JP2001124770A - Method for measuring concentration of substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for measuring a concentration whereby a measurement object can be measured with a higher accuracy and a higher sensitivity by an inexpensive optical system in a simple apparatus constitution by making use of merits of the shift of a bioluminescence energy which is hard to affect by impurities, can be measured by a uniform system and requires no excitation light. SOLUTION: According to the method, the concentration of a substance X is measured with the use of a substance A labeled by an enzyme E for catalyzing a luminous reaction and a substance B labeled by a fluorescent substance F. The concentration of the substance X is measured by using a fluorescence amount of the fluorescent substance F because of the radiationless shift of the emission energy in the luminous reaction catalyzed by the enzyme E as a marker.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a method for measuring the concentration of a substance. More specifically, the invention of this application does not require the operation of removing the released labeling substance when measuring the degree of the remaining labeling substance in the detection system, so that the enzyme immunoassay and the like can be automated. The present invention relates to a new concentration measuring method and a measuring kit for performing the measuring method.

[0002]

2. Description of the Related Art Pathogens and viruses in blood and urine, biological components such as antibodies, tumor markers, cytokines and hormones, drugs such as antibiotics and narcotics, and additives, antibiotics, contaminants, and bacteria in foods The detection and concentration measurement of toxins produced by the company, as well as chemical contaminants such as environmental hormones, dioxins, and PCBs released into the environment are clinical diagnostics,
It is indispensable for preventing infection, controlling food hygiene, and preventing environmental pollution.

[0003] Detection and concentration measurement of these substances include, for example, antigens and antibodies, physiologically active proteins such as hormones and cytokines and receptors, lectins and lectin-binding carbohydrates, and the like.
It is performed by detecting or measuring the concentration of a complex of these substances formed by specific affinity acting between DNA and a DNA-binding protein or the like, or a substance that has not formed a complex. For example, a commonly used immunoassay for measuring an antigen or an antibody is an immunoturbidimetric method for optically detecting an agglutination reaction accompanying an antigen-antibody reaction, which has an affinity for the antigen or antibody to be measured. This is a labeled immunoassay using a labeled antibody or labeled antigen.
Enzyme-labeled immunoassays and fluorescent-labeled immunoassays using antibodies or antigens labeled with enzymes or fluorescent substances are widely used because of their ease of handling.

However, in the case of these immunoassays, since the labeled signal of the labeled antibody or antigen which forms a complex with the antigen or antibody to be measured is measured, unreacted unreacted substances which are not involved in the formation of the complex are measured. Separation and removal of the labeled antibody or antigen from the detection system using a washing solution [Bound / F
ree (B / F) separation]. For this reason, the antigen-antibody reaction needs to be performed on a solid phase, and the number of operation steps for measurement, such as the immobilization operation of the antibody or antigen, the binding reaction operation, and the washing operation, is complicated, and the measurement requires time. There were problems such as. In addition, it has been an obstacle in automating the measurement.

For this reason, as a measurement method which does not require B / F separation, for example, EMIT (Enzyme Multiplied Immunoassa
y Technique) method, ECIA (Enzyme Channeling Immun)
oassay) method, etc. (Yuichi Endo et al., Protein Nucleic Acid Enzyme Supplement No. 31, p13-26, 198)
7). However, these methods require complicated operations, low sensitivity,
Due to disadvantages such as low versatility, it is not widely used.

On the other hand, the inventors of the present application focused on a phenomenon in which the stability of the variable (Fv) region, which is the antigen recognition site of an antibody, changes depending on the presence or absence of binding to an antigen, and focused on the variable region of a single antibody. Were developed using VH and VL constituting the first antibody element and the second antibody element, respectively (Ue
da, H. et al., Nature Biotechnology 14: 1714-1718,
1996). Further, the inventors of the present application have labeled the variable regions VH and VL of the antibody with two types of fluorescent substances having a relationship between a donor and an acceptor of the fluorescent energy transfer, and these labeled VH and VL were combined with the antigen. A method has been proposed in which the concentration of an antigen is specified by measuring the intensity of fluorescence emitted from a fluorescent substance on the receptor side due to the transfer of fluorescent energy generated when a body is formed (Japanese Patent Laid-Open No. 10-78436).
This method uses a conventionally known FRET (Fluorescence Resonance Energy Transfer).
Although this method utilizes the phenomenon of r), it does not require B / F separation, enables measurement in a homogeneous system, and is an excellent immunoassay means for solving the problems of the conventional method.

[0007]

The antigen concentration measuring method utilizing the fluorescence energy transfer phenomenon proposed by the inventors of the present application does not require B / F separation, and enables measurement in a homogeneous system. Yes, it is an excellent immunoassay that solves the problems of the conventional method. However, if there is a contaminant in the sample that is excited by the excitation light of the donor-side fluorescent substance and emits fluorescence of the same wavelength as the acceptor-side fluorescent substance,
Alternatively, if the fluorescent substance itself on the receptor side is slightly excited by the excitation light, this causes a large background, which makes it difficult to measure the concentration of the antigen. In addition, when a foreign substance that strongly absorbs the excitation light is present in the sample, the intensity of the excitation light that excites the fluorescent substance on the donor side changes depending on the concentration of the foreign substance, so that the measurement of the antigen concentration becomes inaccurate. There was also a problem. Furthermore, the measuring device used in this method requires a light source for irradiating the sample with excitation light of a specific wavelength and an optical system for detecting the fluorescence intensity of the specific wavelength. However, there is a disadvantage that the apparatus becomes complicated and the apparatus price becomes expensive.

[0008] The invention of this application has been made in view of the circumstances described above, and is difficult to be affected by contaminants, can be measured in a homogeneous system, and does not require excitation light. Emission energy transfer (Bioluminescence
By utilizing the advantages of resonance energy transfer (BRET), a new concentration measurement method that can measure a measurement target with higher accuracy and sensitivity and that can measure with a simple optical system and a cheap optical system is provided. The challenge is to do.

[0009]

This application provides the following inventions (1) to (21) for solving the above-mentioned problems. (1) a substance A labeled with an enzyme E that catalyzes a luminescence reaction;
A method for measuring the concentration of a substance X using a substance B labeled with a fluorescent substance F, wherein the amount of fluorescence of the fluorescent substance F due to non-radiative transfer of emission energy catalyzed by an enzyme E is used as an index. A concentration measuring method characterized by measuring the concentration. (2) Substance A is substance X or a part thereof, substance B is a substance capable of binding to substance X, or substance B is substance X or a part thereof and substance A is a substance capable of binding to substance X; The method according to the above (1), wherein the concentration of the substance X is measured using the degree of decrease in the amount of fluorescence of the fluorescent substance F due to the inhibition of the formation of the complex A: B due to the presence of the substance X as an index. (3) The substance A and the substance B are both substances having a binding ability to the substance X, and the concentration of the substance X is measured by using the degree of increase in the amount of fluorescence of the fluorescent substance F due to the formation of the complex A: B: X as an index. The method of the above (1). (4) The method according to any one of (1) to (3), wherein the substance A or the substance B is immobilized on a plate. (5) The method according to the above (4), wherein the concentration of the substance X is measured without separating the non-immobilized substance B or A, which is free from binding to the substance X, from the fixed plate. (6) The method according to any one of (1) to (5), wherein the substance X is a biopolymer or a low-molecular compound. (7) Substance A or substance B capable of binding to substance X
Is the antibody, receptor, lectin, peptide, nucleic acid, sugar chain or a part of these molecules, the method according to any of (2), (4) to (6) above. (8) Substance A is an antibody, receptor, lectin, peptide, nucleic acid, sugar chain or a part of these molecules, and substance B binds to substance X at a site different from substance A.
The method according to any one of the above (3) to (6), which is a lectin, peptide, nucleic acid, sugar chain or a part of these molecules. (9) The method according to any one of (1) to (8) above, wherein the enzyme E is an enzyme that catalyzes a bioluminescence reaction or a variant thereof. (10) The method according to any one of (1) to (8) above, wherein the enzyme E is an enzyme that catalyzes a chemiluminescence reaction or a variant thereof. (11) From (1) to (1), wherein the fluorescent substance F is a chemically synthesized fluorescent substance or a fluorescent protein which serves as an acceptor of the non-radiative luminescence energy transfer of the luminescence reaction catalyzed by the enzyme E.
0) Either method. (12) A measurement kit comprising a reagent containing substance A labeled with enzyme E that catalyzes a luminescence reaction, a reagent containing substance B labeled with fluorescent substance F, and a reagent containing a substrate for enzyme E. (13) A measurement kit comprising a plate on which a reagent containing substance A labeled with enzyme E that catalyzes a luminescence reaction, a reagent containing a substrate of enzyme E, and substance B labeled with fluorescent substance F are immobilized. (14) A measurement kit comprising a reagent containing a substance B labeled with a fluorescent substance F, a plate on which a substance A labeled with an enzyme E that catalyzes a luminescence reaction is immobilized, and a reagent containing a substrate of the enzyme E. (15) Substance A is a substance X to be measured or a part thereof, substance B is a substance capable of binding to substance X, or substance B is a substance X or a part thereof and substance A is a substance capable of binding to substance X. Certain measurement kits according to any one of the above (12) to (14). (16) The measurement kit according to any one of the above (12) to (14), wherein both the substance A and the substance B have a binding ability to the substance X to be measured. (17) Substance A or substance B capable of binding to substance X
Is the antibody, receptor, lectin, peptide, nucleic acid, sugar chain or a part of these molecules. (18) Substance A is an antibody, receptor, lectin, peptide, nucleic acid, sugar chain or a part of these molecules, and substance B binds to substance X at a site different from substance A.
The measurement kit according to the above (16), which is a lectin, peptide, nucleic acid, sugar chain or a part of these molecules. (19) The measurement kit according to any one of (12) to (18), wherein the enzyme E is an enzyme that catalyzes a bioluminescence reaction or a variant thereof. (20) The measurement kit according to any one of (12) to (18), wherein the enzyme E is an enzyme that catalyzes a chemiluminescence reaction or a variant thereof. (21) From (12) to (2), wherein the fluorescent substance F is a chemically synthesized fluorescent substance or a fluorescent protein which serves as an acceptor of the non-radiative luminescence energy transfer of the luminescence reaction catalyzed by the enzyme E.
The measurement kit of any of 1).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The invention (1) of this application is a measuring method utilizing the phenomenon of bioluminescence energy transfer (BRET). The energy transfer efficiency in this BRET is defined as follows. The emission lifetime in the excited state of the donor luminescent material in the absence of the fluorescent material serving as an acceptor is τ,
Assuming that the emission lifetime when there is an acceptor and there is excitation energy transfer is τ ′, the non-radiative energy transfer efficiency E is defined by E = 1−τ ′ / τ. In general, assuming that the emission intensity when there is no excitation energy transfer is F and the intensity when there is energy transfer is F ′, τ ′ / τ ≒ F ′ / F is established, so that E = 1−F ′ / F Becomes Here, the distance R between the donor and acceptor is represented as ^{R = Ro (E -1 -1)} 1/6 [Ro = 9786 (κ 2 n -4 Q D J) 1/6] You. κ ² is the relative orientation between the donor and the acceptor, and a value of 2/3 is accepted during random motion by heat. N is the refractive index of the solution. J is the overlap integral of the emission spectrum of the donor and the absorption spectrum of the acceptor. Q _D is the quantum efficiency of the energy donor. Therefore, since Ro is a constant peculiar to the reaction system, the energy transfer efficiency E is given by E = 1 / (1+ (R / Ro) ⁶ ). Therefore, the distance R between the donor and the acceptor is R
In the vicinity of o, the energy transfer efficiency is affected by the sixth power. In other words, the energy transfer efficiency will be very sensitive to the distance between the donor and the acceptor. When a generally used fluorescent dye is used as an acceptor, since Ro = 2 to 8 nm, the donor and / or
Alternatively, in the state where the receptor dye is moving randomly in the solution, no energy transfer occurs. If for some reason the distance between the donor and the acceptor dye falls below about 15 nm, energy transfer occurs and the fluorescence intensity of the acceptor dye changes.

Accordingly, in the invention (1) of this application,
The substance A labeled with the enzyme E that catalyzes the luminescence reaction and the substance B labeled with the fluorescent substance F approach a distance of about 15 nm or less depending on the presence of the substance X to be measured in the sample (complex A). : B) or A and B are about 15 nm
The deviation from the above distance is measured by using the change in the fluorescence intensity of the fluorescent substance F as an index, and thereby the concentration of the substance X is measured.

The method of the invention (1) can be specifically carried out as the invention (2). That is, the method of the present invention (2) is a concentration measurement by a so-called “competition method”, in which the substance A is the substance X to be measured or a part thereof, and the substance B has a binding ability to the substance X, or the substance B Is substance X or a part thereof, substance A is a substance capable of binding to substance X, and formation of complex A: B is substance X
Is a method of measuring the concentration of the substance X using the degree of decrease in the amount of fluorescence of the fluorescent substance F due to inhibition by the presence of the substance X as an index. FIG. 1 is a conceptual diagram showing an example of the method of the present invention (2). In the case of the method illustrated in FIG. 1, the substance A labeled with the enzyme E that catalyzes the luminescence reaction is the substance X to be measured.
And substance B labeled with fluorescent substance F is a substance capable of binding to substance X. When the substance X does not exist, the substance A and the substance B form a complex A: B due to the affinity of each other, and the distance between the enzyme E and the fluorescent substance F is about 15
nm or less. At this time, if the substrate S of the enzyme E exists,
The enzyme E catalyzes the conversion of S → P and transfers the emission energy of the emission wavelength (excitation light wavelength) λ1 to the fluorescent substance F in a non-radiative manner, so that the fluorescent substance F obtains the fluorescence of the wavelength λ2. However, when the substance X is present, two kinds of complexes X: B and A: B are formed due to the respective affinity of the substance X and the substance A and the substance B, so that a complex with the substance B is formed. Without this, the substance A, which is present alone, emits light of the wavelength λ1. As a result, it is possible to measure the concentration of the substance X contained in the sample by measuring the emission intensity and / or the fluorescence intensity of the entire measurement system.

The invention (1) of this application can also be specifically implemented as invention (3). The method of the present invention (3) is a concentration measurement by a so-called “sandwich method”, in which both the substance A and the substance B have a binding ability to the substance X, and the fluorescence due to the formation of the complex A: B: X. In this method, the concentration of the substance X is measured using the degree of increase in the amount of fluorescence of the substance F as an index. FIG. 2 is a conceptual diagram showing an example of the method of the present invention (3). In the case of the method shown in FIG. 2, the substance A labeled with the enzyme E that catalyzes the luminescence reaction does not bind to the substance B by itself, and generates a luminescence signal of the wavelength λ1 due to the presence of the substrate S. . On the other hand, when the substance X is present, the substance A and the substance B form a complex A: B: X of the three via the substance X, the distance between the enzyme E and the fluorescent substance F becomes about 15 nm or less, and the enzyme Due to the transfer of the emission energy from E, fluorescence of wavelength λ2 is obtained from the fluorescent substance F. As a result, it is possible to measure the concentration of the substance X contained in the sample by measuring the emission intensity and / or the fluorescence intensity of the entire measurement system.

The process inventions (1) to (3) as described above can be carried out in a liquid phase system in which each substance is reacted in a solution, or the substance A or B is immobilized on a plate. It can also be carried out in a solid phase system [Invention (4)].
Also, in the measurement using the solid phase system, the concentration of the substance X can be measured without separating the non-immobilized substance B or A in a free state without binding to the substance X from the immobilized plate. )].

When the methods (1) to (3) are carried out in a liquid phase system, the method can be carried out, for example, using the measurement kit of the invention (12). That is, the measurement kit of the invention (12) has a reagent containing the substance A labeled with the enzyme E that catalyzes the luminescence reaction, a reagent containing the substance B labeled with the fluorescent substance F, and a reagent containing the substrate of the enzyme E. At the time of measurement, these reagents may be mixed, a sample solution may be added and reacted for a certain period of time, and then the fluorescence intensity of the reaction solution may be measured using a fluorescence spectrometer or the like.

When the methods of the inventions (1) to (3) are carried out in a solid phase system, they can be carried out using the measurement kit of the invention (13) or (14). For example, the measurement kit of the invention (13) has a plate on which a reagent containing a substance A labeled with an enzyme E that catalyzes a luminescence reaction, a reagent containing a substrate of the enzyme E, and a substance B labeled with a fluorescent substance F are immobilized. After reacting each reagent and sample solution sequentially on the plate to cause a reaction, the fluorescence intensity of the reaction solution on the plate may be measured using a fluorescence spectrometer or the like.

Further, in the assay kits of the inventions (12) to (14), when the competition method of the invention (2) is carried out, the substance A is the substance X to be measured or a part thereof, and the substance B is the substance X In a preferred embodiment, the substance capable of binding to X or the substance B is the substance X or a part thereof, and the substance A is a substance capable of binding to the substance X [Invention (15)]. Also,
When the sandwich method of the invention (3) is carried out, a preferable embodiment is that both the substance A and the substance B are substances capable of binding to the substance X to be measured [invention (16)].

In the methods of the inventions (1) to (5) of this application, the substance X to be measured can be a biopolymer or a low-molecular compound [Invention (6)]. Examples of the biopolymer include proteins, peptides, nucleic acids, sugar chains and the like. When these substances X are to be measured, the invention
In the competition method (2), the substance A or the substance B can be an antibody, a receptor, a lectin, a peptide, a nucleic acid, a sugar chain, or a part of these molecules [Invention (7)]. In the sandwich method, substance A is an antibody, a receptor, a lectin, a peptide, a nucleic acid, a sugar chain or a part of these molecules, and substance B is an antibody that binds to substance X at a site different from substance A;
It can be a receptor, lectin, peptide, nucleic acid, sugar chain or a part of these molecules [Invention (8)]. That is, the relationship between the substance X and the substance A / B is, for example, as follows: antigens and peptides and antibodies, physiologically active proteins and receptors such as hormones and cytokines, lectins and lectin-binding carbohydrates, and DNA and DNA-binding proteins. Any of these can be set as the substance to be measured as the substance X.

Furthermore, in the above inventions (1) to (8), the enzyme E as a donor of luminescence energy transfer may be an enzyme that catalyzes a bioluminescence reaction or a variant thereof [Invention (9) )], And are, for example, luminescent enzymes originating from living organisms such as luminescent bacteria, fireflies, sea fireflies, Japanese click beetle and Renilla. The enzyme E can also be an enzyme that catalyzes a chemiluminescent reaction or a variant thereof [Invention (10)], for example, a peroxidase enzyme. The substrate of these enzymes E may be such that the enzyme E emits light by its catalytic action. For example, luciferin is used for firefly-derived luciferase, and luminol (5- luciferase is used for peroxidase). Amino-2,3-dihydro-1,4-phthalazinedione) and the like can be used.

Further, in the above inventions (1) to (10), the fluorescent substance F, which is a receptor for luminescence energy transfer,
A chemically synthesized fluorescent substance or a fluorescent protein that serves as an acceptor of the non-radiative luminescence energy transfer of the luminescence reaction catalyzed by the enzyme E [Invention (11)].
Those commonly used for fluorescent labeling of biological samples can be used. However, luminescence energy transfer from the enzyme E catalyzing the luminescence reaction to the fluorescent substance F occurs more efficiently as the overlap between the emission spectrum of the enzyme E and the excitation spectrum of the fluorescent substance F increases. It is preferable to use a fluorescent substance having an excitation maximum wavelength close to the emission maximum wavelength of the enzyme E. Further, from the viewpoint of fluorescence detection, a fluorescent substance F having a maximum excitation wavelength and a maximum fluorescence wavelength separated from each other and having as large an absorption coefficient and a quantum yield of fluorescence as possible is preferable. Further, those having various functional groups for labeling the substance B are preferable.

The above-mentioned aspects of the inventions (6) to (11) are also preferable aspects for the measurement kits of the inventions (12) to (14) [inventions (17) to (21)].

Hereinafter, the present invention of this application will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.

[0023]

EXAMPLES Example 1 Concentration of c-Myc peptide by competitive method
Competition method using a c-Myc epitope labeled with firefly luciferase as the substance A for measurement and an anti-Myc antibody labeled with the carbocyanine derivative Cy3 or Cy3.5 as the substance B [Invention
(2)], the concentration of c-Myc peptide was measured. FIG. 3 is a conceptual diagram of this measurement. The measurement was performed in a liquid phase system. a. Preparation of sample a-1: c-Myc peptide Human c-peptide consisting of 31 amino acids shown in SEQ ID NO: 1 in the sequence listing
Myc peptide (Genosis Biotechnology) was used. a-2: Luciferase-labeled c-Myc epitope Luciferase cDNA cut out from pGEM-luc (Promega) and synthetic D encoding c-Myc epitope
After ligating with an NA fragment (SEQ ID NO: 2) to recombine into an expression vector and transforming a host cell (E. coli TG1), the cell is cultured and a luciferase-labeled c-Myc epitope is obtained from the culture. Was. a-3: Cy3 or Cy3.5-labeled anti-c-Myc antibody IgG1 / κ monoclonal antibody 9E10 against a synthetic peptide immunogen obtained from the human c-myc proto-oncogene (Evan,
GI et al., Molecular and Cellular Biology 5: 3610
-3616, 1985). Antibodies are purified from ascites, PB
After replacement with S buffer, a labeling reaction was performed. That is, the fluorescent dye (Cy3 / Cy3.5) and the antibody were mixed at an appropriate molar ratio, and allowed to react at room temperature for 1 hour or more with good stirring. Next, unreacted dye was removed by gel filtration using 50 mM Tris-HCl (pH 8.0) as an eluent. The amount of the fluorescent dye immobilized on the antibody was determined by measuring the protein concentration determined by the BCA assay and the absorbance of each fluorescent substance at the absorption maximum wavelength. b. Measurement of c-Myc concentration b-1: Method Luciferase-labeled c-Myc epitope (0.04 μM), C
y3 or Cy3.5 labeled anti-c-Myc antibody (1.0 μM), and c
-Myc peptide (0.01 to 100 µg / ml) was mixed and reacted at 4 ° C for about 1 hour under light shielding. Next, the following reaction buffer was added, and the change in fluorescence intensity was measured with a fluorometer F2000.

Reaction buffer: 50 mM Tris-HCl (pH 8.0) 5 mM MgSO ₄ 1 mM EDTA 1 mM D-Luciferin Na 5 mM ATP 300 μM CoA 33.3 mM DTT b-2: Result measurement Are as shown in FIG. This FIG.
The measurement was performed five times for each peptide concentration, and the average value of three measurements excluding the maximum value and the minimum value is shown. c-M
As the concentration of the yc peptide (X) increases, the formation of a complex between the luciferase-labeled c-Myc epitope (A) and the anti-c-Myc antibody (B) is inhibited, and the fluorescence of 570 nm (Cy3) relative to 550 nm emission by luciferase or 596nm
The percentage of (Cy3.5) decreased.

From the above results, it was confirmed that the substance of unknown concentration in the sample can be measured with high precision and easily by the method of the present invention. Example 2 Chicken egg white lithochi by sandwich method
Beam antibody variable regions V _H labeled Renilla luciferase as analyte A in (HEL), sandwich method the fluorescent protein EYFP using antibody variable regions V _L labeled as the substance B by [the invention (3)], chicken The concentration of egg white lysozyme (HEL) was measured. a. Sample preparation a-1: Vector construction <pET32 / V _H (HEL) -Rluc> Using a vector having a gene for Renilla luciferase, pRL-null (Promega) as a template, and primers having restriction enzyme sites NotI and XhoI Using Renilla luciferase gene fragment to PC
Amplified by R.

The vector owned by the inventors, pET32 / V _H (H
EL) -EBFP (R.Arai, H.Ueda, K.Tsumoto, WCMahoney,
I. Kumagai, and T. Nagamune, Protein Engineering, vo
l.13, p.369-376 (2000)), cut with NotI and XhoI, and insert the above-mentioned PCR fragment into pET32 / V _H (HEL)-
Rluc was obtained. <PET32 / V _L (HEL) -EYFP> pET3 owned by the inventors
2 / V _L (HEL) -EBFP (R.Arai, H.Ueda, K.Tsumoto, WCMaho
ney, I. Kumagai, and T. Nagamune, Protein Engineerin
g, vol.13, p.369-376 (2000)) The vector was mutated by the Kunkel method to obtain pET32 / _VL (HEL) -EYFP. a-2: Expression E. coli AD494 (DE3) LysS (Novagen) was transformed with the prepared plasmid, and 50 μg / ml ampicillin, 34 μg / ml
Incubated overnight at 37 ° C. on LB plates containing ml chloramphenicol and 15 μg / ml kanamycin.

The obtained colonies were treated with 50 μg / ml ampicillin, 34 μg / ml chloramphenicol, and 15 μg / ml
In a 5 ml LB medium containing kanamycin at 37 ° C. overnight, this culture is subcultured into 100 ml LB medium and
And cultured. IPTG when OD ₆₀₀ reaches around 0.5
(Isopropyl-β-D-thiogalactopyranoside) to a final concentration of 0.1 mM, and the cells were further cultured overnight at 16 ° C. a-3: Purified harvested by centrifugation cultured cells (V _H (HEL) purification of -Rluc), Sonication of _{5ml Buffer (50mM NaH 2 PO 4} , 0.1MN
aCl, pH 7.0). Next, the cells were sonicated and centrifuged to obtain a supernatant. Here, the HEL-immobilized gel was added and left overnight at 4 ° C. to adsorb the target protein to the HEL-immobilized gel. The recovered HEL-immobilized gel is from Sonication Buf
After washing several times with fer, fill the column and add 900 μl of Elution
Elution was performed with Buffer (0.1 M Tris-HCl, 0.5 M NaCl, pH 10). At this time, 1M for neutralization was previously placed in the tube.
100 μl of Tris-HCl (pH 6.8) was stored therein. The eluate was dialyzed against a buffer for measurement. (Purification method of _VL (HEL) -EYFP) Cultured cells were collected by centrifugation, and 5 ml of Sonication Buffer (50 mM NaH ₂ PO ₄ , 300 mM
NaCl, pH 7.0). Next, the cells were sonicated and centrifuged to obtain a supernatant. TALON was added thereto and left overnight, and the target protein was adsorbed to TALON. Collected TAL
After washing the ON several times with the Sonication Buffer, the column is packed into an Elution Buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 15 mM NaCl).
0 mM Imidazole (pH 7.0)). The eluate was measured using a measurement buffer (25 mM Tris-HCl, 0.1 M NaCl, pH7.
Dialysis was performed in 5). b. Measurement of HEL concentration b-1: Method HEL (0.1 μg / ml-100 μg / ml), V
_{H (HEL) -Rluc (0.1μM)} , and V _L (HEL) -EYFP (0.1μ
M) and allowed to react for 5 minutes. After adding coelenterazine, a substrate for renilla luciferase, to a final concentration of 10 μM, the emission spectrum was measured. Measurement is IMUC7000
(Otsuka Electronics). b-2: Result FIG. 5 shows the obtained emission spectrum.

As the HEL concentration increased, the peak near 525 nm, which is the maximum fluorescence wavelength of EYFP, increased in a HEL concentration-dependent manner.

From this, V _H (HEL) -Rluc, V _L (HEL) -EYFP,
It was shown that the concentration of the complex formed by the three members of HEL increased, and the luminescence energy of Renilla luciferase was transferred to EYFP in a non-radiative manner.

FIG. 6 shows the EYF concentration on the horizontal axis of the HEL concentration.
A graph is shown in which the vertical axis represents the ratio (525 m / 475 nm) between the maximum fluorescence wavelength of P and the emission intensity at the maximum emission wavelength of Renilla luciferase.

From this, it was found that when the HEL concentration was 1 μg / ml or more, the HEL concentration could be measured with high accuracy using the sandwich method of the present invention.

[0032]

As described above in detail, the present application makes it possible to carry out measurement by taking advantage of the advantage of bioluminescence energy transfer, which is hardly affected by contaminants, enables measurement in a homogeneous system, and does not require excitation light. There is provided a new concentration measurement method capable of measuring an object with higher accuracy and sensitivity and measuring with a simple device configuration using an inexpensive optical system. This concentration measuring method is particularly useful in clinical examinations, food hygiene examinations, examinations of chemical pollutants such as environmental hormones and the like, which require higher precision and higher sensitivity. In addition, since this measurement method can perform measurement in a uniform system and does not require a light source or the like, it is useful for establishing a simpler measurement system such as application to an automatic analyzer.

[0033]

[Sequence List] <110> Japan Science and Technology Corporation <120> Substance concentration measurement method <130> NP00317-SH <160> 2 <210> 1 <211> 31 <212> PRT < 213> Homo sapience <400> 1 Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Arg Lys Arg 1 5 10 15 Arg Glu Gln Leu Lys His Lys Leu Glu Gln Leu Arg Asn Ser Cys 20 25 30 <210> 2 <211> 33 <212> DNA <213> Artificial sequence <400> 2 gaacaaaagc ttatctcaga agaggatctg aat 33

[Brief description of the drawings]

FIG. 1 is a conceptual diagram in a case where the method of the present application is performed by a competitive method.

FIG. 2 is a conceptual diagram when the method of the present application is performed by a sandwich method.

FIG. 3 is a conceptual diagram of measuring the concentration of c-Myc peptide shown as an example.

FIG. 4 shows the results of measuring the concentration of c-Myc peptide shown in Examples.

FIG. 5 is an emission spectrum as a result of measuring the concentration of HEL shown as an example.

FIG. 6 shows the results of HEL concentration measurement shown as an example.

Claims (21)

[Claims] A substance X labeled with a substance A labeled with an enzyme E that catalyzes a luminescence reaction and a substance B labeled with a fluorescent substance F
A method for measuring the concentration of a substance X, wherein the concentration of a substance X is measured using the amount of fluorescence of a fluorescent substance F due to non-radiative transfer of luminescence energy in a luminescence reaction catalyzed by an enzyme E as an index. Method. 2. Substance A is substance X or a part thereof, substance B is a substance capable of binding to substance X, or substance B is substance X or a part thereof and substance A is a substance capable of binding to substance X. 2. The method according to claim 1, wherein the concentration of the substance X is measured using the degree of decrease in the amount of fluorescence of the fluorescent substance F due to inhibition of the formation of the complex A: B by the presence of the substance X as an index. 3. The substance A and the substance B are both substances capable of binding to the substance X, and the concentration of the substance X is determined based on the degree of increase in the amount of fluorescence of the fluorescent substance F due to the formation of the complex A: B: X. The method of claim 1, wherein the measuring is performed. 4. The method according to claim 1, wherein the substance A or the substance B is immobilized on a plate. 5. The method according to claim 4, wherein the concentration of the substance X is measured without separating the non-immobilized substance B or A, which is free from binding to the substance X, from the immobilized plate. 6. The method according to claim 1, wherein the substance X is a biopolymer or a low molecular compound. 7. The substance A or the substance B capable of binding to the substance X is an antibody, a receptor, a lectin, a peptide, a nucleic acid,
The method according to any one of claims 2, 4 to 6, which is a sugar chain or a part of these molecules. 8. An antibody wherein the substance A is an antibody, a receptor, a lectin, a peptide, a nucleic acid, a sugar chain or a part of these molecules, and the substance B binds to the substance X at a site different from the substance A;
The method according to any one of claims 3 to 6, which is a receptor, a lectin, a peptide, a nucleic acid, a sugar chain, or a part of these molecules. 9. The method according to claim 1, wherein the enzyme E is an enzyme that catalyzes a bioluminescence reaction or a variant thereof. 10. The method according to claim 1, wherein the enzyme E is an enzyme that catalyzes a chemiluminescence reaction or a variant thereof. 11. The method according to claim 1, wherein the fluorescent substance F is a chemically synthesized fluorescent substance or a fluorescent protein that serves as an acceptor of non-radiative luminescence energy transfer of a luminescence reaction catalyzed by the enzyme E. 12. A measurement kit comprising a reagent containing a substance A labeled with an enzyme E that catalyzes a luminescence reaction, a reagent containing a substance B labeled with a fluorescent substance F, and a reagent containing a substrate of the enzyme E. 13. A plate comprising a reagent containing a substance A labeled with an enzyme E that catalyzes a luminescence reaction, a reagent containing a substrate of the enzyme E, and a substance B labeled with a fluorescent substance F immobilized thereon. Measurement kit. 14. A measurement comprising a reagent containing a substance B labeled with a fluorescent substance F, a plate on which a substance A labeled with an enzyme E that catalyzes a luminescence reaction is immobilized, and a reagent containing a substrate of the enzyme E. kit. 15. The substance A is the substance X to be measured or a part thereof, and the substance B is a substance capable of binding to the substance X. Alternatively, the substance B is a substance X or a part thereof, and the substance A is capable of binding to the substance X. The measurement kit according to any one of claims 12 to 14, which is a substance. 16. The measurement kit according to claim 12, wherein both the substance A and the substance B have a binding ability to the substance X to be measured. 17. The assay kit according to claim 15, wherein the substance A or the substance B capable of binding to the substance X is an antibody, a receptor, a lectin, a peptide, a nucleic acid, a sugar chain, or a part of these molecules. 18. The substance A is an antibody, a receptor, a lectin, a peptide, a nucleic acid, a sugar chain or a part of these molecules,
17. The measurement kit according to claim 16, wherein the substance B is an antibody, a receptor, a lectin, a peptide, a nucleic acid, a sugar chain, or a part of these molecules that binds to the substance X at a site different from the substance A. 19. The assay kit according to claim 12, wherein the enzyme E is an enzyme that catalyzes a bioluminescence reaction or a variant thereof. 20. The kit according to claim 12, wherein the enzyme E is an enzyme catalyzing a chemiluminescence reaction or a variant thereof. 21. The measurement kit according to any one of claims 12 to 20, wherein the fluorescent substance F is a chemically synthesized fluorescent substance or a fluorescent protein which serves as an acceptor of non-radiative luminescence energy transfer of a luminescence reaction catalyzed by the enzyme E. .