JP2015163865A - Method for manufacturing cortisol-albumin conjugate, cortisol-albumin conjugate, substrate for cortisol measurement, kit for cortisol measurement, and method for manufacturing cortisol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cortisol-albumin conjugate having high water dispersibility and a method for manufacturing the same, a method for measuring cortisol having a good reproductivity for measurement, and a substrate and a kit for measuring the cortisol.SOLUTION: A method for manufacturing a cortisol-albumin conjugate in which the number of cortisol-albumin bonds is from 7 to 14 to an albumin, includes: a process (1) of manufacturing a cortisol active ester by reacting a compound obtained by introducing a carboxyl group to a cortisol and a condensation agent as well as a carboxylic acid activation reagent; and a process (2) of reacting the albumin and the cortisol active ester. There are provided the method for manufacturing the cortisol-albumin conjugate in which the molar ratio of the cortisol active ester to the albumin is from 15 to 30 and the molar ratio of the condensation agent to the albumin is from 70 to 150; the cortisol-albumin conjugate manufactured by the manufacturing method; a method for measuring the cortisol by using the cortisol-albumin conjugate; and a substrate for measuring the cortisol and a kit for measuring the cortisol.

Description

  The present invention relates to a method for producing a cortisol / albumin conjugate that can be used for immunoassay of cortisol, and a cortisol / albumin conjugate obtained by the production method, and further, a substrate for cortisol measurement using the conjugate and the kit In addition, the present invention relates to a method for measuring cortisol.

  A fluorescence detection method is widely used as a highly sensitive and easy measurement method for quantifying proteins, enzymes, inorganic compounds, and the like. In this fluorescence detection method, the presence of the test substance is detected by detecting the fluorescence emitted when the sample that is considered to contain the test substance that emits fluorescence when excited by the light of the specific wavelength is irradiated with the excitation light of the specific wavelength. It is a method to confirm. In addition, when the test substance is not a fluorescent substance, a substance labeled with a fluorescent dye that specifically binds to the test substance is brought into contact with the sample, and then this binding is detected by detecting fluorescence in the same manner as described above. That is, the existence of the test substance is also widely confirmed.

  In such a fluorescence detection method, in order to improve the detection sensitivity, a method using the effect of electric field enhancement by plasmon resonance is known. In this method, in order to generate plasmon resonance, a sensor chip provided with a metal layer in a predetermined region on a transparent support is prepared, and a metal layer forming surface of the support is formed with respect to the interface between the support and the metal film. Excitation light is incident at a predetermined angle equal to or greater than the total reflection angle from the opposite surface side. The surface plasmon is generated in the metal layer by the irradiation of the excitation light, and the signal / noise ratio (S / N ratio) is improved by enhancing the fluorescence by the electric field enhancing action due to the generation of the surface plasmon. Such a fluorescence detection method based on surface plasmon excitation (hereinafter referred to as “SPF (Surface Plasmon Resonance) method”) is approximately 10 times the fluorescence detection method based on epi-illumination excitation (hereinafter referred to as “epifluorescence method”). Double signal enhancement can be obtained and measurement can be performed with high sensitivity.

  Cortisol, a type of corticosteroid, is a type of glucocorticoid that regulates carbohydrate, fat, and protein metabolism. Among the three types of glucocorticoids, cortisol is the most in vivo. It is known that the secretion of cortisol is also increased by stress, and depending on the amount of secretion, actions such as an increase in blood pressure and blood glucose level or a decrease in immune function may occur. Cortisol (also called hydrocortisone) is clinically used as a steroidal anti-inflammatory drug, for example, acute inflammation, chronic inflammation, autoimmune disease, allergic disease, shock, gout, acute leukemia, transplant rejection It may be used for treatment such as reaction. As an immunoassay method for cortisol, a method utilizing the effect of electric field enhancement by plasmon resonance has been proposed (Patent Document 1).

  When measuring cortisol, which is a low molecular weight compound, a cortisol / albumin conjugate, in which cortisol and albumin are combined, is used by amine coupling by an amidation reaction using a reagent that activates a carboxyl group. Preparation has been done.

  For example, in Patent Document 2, bovine serum albumin (BSA: bovine serum albumin) using isobutyl chloroformate as cortisol-21-hemisuccinate sodium salt as the condensing agent, and the condensing agent / cortisol molar ratio is It is described that bonding is performed at 0.6. Patent Document 3 describes that 17α-hydroxyprogesterone-11-hemisuccinate-BSA is produced by using isobutyl chlorocarbonate with respect to 17α-hydroxyprogesterone-11-hemisuccinate, which is a cortisol-like compound. Patent Document 4 describes that BSA reacts and binds to a cortisol-3-carboxymethyloxime isomer activated in the form of N-hydroxysuccinimide ester. Patent Document 5 describes that BSA is bonded to cortisol-3-carboxymethyloxime using N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC: N, N'-Dicylcyclohexylcarbodiimide). And conjugates with a condensing agent / cortisol molar ratio of 1.6 are described.

  Patent Document 6 describes a method for immunologically measuring cortisol with high sensitivity. In Patent Document 6, first, cortisol and albumin are bound at a certain ratio by utilizing amine coupling by amidation reaction using NHS and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide reagent. A cortisol / albumin conjugate is prepared. The anti-cortisol antibody-labeled fluorescent particles are brought into contact with the anti-cortisol antibody-labeled fluorescent particles on the substrate by bringing the anti-cortisol antibody-labeled fluorescent particles and the test substance cortisol into contact with the obtained substrate on which the cortisol / albumin conjugate is immobilized. Cortisol (cortisol in a cortisol / albumin conjugate) and cortisol as a test substance are competitively bound. Thereafter, by measuring the fluorescence based on the anti-cortisol antibody-labeled fluorescent particles bound to the substrate, cortisol is detected with high sensitivity and immunologically in a clinically meaningful cortisol concentration region (ie, 2 to 30 μg / dL). It is described to measure.

JP 2010-190880 A JP 59-88428 A Japanese Patent Laid-Open No. 3-150464 JP-A-8-333398 JP 2011-148821 A JP2013-83931A

  As described above, the amine coupling method is known as a general method for labeling a low molecular weight compound to a biopolymer (for example, BSA). For example, NHS and 1-ethyl-3- (3-dimethylamino) are known. Amidation reactions using propyl) carbodiimide reagents have been used. Since this amidation reaction is carried out in water, amide bond formation competes with the hydrolysis reaction of the NHS active ester, so usually an excess amount of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and NHS are added. It is made to react in the mode which carries out. However, under the above conditions, the formation of the target polyhapten, cortisol / albumin conjugate, competes with the cross-linking reaction between biopolymers (for example, BSA), and the hardly water-soluble aggregates are also present. At the same time, the dispersibility of cortisol / albumin conjugates in water deteriorates, so in order to increase the production yield, avoid the formation of this hardly water-soluble aggregate, A method for producing a good cortisol / albumin conjugate in a high yield is desired. Further, as a simple and highly sensitive method for measuring cortisol, a fluorescence detection method by surface plasmon excitation is known, but a measurement method with particularly high reproducibility is desired.

  An object of the present invention is to provide a cortisol / albumin conjugate having high water dispersibility and a method for producing the same. Furthermore, the present invention is to provide a cortisol measurement method, a cortisol measurement substrate, and the kit, which have good measurement reproducibility.

  As a result of intensive studies to solve the above problems, the present inventors have made a specific cortisol / albumin by reacting with albumin by using a condensing agent and a cortisol active ester in a molar ratio within a predetermined range. A cortisol / albumin conjugate having a high water dispersibility and a good measurement reproducibility by measuring cortisol using the cortisol / albumin conjugate of the present invention. I found it. The present invention has been completed based on these findings.

That is, according to the present invention,
A step (1) of producing a cortisol active ester by reacting a compound obtained by introducing a carboxyl group into cortisol with a condensing agent and a carboxylic acid activating reagent;
A step of reacting albumin with a cortisol active ester (2),
A method for producing a cortisol / albumin conjugate having a binding number of cortisol to albumin of 7 to 14, comprising:
Provided is a method for producing a cortisol / albumin conjugate, wherein the molar ratio of cortisol active ester to albumin is 15 to 30, and the molar ratio of condensing agent to albumin is 70 to 150.

  Preferably, the compound obtained by introducing a carboxyl group into cortisol is a compound represented by the general formula (1).

In the formula, L ₁ represents a linking group.

Preferably, the number of cortisol bonds to albumin is 8-13.
Preferably, the molar ratio of condensing agent to albumin is 90-130.
Preferably, the molar ratio of cortisol active ester to albumin is 20-25.
Preferably, the condensing agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or its hydrochloride.
Preferably, the carboxylic acid activating reagent is N-hydroxysuccinimide.

Furthermore, according to the present invention, a cortisol / albumin conjugate produced by the method of the present invention is provided.
The cortisol / albumin conjugate of the present invention preferably has a dispersity of 90% or more when 5 mg of cortisol / albumin conjugate is dissolved in 1 mL of phosphate buffer. The degree of dispersion in the present invention refers to 5 mg of cortisol / albumin conjugate that does not precipitate when dispersed in water with 1 mL of phosphate buffer (PBS, Wako Pure Chemical Industries, Ltd., product catalog number: 166-23555, The supernatant after being dissolved in pH 7.1 to 7.3) and centrifuged is collected, and the absorbance at 280 nm is measured using a spectrophotometer at 25 ° C. For this absorbance, a value expressed as a percentage of the absorbance measured with a spectrophotometer of the cortisol / albumin conjugate prepared in the same manner is defined as the degree of dispersion.

Furthermore, according to the present invention, there is provided a substrate for measuring cortisol, which has a metal film on the surface and the cortisol / albumin conjugate of the present invention is immobilized on the metal film.
Furthermore, according to the present invention, there is provided a kit for measuring cortisol, comprising a substrate having a metal film on the surface, the substrate on which the cortisol / albumin conjugate of the present invention is immobilized, and anti-cortisol antibody-labeled fluorescent particles. Provided.
Furthermore, according to the present invention, a test sample containing cortisol and an anti-cortisol antibody-labeled fluorescence are provided on a cortisol measurement substrate having a metal film on the surface and the cortisol-albumin conjugate of the present invention immobilized on the metal film. The step of contacting the particles and binding the anti-cortisol antibody and the cortisol of the cortisol / albumin conjugate by antigen-antibody reaction (3), and the step of measuring the fluorescence based on the anti-cortisol antibody-labeled fluorescent particles bound to the substrate (4 ),
A method for measuring cortisol in a test sample is provided.

  According to the production method of the present invention, a cortisol / albumin conjugate having high water dispersibility can be produced. By using the cortisol / albumin conjugate produced by the production method of the present invention, a cortisol measurement method, a cortisol measurement substrate, and a cortisol measurement kit that have good reproducibility are provided.

FIG. 1 shows the measurement results of the particle size distribution of the cortisol / BSA conjugate.

Hereinafter, the present invention will be described in more detail.
The present invention is a method for producing a cortisol / albumin conjugate having 7 to 14 cortisol bonds to albumin, comprising the following steps (1) and (2), wherein the molar ratio of the condensing agent to albumin is It is 70-150, It is related with the method whose molar ratio of the cortisol active ester body with respect to albumin is 15-30.
A step (1) of producing a cortisol active ester by reacting a compound obtained by introducing a carboxyl group into cortisol with a condensing agent and a carboxylic acid activating reagent; and reacting albumin with the cortisol active ester Step (2)

(Cortisol)
Cortisol is a glucocorticoid having the following structure:

(albumin)
The type of albumin used in the present invention is not particularly limited. For example, albumin derived from animals (bovine, human, etc.), preferably serum albumin derived from animals (bovine, human, etc.), particularly preferably bovine serum albumin (BSA). ) Etc. can be used.

(Active ester)
In the present invention, a compound obtained by introducing a carboxyl group into cortisol is reacted with a condensing agent and a carboxylic acid activating reagent to produce a cortisol active ester. The compound obtained by introducing a carboxyl group into cortisol is preferably a compound represented by the general formula (1).

In the formula, L ₁ represents a linking group. L ₁ is, for example, a linking group represented by -L ₁₁ -N =, and -L ₁₁ -is an alkylene group having 1 to 6 carbon atoms, -O-, -S-, -NH-, -CO-. Or the group which consists of those combination is represented.

  In the present invention, for example, a cortisol oxime derivative which is a compound obtained by introducing a carboxyl group into cortisol is produced by reacting cortisol with (aminooxy) acetic acid hemihydrochloride represented by the following chemical formula. A condensing agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) or its hydrochloride (EDC.HCl) and a carboxylic acid activity such as N-hydroxysuccinimide (NHS) An active ester can be prepared using a crystallization reagent. An active ester represents an intermediate in which cortisol and EDC or other condensing agents and NHS have reacted, and the amidation reaction that forms this intermediate proceeds in water, so amide bond formation and hydrolysis reaction of NHS active ester Will compete. For this reason, condensing agents such as EDC and carboxylic acid activating reagents such as NHS are usually used in large excess. As a result, a hardly water-soluble aggregate is produced. In order to suppress the formation of this aggregate, the reaction is carried out at a molar ratio of the condensing agent to albumin of 70 to 150, preferably 90 to 130. When the molar ratio of the condensing agent and albumin is 150 or less, the ratio of the finished cortisol / albumin conjugate is 14 or less, and the formation of hardly water-soluble aggregates is suppressed. As a result, good water dispersibility is achieved. It can be secured. When the molar ratio of the condensing agent and albumin is 70 or more, the ratio of the cortisol / albumin conjugate becomes 7 or more, and the immunoassay sensitivity can be maintained.

  In addition, since the molar ratio of the condensing agent to albumin is 70 to 150 and the molar ratio of the cortisol active ester body to albumin is 15 to 30, the molar ratio of the condensing agent to the cortisol active ester body is 7/3 to 3/3. It falls within the range of 10/1.

(Condensing agent)
The condensing agent is a reaction reagent for synthesizing a carboxylic acid derivative by an addition / elimination reaction. A water-soluble carbodiimide can be used as a compound preferably used as a condensing agent. Examples of the water-soluble carbodiimide include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) or a hydrochloride thereof (EDC / HCl), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide-meth- Examples thereof include p-toluenesulfonate, but it is preferable to use EDC or EDC · HCl.

(Carboxylic acid activation reagent)
NHS is preferably used as the carboxylic acid activating reagent. In addition to NHS, for example, the nitrogen-containing compounds described in paragraphs 0056 to 0075 of JP-A-2008-096412, or JP-A-2008-096412 Phenol derivatives having an electron-withdrawing group described in paragraph numbers 0076 to 0077 of the publication can also be used.

(Cortisol / albumin conjugate)
In order to maintain the performance of the immunoassay of cortisol and ensure dispersibility in water or an organic solvent to obtain high reproducibility, in the present invention, the molar ratio of cortisol active ester to albumin is 15 to 30. To prepare a cortisol / albumin conjugate. The molar ratio of cortisol active ester to albumin is preferably 20-25. In this reaction process, hydrolysis of the active ester and formation of a cortisol / albumin conjugate occur simultaneously, and the cortisol / albumin ratio of the finally produced cortisol / albumin conjugate is the molar ratio of the cortisol active ester to albumin. Less than. When the molar ratio of the cortisol active ester to albumin is 30 or less, the ratio of the completed cortisol / albumin conjugate is 14 or less, and it is possible to ensure good dispersibility in water. When the molar ratio of cortisol of the active ester to albumin is 15 or more, the ratio of the cortisol / albumin conjugate is 7 or more, and the immunoassay sensitivity can be maintained.

  In the present invention, the number of binding of cortisol to albumin in the final cortisol / albumin conjugate is 7 to 14, preferably 8 to 13, and more preferably 9 to 13. By setting the number of cortisol bonds to albumin to 14 or less, dispersibility in water or an organic solvent can be ensured, and by setting the number of cortisol bonds to albumin to 7 or more, the performance of immunoassay is maintained. be able to.

The number of binding of cortisol to albumin means the ratio of the number of molecules (number of moles), and means the number of molecules of cortisol bound to one molecule of albumin such as BSA. The number of binding of cortisol to albumin can be determined by, for example, MALDI-TOF-MS, and the specific measurement procedure is as follows. Here, the measurement procedure is described for the case where BSA is used as albumin. . A cortisol / BSA conjugate sample is dissolved in 0.1% by mass trifluoroacetic acid (TFA) aqueous solution: acetonitrile = 2/1 (volume ratio), and the concentration of the cortisol / BSA conjugate is adjusted to 1 mg / mL. Mix 4 μL of matrix (SA; sinapinic acid) and 1 μL of sample, and place 1 μL × 4 dots on a gold plate. The sample is then air dried. Insert a gold plate into a matrix-assisted laser desorption / ionization TOF (Time of Flight) mass spectrometer (MALDI-TOF-MS) (Applied BioSystems Voyager) and perform mass information by performing 900 shots for each spot. Is obtained (N = 4). The position of the peak corresponding to the cortisol / BSA conjugate, which is vertically lowered from the center of gravity of the area having an intensity of 50% or more of the maximum value of the peak intensity, is regarded as the molecular weight of the cortisol / BSA conjugate, N = Taking the average value of 4, the number of cortisol relative to BSA is calculated by (molecular weight of cortisol / BSA conjugate−molecular weight of naive BSA) / molecular weight of cortisol derivative (435-18 = 417).

  As a commercially available cortisol / albumin conjugate, SIGMA catalog number: H-2384 HYDROCORTISONE 3- (O-CALBOXY-METHYL) OXYME: BSA (26 mol steroid / mol BSA) is commercially available. The albumin ratio was 26, and the dispersibility in water or an organic solvent was low, and in the measurement of the present invention, a result with good reproducibility could not be obtained.

  The degree of dispersion of the cortisol / albumin conjugate produced by the production method of the present invention and having a binding number of cortisol to albumin of 7 to 14 is preferably 90% or more. Here, the degree of dispersion is measured as follows. That is, 5 mg of cortisol / albumin conjugate, which does not precipitate when dispersed in water, is added to 1 mL of phosphate buffer (PBS, Wako Pure Chemical Industries, Ltd., catalog number: 166-23555, pH 7.1-7). .3) is collected and centrifuged, and the supernatant is collected, and the absorbance at 280 nm is measured at 25 ° C. using SHIMADZU (Shimadzu Corporation) UV-visible spectrophotometer UV-2550. Based on this absorbance, a value expressed as a percentage as the ratio of the absorbance measured in the same manner for each prepared cortisol / albumin conjugate is obtained as the degree of dispersion. The degree of dispersion of the preferred cortisol / albumin conjugate is 90% or more, and more preferably 95% to 100%.

  The cortisol / albumin conjugate of the present invention may be one in which cortisol and albumin are directly bonded, or may be bonded through an appropriate linking group. The thing which is couple | bonded through can be mentioned. For example, as shown in the examples described later, a compound obtained by introducing a carboxyl group into cortisol is produced, and this carboxyl group is activated using EDC, NHS, etc., and bonded to the amino group of albumin. Cortisol / albumin conjugates can be produced.

(Measuring method of particle size distribution)
The particle size distribution of the cortisol / albumin conjugate of the present invention can be evaluated as a coefficient of variation obtained by dividing the standard deviation by the average particle size from the average particle size and standard deviation of the cortisol / albumin conjugate. As an apparatus for measuring the coefficient of variation, it is preferable to use a dynamic light scattering method because of the particle size range and ease of measurement. As a commercially available measuring device using dynamic light scattering, Nanotrac UPA (made by Nikkiso Co., Ltd.), dynamic light scattering type particle size distribution measuring device LB-550 (made by HORIBA, Ltd.), concentrated particles Diameter analyzer FPAR-1000 (made by Otsuka Electronics Co., Ltd.), ZETA SIZER (made by Malvern), etc. are mentioned. In the present invention, ZETA
Using SIZER (manufactured by Malvern), diluted with PBS (800 μL Wako Pure Chemical Industries, Ltd. product catalog number: 166-23555, pH 7.2) so that the albumin concentration is 1 mg / mL, and at 25 ° C. From the median diameter (d = 50) measured under the conditions of viscosity 0.8872 CP and water refractive index 1.330, the average particle diameter and standard deviation are measured, and the coefficient of variation is calculated from the results. Regarding the cortisol / albumin conjugate of the present invention, the coefficient of variation is preferably small, and specifically, it is preferably 1.0 or less.

(Method for measuring cortisol in test sample)
According to the present invention, a test sample containing cortisol and an anti-cortisol antibody-labeled fluorescent particle are brought into contact with the cortisol measurement substrate on which the cortisol / albumin conjugate of the present invention is immobilized, and an anti-antibody reaction is carried out. A step of binding the cortisol antibody and cortisol of the cortisol / albumin conjugate (3), and a step of measuring fluorescence based on the anti-cortisol antibody-labeled fluorescent particles bound to the substrate (4),
A method for measuring cortisol in a test sample is provided.

(substrate)
According to the present invention, a substrate for cortisol immunoassay is provided by immobilizing a cortisol-albumin conjugate having 7 to 14 cortisol bonds to albumin on the substrate. The type of the substrate is not particularly limited as long as surface plasmon fluorescence measurement can be performed, and any substrate can be used. As described later, a metal film formed on the surface of the substrate is used. By immobilizing the cortisol / albumin conjugate on the opposite surface, it is possible to obtain a cortisol immunoassay substrate used for surface plasmon fluorescence detection. Cortisol / albumin conjugate is dispersed in a buffer solution, spotted on a substrate, preferably a metal film formed on the substrate, left for a certain period of time, and then the supernatant is sucked and then dried. The method allows bonding to a substrate, preferably a metal film formed on the substrate.

  As a substrate in the case of performing surface plasmon fluorescence measurement, it is preferable to use a substrate having a metal film on the surface. The metal constituting the metal film is preferably a metal that can cause surface plasmon resonance, and specifically includes metals such as gold, silver, copper, aluminum, platinum, among which gold is particularly preferable. preferable. These metals can be used alone or in combination. The thickness of the metal film here is preferably such that it can cause surface plasmon. Specifically, the thickness is preferably in the range of 0.1 nm to 500 nm, and more preferably in the range of 1 nm to 200 nm. If it is 500 nm or less, the surface plasmon phenomenon of the medium can be sufficiently detected. In consideration of adhesion to the substrate, an intervening layer made of chromium or the like may be provided between the substrate and the metal layer so as not to affect the surface plasmon resonance generated in the metal film. Is possible. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably in the range of 0.1 nm or more and 10 nm or less.

  The metal film can be formed by an ordinary method, for example, a sputtering method, a vapor deposition method, an ion plating method, an electroplating method, an electroless plating method, or the like. Among these, it is preferable to form the metal film by a sputtering method in order to achieve good adhesion to the substrate with which the metal film contacts or the material of the intervening layer.

  Examples of the material of the substrate that can be used in the present invention include optical glass such as BK7 (borosilicate glass), which is a kind of general optical glass, or a synthetic material when considering application to a surface plasmon resonance biosensor. Resins, specifically, those made of a material transparent to laser light, such as polymethyl methacrylate (PMMA), polyethylene terephthalate, polycarbonate, cycloolefin polymer, can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  As an example of the substrate for SPF detection, a substrate in which a gold film is provided on PMMA by a sputtering method can be given as a specific embodiment.

(Fluorescent particles)
In the present invention, the above-mentioned cortisol immunoassay substrate is brought into contact with a test sample containing cortisol and anti-cortisol antibody-labeled fluorescent particles, and then the anti-cortisol antibody-labeled fluorescent particles bound to the cortisol / albumin conjugate on the substrate. By measuring the fluorescence to be measured, cortisol in the test sample can be measured.

  As the fluorescent particles that can be used in the present invention, fluorescent colored particles are preferably used. For example, fluorescent polymer particles such as fluorescent polystyrene beads and fluorescent glass particles such as fluorescent glass beads can be used. Specific examples of the material of the fluorescent particles include polymers using monomers such as styrene, methacrylic acid, glycidyl (meth) acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, and butyl methacrylate. There are synthetic polymer powders such as copolymers using two or more monomers, and latexes in which these are uniformly suspended are preferred. Other organic polymer powders and inorganic substance powders, microorganisms, blood cells, cell membrane fragments, liposomes and the like can be mentioned.

  When latex particles are used, specific examples of latex materials include polystyrene, styrene-acrylic acid copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, and styrene-styrene sulfonic acid. Examples thereof include a salt copolymer, a methacrylic acid polymer, an acrylic acid polymer, an acrylonitrile-butadiene-styrene copolymer, a vinyl chloride-acrylic acid ester copolymer, and a polyvinyl acetate acrylate. As the latex, a copolymer containing at least styrene as a monomer is preferable, and a copolymer of styrene and acrylic acid or methacrylic acid is particularly preferable. The method for producing the latex is not particularly limited, and the latex can be produced by any polymerization method. However, when a surfactant is present at the time of antibody labeling, antibody immobilization becomes difficult. Therefore, non-emulsifier emulsion polymerization, that is, emulsion polymerization without using an emulsifier such as a surfactant, is preferable for producing the latex.

  When the latex itself obtained by polymerization is fluorescent, it can be used as fluorescent latex particles as it is. When the latex obtained by polymerization is non-fluorescent, fluorescent latex particles can be produced by adding a fluorescent substance (fluorescent dye or the like) to the latex. That is, the fluorescent latex particles can be produced by adding a fluorescent dye to a latex particle solution containing water and a water-soluble organic solvent and stirring the solution.

  Liposomes and microcapsules containing fluorescent dyes can also be used as fluorescent particles. Fluorescent color development is not particularly limited as long as it is excited by absorbing ultraviolet light or the like and emitted upon returning to the ground state. For example, yellow-green (excitation wavelength 505 nm / emission wavelength 515 nm, the same applies hereinafter) ), Blue (350-356 nm / 415-440 nm), red (535-580 nm / 575-605 nm), orange (540 nm / 560 nm), red orange (565 nm / 580 nm), crimson (625 nm / 645 nm), dark red ( Fluorescence development such as 660 nm / 680 nm) can be used. These fluorescent particles that emit fluorescence are available from, for example, Invitrogen, and are commercially available under the trade name of FluoSpheres (registered trademark).

(Measuring method of average particle diameter of fluorescent particles)
The average particle diameter of the fluorescent particles varies depending on the particle material, the concentration range for quantifying the test substance, the measuring instrument, etc., but the range of 0.001 to 10 μm (more preferably 0.001 to 1 μm) is preferable. The average particle diameter of the fluorescent particles that can be used in the present invention can be measured with a commercially available particle size distribution meter or the like. Measurement methods of particle size distribution include optical microscopy, confocal laser microscopy, electron microscopy, atomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, electric pulse Measurement methods, chromatography methods, ultrasonic attenuation methods, and the like are known, and devices corresponding to the respective principles are commercially available. Among these measurement methods, it is preferable to measure the average particle diameter of the fluorescent particles using a dynamic light scattering method because of the particle diameter range and ease of measurement. As a commercially available measuring device using dynamic light scattering, Nanotrac UPA (Nikkiso Co., Ltd.), dynamic light scattering type particle size distribution measuring device LB-550 (Horiba, Ltd.), a concentrated particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.) etc. are mentioned. In the present invention, the average particle diameter is determined as a median diameter (d = 50) measured at 25 ° C. under conditions of a viscosity of 0.8872 CP and a refractive index of water of 1.330.

(Anti-cortisol antibody)
As the anti-cortisol antibody to be bound to the fluorescent particles, an antibody having specificity for cortisol can be used. Examples of the anti-cortisol antibody include antiserum prepared from the serum of an animal immunized with cortisol, an immunoglobulin fraction purified from the antiserum, and a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with cortisol. An antibody or a fragment thereof [for example, F (ab ′) 2, Fab, Fab ′, or Fv] and the like can be used. These antibodies can be prepared by a conventional method. Furthermore, the antibody may be modified as in the case of a chimeric antibody, or may be a commercially available antibody or an antibody prepared from animal serum or culture supernatant by a known method.

  The antibody can be used regardless of the animal species or subclass. For example, antibodies that can be used in the present invention are antibodies derived from organisms that can cause an immune reaction such as mice, rats, hamsters, goats, rabbits, sheep, cows, chickens, specifically mouse IgG, mouse IgM. Rat IgG, rat IgM, hamster IgG, rabbit IgG, rabbit IgM, goat IgG, goat IgM, sheep IgG, sheep IgM, bovine IgG, bovine IgM, avian IgY, etc., applicable to both polyclonal and monoclonal . A fragmented antibody is a molecule derived from a complete antibody having at least one antigen-binding site, and specifically, Fab, F (ab ') 2, and the like. These fragmented antibodies are molecules obtained by enzyme or chemical treatment or using genetic engineering techniques.

  Methods for immobilizing binding substances such as antibodies and antigens on particles are described in, for example, the protocol attached to JP 2000-206115 A and Molecular Probes FluoSpheres (registered trademark) polystyrene microsphere F8813. Any known method for preparing a reagent for immunoaggregation reaction can be used. In addition, as a principle for immobilizing an antibody as a binding substance on a particle, any of physical adsorption and chemical bonding by covalent bond can be adopted. As a blocking agent that covers the particle surface that is not coated with the antibody after the antibody is fixed to the particle, known substances such as BSA (bovine serum albumin), skim milk, casein, soybean-derived component, fish-derived component, polyethylene glycol, etc. In addition, a commercially available blocking agent for immune reaction containing these substances or substances having the same properties as these can be used. These blocking agents can be subjected to pretreatment such as partial modification with heat, acid, alkali, or the like, if necessary.

  A specific method for immobilizing the antibody on the particles is exemplified below. An antibody aqueous solution adjusted to a concentration of 0.01 to 20 mg / mL is added to and mixed with a liquid dispersed in water so that the solid content concentration of the particles becomes 0.1 to 10% by mass. Stirring is continued for 5 minutes to 48 hours under conditions of a temperature of the aqueous dispersion of 4 to 50 ° C. Subsequently, the particles and the solution are separated by centrifugation or other methods to sufficiently remove the antibodies that have not been bound to the particles and remain in the solution. Thereafter, the operation of washing the particles with a buffer solution is repeated 0 to 10 times. After performing the operation of mixing the particles and the antibody and binding the antibody to the particles, a component that does not participate in the antigen-antibody reaction, preferably a protein, more preferably BSA (bovine serum albumin), Block Ace (registered trademark), It is desirable to use a blocking agent such as skim milk and casein to protect the unbound portion of the particle surface antibody.

  When immobilizing antigens or antibodies on the particles, stabilizers can be added as necessary. The stabilizer is not particularly limited as long as it stabilizes an antigen or antibody, such as a synthetic or natural polymer such as sucrose or polysaccharide, and a commercially available one such as Immunoassay Stabilizer (ABI) can also be used. Is

(Measuring method)
The measurement method of the present invention is interpreted as the broadest concept including detection of the presence or absence of cortisol, measurement of the amount of cortisol (ie, quantification), and the like. A specific embodiment of the measurement method of the present invention is a competition method.

  In the competitive method, first, a test sample containing cortisol and anti-cortisol antibody-labeled fluorescent particles are placed on a cortisol immunoassay substrate on which a cortisol-albumin conjugate having a binding number of cortisol of 7 to 14 is immobilized. Make contact. When cortisol is not present in the test sample, an anti-cortisol antibody-labeled fluorescent particle and cortisol on the substrate (that is, cortisol in the cortisol / albumin conjugate) cause an antigen-antibody reaction on the substrate. On the other hand, when cortisol is present in the test sample, an antigen-antibody reaction occurs between the cortisol in the test sample and the anti-cortisol antibody-labeled fluorescent particles, and the anti-cortisol antibody-labeled fluorescent particles and the cortisol on the substrate The antigen-antibody reaction with (that is, cortisol in the cortisol / albumin conjugate) is inhibited. After the above reaction is completed, anti-cortisol antibody-labeled fluorescent particles that have not bound to albumin on the substrate are removed. Next, a test sample is detected by detecting the degree of formation of an immune complex on the substrate (ie, a complex of anti-cortisol antibody-labeled fluorescent particles and cortisol in the cortisol / albumin conjugate on the substrate) as fluorescence intensity. The concentration of cortisol in the medium can be measured.

  In the competition method, the form of fluorescence measurement may be a plate reader measurement or a flow measurement. For example, the measurement can be performed by the following method. A plurality of samples with known cortisol amounts having different cortisol concentrations are prepared in advance, and this sample and anti-cortisol antibody-labeled fluorescent particles are mixed in advance. This mixed solution is brought into contact with the region where the cortisol / albumin conjugate is immobilized. The fluorescence signal from the region where the cortisol / albumin conjugate is immobilized is measured while the mixed solution is in contact with the conjugate at a specific time interval. From the plurality of fluorescence signals, the temporal change (slope) of the fluorescence amount is obtained at each cortisol concentration. This time change is plotted as the Y axis and the cortisol concentration is plotted as the X axis, and a relational expression of the cortisol concentration with respect to the time change of the fluorescence amount is obtained by using an appropriate fitting method such as a least square method. Based on the relational expression thus obtained, the amount of cortisol contained in the test sample can be quantified using the result of temporal change in the fluorescence amount using the test sample to be examined.

  The measurement by the competitive method is a preferable measurement method because a result can be obtained in a short time. In addition, the measurement time from the time when the test sample and anti-cortisol antibody-labeled fluorescent particles mixed in advance to the region where the cortisol / albumin conjugate is immobilized is contacted is determined It is preferably within 10 minutes, more preferably within 8 minutes, and even more preferably within 6 minutes.

(Surface plasmon fluorescence measurement)
Fluorescence detection methods include, for example, fluorescence using an apparatus capable of detecting fluorescence intensity, specifically, a microplate reader or a biosensor used for fluorescence detection method (SPF) by surface plasmon excitation. It is preferable to detect the intensity. Since the fluorescence detection method (SPF) by surface plasmon excitation can be measured with higher sensitivity than the epifluorescence method, it is a more preferable measurement method.

  The biosensor used in the fluorescence detection method (SPF) by surface plasmon excitation described above is formed of a material that transmits excitation light of a predetermined wavelength as described in, for example, Japanese Patent Application Laid-Open No. 2008-249361. An optical waveguide, a metal film formed on one surface of the optical waveguide, a light source for generating a light beam, and passing the light beam through the optical waveguide to generate surface plasmons at the interface between the optical waveguide and the metal film It is possible to use a sensor that includes an optical system that is incident at an incident angle and a fluorescence detection unit that detects fluorescence generated by excitation by an evanescent wave enhanced by surface plasmons.

  Fluorescence detection method (SPF) by surface plasmon excitation using fluorescent particles is an assay method for detecting fluorescence from a fluorescent substance depending on the amount of a test substance immobilized on a metal thin film on a substrate. This method is different from the so-called latex agglutination method, in which a change in optical transparency is detected as turbidity as the reaction proceeds in solution. The latex agglutination method uses the property that antibody-sensitized latex in latex reagents and antigens in specimens bind and aggregate due to antibody reaction, and the size of aggregation that increases with time is irradiated with near infrared light. In this method, the antigen concentration is quantified by measuring the change in absorbance per unit time. The fluorescence detection method (SPF) by surface plasmon excitation can provide a detection method of a test substance that is very simple and highly sensitive as compared with the latex agglutination method.

(Cortisol measurement board and kit)
According to the present invention, a kit for measuring cortisol comprising the cortisol / albumin conjugate of the present invention is provided. The kit of the present invention is used in a method for measuring cortisol, and is a cortisol measurement substrate using a cortisol / albumin conjugate prepared by the production method of the present invention and a cortisol measurement kit using the same. In carrying out cortisol measurement in the present invention, it includes a substrate on which a cortisol / albumin conjugate is fixed, a member holding fluorescent particles, and is used for measuring cortisol, such as a surface plasmon excitation device and a fluorescence measurement device. Various kinds of equipment, materials or equipment can also be included. Furthermore, as one element of the kit, a set of necessary materials, such as a test sample whose test substance amount is known and an instruction manual, may be included.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1
(1) Preparation of Cortisol / BSA Conjugate (1-1) Synthesis of Cortisol-Oxime Derivative 1 Cortisol is enaminated and reacted with a hydroxylamine derivative according to the literature (STEROIDS, 1974, Jan. 49-64). -Oxime derivative 1 was obtained. The reaction route is shown below.

  One equivalent of pyrrolidine (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to a methanol (5 mL) suspension of cortisol (1 g, manufactured by Wako Pure Chemical Industries, Ltd.) and stirred at room temperature for 5 minutes. Enamine was formed in the reaction system, and the solution turned red. Thereafter, 1 equivalent of pyrrolidine and (aminooxy) acetic acid hemihydrochloride (0.66 g, manufactured by Wako Pure Chemical Industries, Ltd.) represented by the above chemical formula were added, and the reaction temperature was stirred at 55 ° C. for 5 minutes. Then, oximation was performed. Oxidation proceeded while gradually erasing the solution, and cortisol-oxime derivative 1 (1.1 g: 84%, a compound obtained by introducing a carboxyl group into cortisol of the present invention) was obtained.

(1-2) Preparation of Cortisol / BSA Conjugate-1 To water-free dehydrated DMF (dehydrated NN-dimethylformamide, 1.2 mL, manufactured by Wako Pure Chemical Industries, Ltd.), (1-1) Cortisol-oxime derivative 1 (50 mg) and NHS (67 mg, manufactured by Wako Pure Chemical Industries, Ltd.) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl, 110 mg sum) Koto Pure Chemical Industries, Ltd.) was dissolved and reacted to obtain an active esterified cortisol-oxime derivative 2. Thereafter, a phosphate buffer (PB) of BSA (bovine serum albumin, manufactured by Wako Pure Chemical Industries, Ltd., 322 mg), which is a kind of albumin, is used.
S, Wako Pure Chemical Industries, Ltd., product catalog number: 166-23555, pH 7.1-7.3) Cortisol oxime derivative 2 that was activated esterified to 65 mL was added dropwise. After completion of the reaction, acetonitrile (manufactured by Wako Pure Chemical Industries, Ltd.) / Water = 1/1 (volume ratio) (in water, 0.1% by mass trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, Ltd.) The product was purified by dialysis using 1 L of the solution containing). After purification by dialysis, lyophilization was performed to obtain a white solid cortisol / BSA conjugate-1.

(1-3) Preparation of Cortisol / BSA Conjugate-2 Prepared with (1-1) in water-free dehydrated DMF (N, N-dimethylformamide, 9 mL, manufactured by Wako Pure Chemical Industries, Ltd.) Cortisol-oxime derivative 1 (389 mg) and NHS (523 mg, manufactured by Wako Pure Chemical Industries, Ltd.) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC / HCl, 855 mg, Wako Pure Chemical) Kogyo Co., Ltd.) was dissolved and reacted to obtain an active esterified cortisol-oxime derivative. Thereafter, active ester was added to 570 mL of PBS (Wako Pure Chemical Industries, Ltd., product catalog number: 166-23555) of BSA (bovine serum albumin, manufactured by Wako Pure Chemical Industries, Ltd., 2.85 g) which is a kind of albumin. All the converted cortisol oxime derivatives were added dropwise. After completion of the reaction, a solution of acetonitrile (manufactured by Wako Pure Chemical Industries, Ltd.) / Water = 1/1 (volume ratio, containing 0.1 mass% TFA (made by Wako Pure Chemical Industries, Ltd.) in water) Purification was performed by dialysis using 1 L. After purification by dialysis, lyophilization was performed to obtain a white solid cortisol / BSA conjugate-2.

(1-4) Preparation of Cortisol / BSA Conjugate-3 To water-free dehydrated DMF (9 mL), cortisol-oxime derivative 1 (389 mg) prepared in (1-1), NHS (523 mg) and 1-ethyl -3- (3-Dimethylaminopropyl) carbodiimide hydrochloride (ED
C.HCl, 855 mg) was dissolved and reacted to obtain an active esterified cortisol-oxime derivative. Thereafter, the entire amount of cortisol oxime derivative esterified with active ester was added dropwise to 570 mL of PBS of BSA (1.5 g) which is a kind of albumin. After completion of the reaction, purification was performed by dialysis using 1 L of a solution of acetonitrile / water = 1/1 (volume ratio, containing 0.1% by mass of TFA in water). After purification by dialysis, lyophilization was performed to obtain a white solid cortisol / BSA conjugate-3.

(1-5) Preparation of Cortisol / BSA Conjugate-4 to 7 Similar to the preparation of Cortisol / BSA Conjugate-3, the molar ratio of EDC / BSA is 30, 60, 240, 300. Cortisol / BSA conjugates 4 to 7 were prepared by changing the ratio of EDC / HCl and BSA.

(1-6) Measurement of the number of cortisol bonds to albumin (BSA) by MALDI-TOF-MS (measurement procedure)
Cortisol and BSA conjugates -1 to 5 prepared in (1-2) to (1-5) were dissolved in a solution of 0.1% by mass TFA: acetonitrile = 2/1 (volume ratio), and 1 mg / mL The concentration was adjusted. 1 μL of this solution and 4 μL of matrix (SA; sinapinic acid) were mixed, and 1 μL of the obtained mixed solution was spotted four times on a gold plate. After natural drying, a gold plate was inserted into a MALDI-TOF-MS apparatus (Applied Bio Systems Voyager), and 900 shots were integrated for each spot to obtain data as mass information (N = 4). Using this data, the molecular weight center value of the peak corresponding to the cortisol / BSA conjugate at 50% intensity of the maximum peak intensity was adopted as the peak of the BSA conjugate, and the peak value was lowered vertically from this peak value. The position was regarded as the molecular weight of the cortisol / BSA conjugate, and an average value of N = 4 was obtained. The number of cortisol bonds to BSA was calculated using (molecular weight of cortisol / BSA conjugate-molecular weight of native BSA) / molecular weight of cortisol derivative (435-18 = 417). Table 1 shows the cortisol / BSA ratio of the obtained conjugates-1, 2, and 3.

(1-7) Measurement of degree of dispersion of cortisol / BSA conjugate-1 to 7 in water The white solids of cortisol / BSA conjugate-1 to 7 prepared in (1-2) to (1-5) 5 mg was measured and dissolved in 1 mL of PBS (Wako Pure Chemical Industries, Ltd., product catalog number: 166-23555). The mixture was lysed with a vortex mixer for 20 minutes to dissolve and then centrifuged (15,000 rpm, 10 minutes, 4 ° C.) to settle insoluble matters and the supernatant was sampled. The sampled supernatant was placed in a 1 cm quartz cell, and the absorbance was measured using a UVIMAV spectrophotometer UV-2550 manufactured by SHIMADZU (Shimadzu Corporation).
As a standard sample, 1 mg of sampled sample was dissolved in 1 mL of PBS using cortisol / BSA conjugate-1, and centrifuged (15,000 rpm, 10 minutes, 4 ° C.) to precipitate insoluble matter. The supernatant was sampled. The sampled supernatant was placed in a 1 cm quartz cell and the absorbance was measured. From these data, when the absorbance at 280 nm is 0.72, the cortisol / BSA conjugate is defined as 1 mg / 1 mL, and the concentration of cortisol / BSA conjugate in the supernatant of cortisol / BSA conjugate-2 to 7 is calculated. Then, the degree of dispersion was calculated according to the following equation.
Dispersity: (cortisol / BSA conjugate concentration in the supernatant mg / mL (quantitative from absorbance) / 5) × 100

(1-8) Measurement of particle size distribution of cortisol / BSA conjugate Regarding the supernatant of cortisol / BSA conjugate-1 and -3 obtained in (1-7) and unbound BSA, ZETA SIZER (Malvern) The particle size distribution was measured with Nano series. The sample was diluted with PBS (800 μL Wako Pure Chemical Industries, Ltd. product catalog number: 166-23555) so that the BSA concentration was 1 mg / mL, and at 25 ° C., the viscosity was 0.8872 CP, and the refractive index of water was 1. Measured at .330 conditions. The measurement results are shown in FIG.
From the results of FIG. 1, it can be seen that the dispersibility of the cortisol / BSA conjugate-1 is good and the particle size distribution of the cortisol / BSA conjugate-3 is widely aggregated.

(2) Preparation of fluorescent particles labeled with anti-cortisol antibody Fluorescent particles labeled with an anti-cortisol antibody were prepared as follows.
2% by mass (solid content concentration) fluorescent latex particle aqueous solution (Invitrogen, average particle size 200 nm) 250 μL, 50 mmol / L 2-morpholinoethanesulfonic acid monohydrate (MES, Wako Pure Chemical Industries, Ltd.) 250 μL of buffer (pH 6.0) solution was added, and 5 mg / mL anti-cortisol monoclonal antibody (manufactured by Hytest; Anti-Cortisol)
MAb XM210) 100 μL was added and stirred at room temperature for 15 minutes. Thereafter, 5 μL of 10 mg / mL EDC / HCl aqueous solution was added, and the mixture was stirred at room temperature for 2 hours. After adding 25 μL of 2 mol / L aqueous solution of Glycine (Wako Pure Chemical Industries, Ltd.) and stirring for 30 minutes, centrifugation (15,000 rpm, 4 ° C., 15 minutes) is performed to precipitate fluorescent latex particles. It was. Thereafter, the supernatant was removed, 500 μL of PBS (Wako Pure Chemical Industries, Ltd. product catalog number: 166-23555) was added, and the fluorescent latex particles were redispersed with an ultrasonic cleaner. After centrifugation again (15,000 rpm, 4 ° C., 15 minutes) to remove the supernatant, 500 μL of PBS (Wako Pure Chemical Industries, Ltd. product catalog number: 166-23555) containing 1% by mass BSA was added. In addition, a 1% by weight dispersion of anti-cortisol antibody-bound fluorescent latex particles was prepared by redispersing the fluorescent latex particles.

(3) Fabrication of flow path type sensor chip (3-1) Fabrication of substrate A polymethylmethacrylate (PMMA) substrate (manufactured by Mitsubishi Rayon Co., Ltd., Acrypet VH) is prepared, and the thickness is measured by magnetron sputtering. A 40-nm gold film was produced on one side of the substrate so as to have a width of 4 mm, thereby producing a chip for constituting the substrate. A liquid containing cortisol / BSA conjugate-1 (Example), cortisol / BSA conjugate-2 (Example 2), or cortisol / BSA conjugate-3 (Comparative Example 1) on the gold film surface of the chip. (Concentration: 50 μg / mL in 50 mmol / L MES buffer pH 6, 150 mmol / L NaCl) was spotted and dried to prepare a plurality of substrates 1 to 3 on which each conjugate was immobilized.

(3-2) Substrate cleaning Before attaching the three types of substrates prepared in this way to the flow path of the sensor chip, a cleaning solution (0.05 mass% Tween 20 (polyoxyethylene (20) prepared in advance) was prepared in advance. Washing was repeated three times using 300 μL of a PBS solution (pH 7.1 to 7.3) containing sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.).

(3-3) Fabrication of flow path type sensor chip In order to obtain the configuration of the second embodiment of Japanese Patent Application Laid-Open No. 2010-190880, a plurality of fabricated types of substrates are sealed in the flow path, and the flow path type A sensor chip was produced.

(4) Measurement with a fully automatic immunochemiluminescence measuring device Using a Siemens IMMLYZE1000 fully automatic immunochemiluminescence measuring device, which is a large machine widely used by those skilled in the art for immunoassay, test according to the instruction manual. A test sample with a known substance was measured to obtain a measured value of the test substance.

(5) Immunoassay of cortisol using fluorescent particles The test sample with the known test substance used in (4) is stirred for 10 minutes in advance with the fluorescent particles labeled with the anti-cortisol antibody prepared in (2). Mixed. Next, each of the flow path type sensor chips enclosing the substrates 1 to 3 prepared in (3-3) was spotted. After spotting, the liquid mixture is allowed to flow down at a rate of 10 μL / min while pumping, and the liquid intensity is continued for 1.5 minutes after contacting the gold film surface on which the cortisol / BSA conjugate is fixed. Was measured. The rate of increase of the fluorescence intensity obtained in each substrate per unit time was determined as the fluorescence signal value. In addition, a sample with a cortisol concentration of 0 was prepared, and the fluorescence signal value obtained in the same manner was B0, and when the signal value of each sample containing cortisol was B, the signal value was standardized by obtaining B / B0. Turned into.

(6) Preparation of calibration curve From (1-2), using the fluorescence signal value of the test sample with the known test substance determined in (5) and the measured value of the test substance determined in (4) Calibration curves were prepared for the substrates using the cortisol / BSA conjugates -1 to 3 prepared in (1-4).

(7) Confirmation of reproducibility The test sample M having a known test substance amount and the substrate using the cortisol / BSA conjugates 1-3 prepared in (1-2) to (1-4) H (M is a cortisol concentration of 6.0 to 17.0 μg / dL. H was selected from those having a cortisol concentration of 17.0 to 30.0 μg / dL. These values were determined with the control device IMMULYZE1000. 10) each was prepared, and the same measurement was repeated 10 times to confirm reproducibility. The standard deviation (CV) of each result was expressed as a percentage. The results are shown in Table 3.

  It can be seen that the repetitive reproducibility when measured with the flow path type sensor chip in which the cortisol / BSA conjugate-1 or -2 of the present invention is immobilized is excellent, and the accuracy of the cortisol measurement is high, and the effect of the present invention is confirmed. It was.

(Example 2)
(8) Preparation of test sample As the serum for dogs, the serum of Toyo Beagle dog purchased from Kitayama Labes Co., Ltd. was used, and two test samples were prepared.

(9) Immunoassay of cortisol using fluorescent particles For each of the test samples (dog serum) No. 1 and No. 2 prepared in (8), 20 μL of serum is subdivided and diluted with 80 μL of physiological saline, and 50 μmol of magnesium chloride is sufficient. A plurality of mixed samples 1 and 2 were prepared. This mixed sample was mixed with the fluorescent particles labeled with the anti-cortisol antibody prepared in (2) of Example 1 with stirring for 10 minutes. Next, each of the flow path type sensor chips enclosing the substrates 1 to 3 produced in (3-3) of Example 1 was spotted. After spotting, the liquid mixture is allowed to flow down at a rate of 10 μL / min while pumping, and the liquid intensity is continued for 1.5 minutes after contacting the gold film surface on which the cortisol / BSA conjugate is fixed. Was measured. The rate of increase of the fluorescence intensity obtained in each substrate per unit time was determined as the fluorescence signal value. In addition, a sample with a cortisol concentration of 0 was prepared, and the fluorescence signal value obtained in the same manner was B0, and when the signal value of each sample containing cortisol was B, the signal value was standardized by obtaining B / B0. Turned into. In the same manner as in Example 1, a calibration curve was prepared in consideration of the dilution rate, and the amount of cortisol contained in the dog serum was determined. Similar to Example 1, these measurements were repeated 10 times to confirm reproducibility. The results are shown in Table 4.

  Similar to Example 1, even in measurement using canine serum, the reproducibility is excellent when measured with a flow path type sensor chip on which Cortisol / BSA conjugate-1 or -2 of the present invention is immobilized, It was found that the accuracy of cortisol measurement was high, and the effect of the present invention was confirmed.

Claims (12)

A step (1) of producing a cortisol active ester by reacting a compound obtained by introducing a carboxyl group into cortisol with a condensing agent and a carboxylic acid activating reagent;
A step of reacting albumin with the cortisol active ester (2),
A method for producing a cortisol / albumin conjugate having a binding number of cortisol to albumin of 7 to 14, comprising:
The method for producing a cortisol / albumin conjugate, wherein the cortisol active ester body to albumin has a molar ratio of 15 to 30 and the condensing agent to albumin has a molar ratio of 70 to 150. The manufacturing method of Claim 1 whose compound obtained by introduce | transducing a carboxyl group into the said cortisol is a compound represented by General formula (1).

In the formula, L ₁ represents a linking group. The manufacturing method of Claim 1 or 2 whose number of coupling | bonding of cortisol with respect to albumin is 8-13. The manufacturing method as described in any one of Claim 1 to 3 whose molar ratio of the said condensing agent with respect to the said albumin is 90-130. The manufacturing method as described in any one of Claims 1-4 whose molar ratio of the said cortisol active ester body with respect to the said albumin is 20-25. The manufacturing method according to any one of claims 1 to 5, wherein the condensing agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or a hydrochloride thereof. The production method according to claim 1, wherein the carboxylic acid activating reagent is N-hydroxysuccinimide. A cortisol / albumin conjugate produced by the method according to claim 1. The cortisol / albumin conjugate according to claim 8, which has a dispersity of 90% or more when 5 mg of cortisol / albumin conjugate is dissolved in 1 mL of phosphate buffer. A substrate for cortisol measurement, comprising a metal film on the surface, wherein the cortisol / albumin conjugate according to claim 8 or 9 is immobilized on the metal film. A kit for cortisol measurement, comprising the cortisol measurement substrate according to claim 10 and anti-cortisol antibody-labeled fluorescent particles. A step of bringing a test sample containing cortisol and anti-cortisol antibody-labeled fluorescent particles into contact with the cortisol-measuring substrate according to claim 10 and binding the anti-cortisol antibody and cortisol of the cortisol-albumin conjugate by an antigen-antibody reaction. (3) and a step of measuring fluorescence based on the anti-cortisol antibody-labeled fluorescent particles bound to the substrate (4),
A method for measuring cortisol in a test sample.

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