buffer. Pepsin was added to a concentration of 3% and the mixture was heated at 37 ° C. for 3 hours, and then adjusted to pH 7 with 4N NaOH. This mixed reaction solution was subjected to gel filtration using a column packed with Sephadex G-200 to obtain F (ab ′) 2.

  After concentrating the F (ab ′) 2 fraction to 1 ml, 0.1 ml of 22 mM 2-mercaptoethanol is added and heated at 37 ° C. for 1 hour. This mixed reaction solution was subjected to gel filtration using a column packed with Sephadex G-200 to obtain F (ab ′). Dissolve 100 mg of alkaline phosphatase (ALP) in 1 ml of 10 mM PBS (pH 6.0), add 0.1 ml of a 20 mM concentration dimethylformamide solution of N- (6-Maleimidocaproyloxy) succinimide (EMCS) at 25 ° C. The reaction was carried out at a temperature of 30 minutes. Next, this reaction mixture was subjected to gel filtration with a 0.1 M phosphate buffer (pH 6.0) using a column packed with Sephadex G-25 to separate maleimidated ALP and unreacted EMCS.

  Next, the F (ab ′)-modified monoclonal antibody and maleimidated ALP are mixed and left overnight at 4 ° C., followed by gel filtration using a column filled with Sephadex G-200 to obtain an ALP-labeled monoclonal antibody. It was.

Reference example 2
Preparation of insoluble carrier-immobilized monoclonal antibody A solution obtained by dissolving a monoclonal antibody derived from a mouse having specific reactivity with an antigen in 10 mM PBS (pH 7.4) at a concentration of 1 mg / ml is a flat-bottom microplate (NUNC). Add 0.1 ml to each well, leave at room temperature for 1 hour, wash with PBS, add 0.3 ml of 2% bovine serum albumin (BSA) aqueous solution, and leave at room temperature for 2 hours for blocking treatment Thus, a monoclonal antibody-immobilized microplate was obtained.

Example 4 Measurement of Pumilio by one-step method
Reaction reagent
200 mM Glycine buffer pH 8.8
1.5 mM Thio NAD
1.0 mM NADH
0.5 mM Androsterone 3-phosphate
20 units / ml 3α-Hydroxysteroid dehydrogenase

Measurement sample
1, 10, 100, 200 ng / ml Pumilio

Measurement Method A 0.1% BSA-containing TBS solution (pH 7) containing purified Pumilio (standard substance) in the range of 0 to 200 ng / ml on a microplate immobilized with the anti-Pumilio mouse monoclonal antibody prepared by the method of Reference Example 2. .5) 50 μl was added and incubated for 1 hour at room temperature. Next, after removing the solution in the well by suction, the well is washed with physiological saline, and then contains an ALP-labeled anti-Pumilio mouse monoclonal antibody prepared by the method of Reference Example 1 at a concentration of about 3 μg / ml. 50% of 0.1% BSA-1 mM MgCl ₂ -0.1 mM ZnCl ₂ -containing TBS solution (pH 7.5) was added and incubated at room temperature for 1 hour. Next, after removing the solution in the well by suction, the well is washed with physiological saline, and then 100 μl of the reaction reagent is added to each well and heated at 37 ° C. while being heated at 37 ° C. 800AFC), using a 405 nm filter, measure the absorbance for 30 minutes, and plot the change in absorbance for 30 minutes against the concentration of the standard substance. Got.

Example 5 Measurement of Pumilio by 2-step method
Reaction reagent A
100 mM Tris-HCl (pH 8.0)
0.2 mM Androsterone 3-phosphate

Reaction reagent B
200 mM Glycine buffer pH 8.8
3.0 mM Thio NAD
2.0 mM NADH
40 units / ml 3α-Hydroxysteroid dehydrogenase

Measurement sample
0.1, 0.5, 1, 2, 5 ng / ml Pumilio

Measurement Method A 0.1% BSA-containing TBS solution (pH 7) containing purified Pumilio (standard substance) in the range of 0 to ng / ml in a microplate on which the anti-Pumilio mouse monoclonal antibody prepared by the method of Reference Example 2 was immobilized. .5) 50 μl was added and incubated for 1 hour at room temperature. Next, after removing the solution in the well by suction, the well is washed with physiological saline, and then contains an ALP-labeled anti-Pumilio mouse monoclonal antibody prepared by the method of Reference Example 1 at a concentration of about 3 μg / ml. 50% of 0.1% BSA-1 mM MgCl ₂ -0.1 mM ZnCl ₂ -containing TBS solution (pH 7.5) was added and incubated at room temperature for 1 hour. Next, the solution in the well was removed by suction, and the well was washed with physiological saline. Then, 50 μl of the reaction reagent A was added to each well and incubated for 1 hour. Next, add 50 μl of reaction reagent B to each well, measure the absorbance for 30 minutes using a 405 nm filter with a microplate reader (Corona's MTP-800AFC) while heating at 37 ° C. By plotting the change in absorbance against the concentration of the standard substance, a calibration curve having a good concentration dependency was obtained up to the low concentration shown in FIG.

  The present invention can be suitably used in clinical inspection fields and food inspection fields where high sensitivity and simple measurement are required.

Measurement principle of enzyme immunoassay (ELISA) The activity measurement result about Fumarase obtained in Example 1. The activity measurement result about Fumarase obtained in Comparative Example 1. The activity measurement result about Phosphoenolpyruvate carboxylase obtained in Example 2. FIG. The activity measurement result about Phosphoenolpyruvate carboxylase obtained in Comparative Example 2. The activity measurement result about Alkaline phosphatase obtained in Example 3. FIG. The activity measurement result about Alkaline phosphatase obtained in Comparative Example 3. Calibration curve obtained in Example 4. Calibration curve obtained in Example 5.

Claims (15)

An enzyme measurement method using an antibody-enzyme complex,
The reaction product of the antibody-enzyme complex by the enzyme is a compound having an androsterone skeleton (provided that the androsterone skeleton has a hydroxy group at the 3α position and a carbonyl group at the 17 position), and has the androsterone skeleton. Compound quantification is performed using NADH and / or NADPH, thioNAD and / or thioNADP, and 3α-hydroxysteroid dehydrogenase ( 3α-HSD ) to generate thioNADH and / or thioNADPH by enzyme cycling reaction. The method is carried out by measuring the amount of thio-NADH and / or thio-NADPH, or measuring the color change caused by thio-NADH and / or thio-NADPH. The method according to claim 1, wherein the antibody-enzyme complex comprises an antibody specific for a target protein antigen and an enzyme labeled with the antibody , and is used for measurement of the target protein. A nucleic acid probe measurement method using an enzyme-labeled nucleic acid probe,
The enzyme reaction product of the enzyme-labeled nucleic acid probe is a compound having an androsterone skeleton (provided that the androsterone skeleton has a hydroxy group at the 3α position and a carbonyl group at the 17 position), and has the androsterone skeleton. Compound quantification is performed using NADH and / or NADPH, thioNAD and / or thioNADP, and 3α-hydroxysteroid dehydrogenase ( 3α-HSD ) to generate thioNADH and / or thioNADPH by enzyme cycling reaction. The method is carried out by measuring the amount of thio-NADH and / or thio-NADPH, or measuring the color change caused by thio-NADH and / or thio-NADPH. The method according to claim 3, wherein the enzyme-labeled nucleic acid probe comprises a nucleic acid probe that specifically binds to a target nucleic acid and an enzyme that is labeled on the nucleic acid probe, and is used for measurement of the target nucleic acid. The method according to any one of claims 1 to 4, wherein the amount of thio-NADH and / or thio-NADPH produced is measured by measuring absorbance at the absorption wavelength of thio-NADH and / or thio-NADPH. The method according to any one of claims 1 to 4, wherein the color change due to the generated thio-NADH and / or thio-NADPH is measured visually. The method according to any one of claims 1 to 6, wherein the enzyme (labeled enzyme) of the antibody-enzyme complex or the enzyme-labeled nucleic acid probe is alkaline phosphatase . The method according to claim 7, wherein the substrate of the enzyme (labeled enzyme) of the antibody-enzyme complex or enzyme-labeled nucleic acid probe is androsterone 3-phosphate, and the compound having an androsterone skeleton is androsterone. Reaction between the enzyme-enzyme complex or enzyme-labeled nucleic acid probe enzyme (labeled enzyme) and a substrate to obtain a compound having an androsterone skeleton as a reaction product, and generation of thio-NADH and / or thio-NADPH The method according to claim 1, which is performed sequentially. Reaction between the enzyme-enzyme complex or enzyme-labeled nucleic acid probe enzyme (labeled enzyme) and a substrate to obtain a compound having an androsterone skeleton as a reaction product, and generation of thio-NADH and / or thio-NADPH The method according to claim 1, which is performed in parallel. A kit for enzyme immunoassay comprising the following reagents:
An antibody specific for a target protein antigen is labeled , and a compound having an androsterone skeleton (provided that the androsterone skeleton has a hydroxy group at the 3α position and a carbonyl group at the 17 position) is phosphorylated 3α- An enzyme capable of converting a hydroxy group to a 3α-hydroxy group ,
A compound having an androsterone skeleton in which a 3α-hydroxy group which is a substrate of the enzyme is phosphorylated,
3α-hydroxysteroid dehydrogenase ( 3α-HSD),
NADH and / or NADPH,
Thio NAD and / or Thio NADP A kit for measuring a nucleic acid probe comprising the following reagents:
A nucleic acid probe that specifically binds to the target nucleic acid is labeled , and a compound having an androsterone skeleton (however, the androsterone skeleton has a hydroxy group at the 3α position and a carbonyl group at the 17 position) is phosphorylated. An enzyme capable of converting a 3α-hydroxy group to a 3α-hydroxy group ,
A compound having an androsterone skeleton in which a 3α-hydroxy group which is a substrate of the enzyme is phosphorylated,
3α-hydroxysteroid dehydrogenase ( 3α-HSD )
NADH and / or NADPH,
Thio NAD and / or Thio NADP The kit according to claim 11 or 12 , wherein the enzyme (labeled enzyme) of the antibody-enzyme complex or the enzyme-labeled nucleic acid probe is alkaline phosphatase . The kit according to any one of claims 11 to 13, wherein the compound having an androsterone skeleton in which the 3α-hydroxy group is phosphated is androsterone 3-phosphate, and the compound having an androsterone skeleton is androsterone. . The kit according to any one of claims 11 to 14, which is a kit for use in the method according to any one of claims 1 to 10.