Sample solution preparation method, measurement unit, measurement kit, and preparation device

Abstract

A sample solution preparation method, a measurement unit, a measurement kit, and a sample solution preparation device for measuring a target compound contained in a food by using a fluorescent immunoassay method. The features of the present disclosure include a dialysis step for bringing a target compound-containing solution including a target compound into contact with a dialysate through a dialysis membrane to transfer the target compound to the dialysate, wherein the molecular weight cut-off of the dialysis membrane is in the range of 2 x 10 2 to 2 x 10 5 , and the volume of the dialysate is relatively smaller than the volume of the target compound-containing solution.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of Japanese Patent Application No. 2020-068486, filed on April 6, 2020, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This disclosure relates to a sample solution preparation method, a measurement unit, a measurement kit, and a sample solution preparation apparatus for measuring the concentration of a target compound contained in food using a fluorescence immunoassay method. Background Technology

[0004] Fluorescence polarization immunoassay (FPIA) is a known immunoassay method that uses fluorescence and antigen-antibody reactions to detect target substances. Unexamined Japanese Patent Application Publication No. H03-103765 ​​describes the principle of FPIA, in which a sample is irradiated with excitation light, and among the polarization components emitted from the sample, the polarization component parallel to the incident light and the polarization component perpendicular to the incident light are measured, and the degree of polarization is calculated using both polarization components. Since the degree of fluorescence polarization is proportional to the effective volume of the target substance, this value increases with the molecular weight of the substance. Therefore, the target substance preferably has a low molecular weight. Unexamined Japanese Patent Application Publication No. H11-503521 (translation of a PCT application) discloses a method for measuring vancomycin by FPIA, which avoids measurement errors caused by the mixing of vancomycin analogs by setting vancomycin, whose molecular weight is lower than that of the antigen protein, as the target substance and preparing a monoclonal antibody that specifically binds to vancomycin.

[0005] Substances present in food and causing problems based on their concentrations include mycotoxins, shellfish toxins, chemicals used in animal feed, and chemicals used in crop cultivation. Preventing food poisoning and assessing agricultural chemical residues in food are crucial for ensuring food safety. Deoxynivalenol (DON), a mycotoxin that infects wheat and other grains, is routinely measured by liquid chromatography-tandem mass spectrometry (LC / MS); however, if a corresponding antibody can be prepared, it can be measured using FPIA, similar to vancomycin. While blood vancomycin concentrations are measured in a laboratory specializing in medical sample processing after blood sampling, food contamination is preferably measured in situ. Because food contains high levels of fat, sugar, protein, and other impurities, it is necessary to extract mycotoxin-containing components from various foods and prepare samples sufficiently concentrated for measurement by FPIA. Furthermore, target compounds considered problematic under food safety laws are not limited to mycotoxins but also include agricultural chemical residues. Additionally, in livestock and fisheries, there are products claiming to be free of antibiotics and antimicrobials, marketed under the slogan of "healthy farming." Equally important is measuring the concentrations of antibiotics and antimicrobial agents included in meat and fish grown in ways that resemble target compounds, and assessing the safety of the meat and fish. Therefore, a method for sample preparation is needed that allows for the in-situ detection of target compounds, such as mycotoxins and agrochemical residues in food, using fluorescent immunoassay methods.

[0006] Besides FPIA, immunoassays using fluorescence include fluorescence enzyme immunoassay (FEIA), in which an antigen-antibody reaction is performed using an enzyme-labeled antigen or antibody as a labeling substance, and fluorescence intensity is measured by adding a fluorescent substance; and fluorescent antibody (FA) methods, in which antibodies that specifically recognize antigens are used to examine antigen distribution. Because immunoassays using fluorescence can detect trace elements, the need for in-situ sample preparation is not limited to FPIA, but also applies to FEIA, FA, and other immunoassays using fluorescence.

[0007] In view of the above-described status quo, the purpose of this disclosure is to provide a method for preparing a sample solution for measuring the concentration of a target compound contained in food using a fluorescence immunoassay method.

[0008] Another object of this disclosure is to provide a measuring unit, measuring kit, and preparation apparatus for measuring the concentration of a target compound contained in food using a fluorescence immunoassay method. Summary of the Invention

[0009] To achieve the objectives described above, the sample solution preparation method is a method for measuring the concentration of a target compound contained in food using a fluorescence immunoassay method, the sample solution preparation method comprising:

[0010] A dialysis step is used to contact a solution containing the target compound, which is then passed through a dialysis membrane to a dialysate, thereby transferring the target compound to the dialysate.

[0011] The molecular weight cutoff of the dialysis membrane is 2×10⁻⁶. 2 Up to 2×10 5 Within the range, and

[0012] The volume of the dialysate is relatively smaller than the volume of the solution containing the target compound.

[0013] The measuring unit is used to measure the concentration of a target compound contained in food using a fluorescence immunoassay method, and includes:

[0014] The measurement area includes components that transmit wavelengths in the range of 300 to 800 nm; and

[0015] Dialysis membrane, having a capacity of 2×10 2 Up to 2×10 5 The molecular weight cutoff within the specified range.

[0016] The assay kit is used to measure the concentration of a target compound contained in food using a fluorescent immunoassay method. The target compound is selected from the group consisting of mycotoxins, shellfish toxins, chemicals used to feed animals, and chemicals used to grow crops. The assay kit includes:

[0017] The aforementioned measurement unit;

[0018] Dialysis fluid to be filled into the measuring unit;

[0019] Antibodies that specifically bind to the target compound; and

[0020] Fluorescent labeling of target compound derivatives.

[0021] The sample solution preparation device is a sample solution preparation device used in fluorescence immunoassay methods, and includes:

[0022] A container that holds a solution containing the target compound; and

[0023] One or more dialysis cartridges are placed in a container.

[0024] The ratio of the total capacity of the dialysis cartridges to the capacity of the container (total capacity of the dialysis cartridges / capacity of the container) is 1 / (1×10⁻⁶).4 The range is from 1 / 10 to 1 / 10.

[0025] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory, and do not limit this disclosure.

[0026] Samples can be prepared for measuring target compounds in food using a fluorescence-based immunoassay method. Attached Figure Description

[0027] A more complete understanding of this application can be obtained by considering the following detailed description in conjunction with the accompanying drawings, in which:

[0028] Figure 1 This is a schematic diagram showing a dialysis cartridge filled with dialysis fluid being immersed in an extraction container containing a solution of the target compound.

[0029] Figure 2 It is a schematic diagram showing a mode in which a ground food sample is placed in a filter bag and immersed in a solvent to prepare a solution containing the target compound, which is simultaneously dialyzed by a dialysis chamber;

[0030] Figure 3 It is a diagram depicting a dialysis cartridge equipped with a heater;

[0031] Figure 4 It is a diagram depicting a pattern in which a solution containing the target compound with a higher specific gravity is placed in the lower layer, the dialysis membrane is placed on top of the lower layer, and the dialysate is filled onto the upper surface of the dialysis membrane.

[0032] Figure 5 The diagram illustrates a mode in which a solution containing the target compound is contained in a container placed on a magnetic stirrer, a dialysis cartridge is immersed in the solution, and iron oxide particles containing silver particles are added to both the solution and the dialysate; when the magnetic stirrer is activated, the iron oxide particles containing silver particles rotate according to the rotation of the motor, thereby stirring both the solution and the dialysate.

[0033] Figure 6 It is a diagram depicting a pattern in which a dialysis box is immersed in a solution containing the target compound contained in a container, while the adsorbent is placed on the bottom of the container;

[0034] Figure 7A This is an example of a preferred configuration for a rectangular measurement unit; a schematic diagram showing that the measurement area for transmitted excitation light is arranged above the measurement unit and the dialysis membrane is arranged below the measurement unit.

[0035] Figure 7BThis is a diagram illustrating how the measuring unit is used; the dialysate is filled into the measuring unit, which is then immersed in a solution containing the target compound.

[0036] Figure 8 This is a diagram illustrating an example of a sample solution preparation apparatus that includes a container, a dialysis chamber, and a stirring device;

[0037] Figure 9A It is a diagram depicting a sample solution preparation apparatus comprising multiple dialysis cartridges arranged in a container and the dialysis cartridges being equipped with stoppers or lids;

[0038] Figure 9B It is a diagram depicting a sample solution preparation apparatus comprising multiple dialysis cartridges arranged in a container and connected to each other by connecting members;

[0039] Figure 10 It is a diagram illustrating a pattern in which a sample solution preparation apparatus includes a dialysis cartridge with a long tube shape arranged in a container, and the dialysis cartridge includes components with dialysis capability and components without dialysis capability.

[0040] Figure 11 This is a graph showing the results of Example 1;

[0041] Figure 12 This is a graph showing the results of Example 2; and

[0042] Figure 13 This is a graph showing the results of Example 3. Detailed Implementation

[0043] The first embodiment of this disclosure is a sample solution preparation method for measuring the concentration of a target compound contained in food using a fluorescence immunoassay method. The sample solution preparation method includes...

[0044] A dialysis step is used to contact a solution containing the target compound, which is then passed through a dialysis membrane to a dialysate, thereby transferring the target compound to the dialysate.

[0045] The molecular weight cutoff of the dialysis membrane is 2×10⁻⁶. 2 Up to 2×10 5 Within the range, and

[0046] The volume of the dialysate is relatively smaller than the volume of the solution containing the target compound.

[0047] Immunoassays using fluorescence include FEIA, FA, and FPIA, all of which are within the scope of this disclosure. For ease of explanation, FPIA will be described below.

[0048] FPIA is based on the principle of competitive binding immunoassay. It uses two reagents: a fluorescently labeled compound obtained by labeling the same molecule as the target molecule with a fluorescent material; and an antibody that specifically binds to the target molecule. These fluorescently labeled compounds and antibodies, along with the target solution, are added to a measurement unit. The unit is then illuminated with excitation light, and the polarization components (Ih) emitted from the sample parallel to the incident light and (Iv) perpendicular to the incident light are measured. The fluorescence polarization degree P is calculated as (Ih - Iv) / (Ih + Iv). Since the fluorescence polarization degree P varies with the concentration of the target molecule, the concentration of the target molecule can be detected, for example, using a pre-prepared calibration curve. Because FPIA uses a specific reaction with the antibody, it reduces measurement errors caused by analogs such as metabolites compared to conventional liquid LC / MS ( / M) methods.

[0049] The target compound in this disclosure can be any compound contained in food and can be measured using a fluorescent immunoassay method without particular limitation. However, since the use of a fluorescent immunoassay method allows the detection of compounds at low concentrations, this method is preferably used to measure compounds contained in food at low concentrations that affect organisms, such as mycotoxins, shellfish toxins, chemicals used in animal feed, chemicals used in crop cultivation, etc.

[0050] Mycotoxins include fungal toxins such as aflatoxin, ochratoxin, deoxynivalenol, zearalenone, fumonisin, patulin, citrinin, T-2 toxin, and HT2 toxin, as well as alkaloids (such as ergot alkaloids and hyoscyamine alkaloids).

[0051] Shellfish toxins include okadaic acid, dominoic acid, and salicornic acid.

[0052] Furthermore, chemicals used in animal husbandry include: tetracycline antibiotics, such as tetracycline, oxytetracycline, chlortetracycline, and lysine; nitrofurans, such as nitrofurazone, nitrofurantoin, furazolidone, and furazolidone; antibiotics, such as chloramphenicol; antibacterial agents, such as malachite green, gentian violet, and enrofloxacin; and growth promoters, such as lactopamine, clenbuterol, diethylstilbestrol, and salbutamol.

[0053] In addition, chemicals used in crop cultivation include agricultural chemicals such as imidacloprid, fenitrothion, bromonitrile, and tetrachloroisophthalonitrile.

[0054] Foods include: grains such as rice, wheat, barley, and beans; vegetables such as spinach, tomatoes, and carrots; frozen foods such as frozen spinach, frozen green soybeans, and frozen carrots; beverages such as milk, water, and alcohol; condiments such as soy sauce and other sauces; meats such as chicken and pork; fish such as silverfish, sea bream, tuna, and eel; shellfish such as oysters and scallops; and processed foods containing these.

[0055] In the sample solution preparation method disclosed herein, a solution containing the target compound is referred to as a target compound-containing solution, and this target compound-containing solution is contacted with a dialysis membrane to transfer the target compound into the dialysate. The target compound-containing solution can be the food itself, or it can be a solution obtained by mixing the food or ground food with a solvent. Liquid foods (such as beverages and liquid condiments) can be directly contacted with the dialysis membrane. However, if the food is solid or semi-solid, it is preferable to extract the target compound in the solvent beforehand. For example, solutions obtained by adding a solvent to the food and mixing the solvent and food, solutions obtained by pre-grinding the food and mixing the ground food with a solvent, and solutions obtained by adding a solvent to the food and homogenizing the solvent and food can be used as target compound-containing solutions. The degree of agglomeration used for grinding the food can be, for example, the degree of agglomeration applied when preparing samples by LC / MS ( / MS) methods. The extraction time from food to solvent varies depending on the food, the target compound, and the solvent used, and therefore, optimal conditions can be selected by pre-measuring the extraction time and extraction rate. If the solution containing the target compound contains insoluble substances such as cellulose, chitin, and deacetylated chitosan, these insoluble substances can be removed beforehand by centrifugation, static separation, filtration, etc.

[0056] Examples of solvents that can be added to food include water; alcohols such as methanol, ethanol, and butanol; ketones such as acetone, diethyl ketone, and methylpentyl ketone; alkanes such as hexane and heptane; ethers such as diethyl ether; methyl sulfoxide; acetonitrile; chloroform; and mixtures of these. As solvents for extracting target compounds, the solvents used for measurement by LC / MS ( / MS) methods can be selected and used as needed. For example, to prepare a sample for measuring DON contained in wheat, a mixture of acetonitrile:water:methanol = 5:95:5 can be used, and methanol can be used to extract chloramphenicol as a chemical for animal feed. It should be noted that solvents different from those used to prepare samples by conventional LC / MS ( / MS) methods can also be used.

[0057] The dialysis membrane used in this disclosure can have a molecular weight cutoff of 2×10⁻⁶. 2 Up to 2×10 5 The membrane. The preferred molecular weight cutoff is 2 × 10⁻⁶.2 Up to 1.4×10 5 Within the range, more preferably within 1.5 × 10 4 Up to 1.4×10 5 Within the range. Solutions containing the target compound may contain salts (such as Tris and PBS), reducing agents (such as DTT and β-mercaptoethanol), and preservatives (such as sodium azide and timerosar), which may act as inhibitors of measurement errors during measurements performed via FPIA. Therefore, the lower limit of the molecular weight cutoff was set at 2 × 10⁻⁶. 2 In order to eliminate these compounds. On the other hand, because the molecular weight is greater than 2 × 10 5 The substance exhibits light-scattering properties, which could potentially introduce measurement errors in FPIA. Therefore, the upper limit for the molecular weight cutoff is set at 2 × 10⁻⁶. 5 Examples of such dialysis membranes include dialysis tubing made of cellulose esters and dialysis tubing made of synthetic polymers such as polymethyl methacrylate and polysulfone.

[0058] In addition to the molecular weight cutoff, the dialysis membrane used in this disclosure may preferably be a membrane resistant to organic solvents.

[0059] The volume of the dialysate is characteristically smaller than the volume of the solution containing the target compound. For example, when the solubility of the target compound is equal in both the solution and the dialysate, and the target compound migrates uniformly to both sides, the concentration of the target compound in the dialysate can be close to the concentration in the solution by making the ratio of the dialysate volume to the target compound solution volume (dialysate volume / target compound solution volume) smaller. For example, if the ratio between the dialysate and the target compound solution is 1 / 100, the concentration of the target compound in the dialysate at dialysis equilibrium becomes 0.99 times the concentration of the target compound in the solution, where samples for measurement can be prepared with almost no dilution. The ratio of the dialysate volume to the target compound solution volume (dialysate volume / target compound solution volume) is within 1 / (1×10⁻⁶). 4 The range is from 1 / 10 to 1 / 10, preferably in the range of 1 / 1 × 10. 2 To 1 / 1×10 4 More preferably within the range of 1 / 1×10 2 To 1 / 1×10 3 Within the range.

[0060] Dialysis time varies depending on the dialysis membrane used, the solvent used, and the target compound; therefore, optimal conditions can be selected by pre-measuring dialysis time and dialysis rate.

[0061] As such a dialysis membrane, for example, a dialysis cartridge equipped with a dialysis membrane on its outer periphery can be used. An example of this model is... Figure 1 As shown in the diagram, a dialysis cartridge 5 filled with dialysis fluid 7 is immersed in an extraction container 1 containing a solution 3 containing the target compound. The target compound contained in the solution 3 is transferred through a dialysis membrane to one side of the dialysis fluid 7, while salts and the like cannot be transferred to that side of the dialysis fluid, thus obtaining a dialysis fluid 7 from which salts and light-diffusing substances have been removed. Furthermore, the ratio of the volume of the dialysis fluid to the volume of the solution containing the target compound can be easily adjusted using the dialysis cartridge 5. Figure 1 The diagram illustrates a pattern in which the volume of solution 3 containing the target compound is significantly larger than the volume of dialysate 7. This allows for the preparation of sample solutions in which the concentration of the target compound contained in the solution containing the target compound is maintained.

[0062] Figure 2 The diagram illustrates a mode in which a bag filter 9 receives a ground food sample and then immerses it in a solvent to prepare a solution 3 containing the target compound. Extraction of the target compound from the food and dialysis via a dialysis cartridge 5 can be performed simultaneously, thereby reducing sample solution preparation time.

[0063] Dialysis can also be performed with the dialysate at a temperature higher than that of the solution containing the target compound. Generally, solubility increases with increasing solvent temperature. Therefore, raising the temperature of the dialysate above that of the solution containing the target compound allows for the transfer of a high concentration of the target compound to the dialysate side. Figure 3 An example of a mode in which the dialysate 7 in dialysis cartridge 5 is heated by heater 11 is shown. For example, the fluid temperature of the dialysate 7 can be increased by inserting a heating device into dialysis cartridge 5. The temperature difference between the target compound solution 3 and the dialysate 7 varies depending on the target compound, the solvent used, the type of dialysis membrane, and the dialysate 7, and therefore, optimal conditions can be selected by pre-measuring the dialysis time and dialysis rate at various temperature differences.

[0064] In this disclosure, a solvent having a different solubility for the target compound than that of the dialysate can be used as the solvent when mixing food or its ground form with a solvent. For example, if solvent A has a lower solubility for the target compound than solvent B, solvent A is used to prepare a solution containing the target compound, and solvent B is used as the dialysate. Since water containing ethanol has a higher solubility for chloramphenicol than water, water containing ethanol is used as the dialysate when detecting chloramphenicol as the target compound from milk. Moreover, when dialysis is performed by setting the ratio of the volume of the dialysate to the volume of the solution containing the target compound (volume of dialysate / volume of solution containing the target compound) to 1 / 100, the concentration of the target compound in the dialysate can be 0.99 times or more than the concentration of the target compound in the solution containing the target compound at dialysis equilibrium.

[0065] The contact between the target compound solution and the dialysate through the dialysis membrane is not limited to using the dialysis cartridge described above. In particular, when the target compound solution and the dialysate have different specific gravities, the fluid with the higher specific gravity can be placed in the lower layer, the dialysis membrane can be placed on top of the lower layer, and the fluid with the lower specific gravity can be poured onto the dialysis membrane to transfer the target compound into the dialysate. Figure 4 An example of a pattern is shown in which a solution 3 containing the target compound is placed in the lower layer, and dialysate 7 is filled onto the upper surface of the dialysis membrane 13. For example, when doxycycline is determined to be the target compound, the solution 3 containing the target compound is prepared, for example, by mixing ground pork (as a food source) with water, and if chloroform is used as the dialysate 7, chloroform is placed in the lower layer, allowing the dialysis membrane 13 to be placed on the upper surface of the lower layer, and the solution 3 containing the target compound is filled on top of the dialysis membrane 13. This pattern is effective for this target compound because chloroform has a higher solubility for doxycycline than water.

[0066] In this disclosure, the dialysis step can be performed simultaneously with stirring the solution containing the target compound and the dialysate using a stirrer blade comprising iron oxide particles containing silver particles. Stirring fluids by rotating the stirrer blade using magnetic force is a well-known technique. Stirring the solution containing the target compound and the dialysate using iron oxide particles containing silver particles can shorten the dialysis equilibration time. These silver-containing iron oxide particles include silver-plated iron oxide particles and silver-iron oxide particles. The particle size is not limited, but preferably has a diameter in the range of 0.1 to 10 μm, more preferably 1 to 10 μm. In particular, it has been found that when iron oxide particles containing silver particles are used as the stirrer blade, fluorescent substances (such as riboflavin) contained in the solution containing the target compound and the dialysate can be adsorbed. Riboflavin has a molecular weight of 376, which is close to the molecular weight of the target compound. Thus, even when riboflavin permeates the dialysis membrane, the riboflavin contained in the solution is adsorbed by the iron oxide particles containing silver particles, thereby enabling the preparation of sample solutions with smaller measurement errors. Figure 5 The diagram illustrates a configuration in which a container 1, placed on the upper surface of a magnetic stirrer 17, contains a solution 3 containing the target compound. A dialysis cartridge 5 is immersed in this solution, and iron oxide particles 15 containing silver particles are added to both the solution and the dialysate. When the magnetic stirrer 17 is activated, the iron oxide particles 15 containing silver particles aggregate, and when the magnetic stirrer 17 is stopped, the iron oxide particles 15 containing silver particles disperse, thereby stirring both the solution and the dialysate.

[0067] In this disclosure, the dialysis step can be performed by adding an impurity adsorbent to a solution containing the target compound. These impurities include neutral fats, cholesterol, phospholipids, free fatty acids, fluorescent substances (such as vitamin B2), and light-scattering materials (such as protein microparticles) having 5 to 30 carbon atoms. Examples of such adsorbents include gelatin, lime, protein, and bentonite. The optimal amount of adsorbent used can be selected by pre-assessing the type and concentration of the impurities to be removed. Figure 6 The diagram shows a configuration in which the dialysis cartridge 5 is immersed in a target compound solution 3 contained in a container 1, and the adsorbent 19 is placed at the bottom of the container 1. The adsorbent can be, for example, granular particles with a particle size of 0.1 to 5 mm, or it can be a mixture of granular particles and the target compound solution. With this particle size, the adsorbent will not permeate the dialysis membrane and can effectively adsorb impurities.

[0068] A second embodiment of this disclosure is a measuring unit for measuring the concentration of a target compound contained in food using a fluorescence immunoassay method, wherein a measuring region is arranged with components that transmit wavelengths in the range of 300 to 800 nm and a molecular weight cutoff of 2 × 10⁻⁶. 2 Up to 2×10 5The dialysis membrane. Examples of using a dialysis cartridge as a dialysis membrane have been described in this disclosure. This is because if such a dialysis cartridge can be used as a measurement unit, sample preparation and measurement can be performed through a series of operations. The measurement unit according to this disclosure is characterized by including a dialysis membrane useful for sample preparation and a measurement region useful for fluorescence measurement.

[0069] The shape of the measuring unit can be selected so that it can be attached to the device to be used. Figure 7A An example of a preferred mode for the measuring unit is shown, and Figure 7B An example of a sample solution preparation method using a measuring unit is shown. For ease of explanation, the body 27 of the measuring unit 21 is defined as rectangular, wherein two front and rear surfaces with a width of 32 mm and a height of 50 mm are connected by two side and bottom surfaces with a thickness of 5 mm, and excitation light is transmitted through the front and rear surfaces with a thickness of 1 mm.

[0070] The material of the main body 27 can be resin, such as polypropylene, polyethylene, polymethylpentene, ethylene-tetracyclododecene copolymer, polyacetal, acrylonitrile-butadiene-styrene resin, hydroxybenzoic acid ester polyester, polyetherimide, methacrylic acid resin, polyethylene terephthalate, polyethylene terephthalate, polybutylene terephthalate, polycyclohexanediol terephthalate, polyethylene naphthalate, polyacrylonitrile, polystyrene, polyamide, polycarbonate, polyvinyl alcohol or polylactic acid, glass, quartz, etc., without particular restrictions.

[0071] However, the measuring unit 21 has a measuring region 23, which is made of a component that transmits wavelengths in the range of 300 to 800 nm, preferably 400 to 700 nm. If the wavelength transmission is within this range, the fluorescence polarization can be measured without error. Examples of such components include glass, quartz, PMMA, PC, COP, COC, and PS.

[0072] Furthermore, the measuring unit 21 is equipped with a molecular weight cutoff of 2×10⁻⁶. 2 Up to 2×10 5 Preferably 1.5×10 4 Up to 1.4×10 5 The dialysis membrane 25. By using the dialysis membrane 25 with a molecular weight cutoff within the range described above, salts, light-scattering materials, and other impurities can be effectively removed. Commercially available products can also be used as this dialysis membrane 25. For example, dialysis discs or membranes can be used.

[0073] In the measurement unit 21, the position of the measurement region 23 can be formed corresponding to the incident position of the excitation light of the device used. Since the excitation light passes through the measurement unit 21, the measurement region 23 is formed on a portion of the front surface and a portion of the rear surface opposite the corresponding portion of the front surface. Furthermore, the dialysis membrane 25 can be arranged in the side surface or the bottom surface, not limited to the front and rear surfaces, as long as the dialysis membrane 25 does not overlap with the measurement region 23. For example, the measurement unit body 27 can be pre-prepared, in which the position for arranging the measurement region 23 and the dialysis membrane 25 is formed as a cavity, and the measurement region 23 and the dialysis membrane 25 can be mounted on the measurement unit 21 by bonding, welding, assembly or other methods.

[0074] like Figure 7B As shown, dialysate 7 is filled into the measuring unit 21, which is then immersed in a solution 3 containing the target compound. After a certain period of time, the measuring unit 21 is removed from the solution 3 containing the target compound, and the solution 3 adhering to the outer periphery of the measuring unit 21 is removed. Furthermore, the outer periphery of the dialysis membrane 25 may be covered with a waterproof film if necessary. To measure the fluorescence polarization degree P, fluorescently labeled compounds are prepared in advance, wherein molecules identical to the target compound are labeled with a fluorescent substance and an antibody that specifically binds to the target compound. These fluorescently labeled compounds and antibodies are added to the measuring unit 21 before it is attached to the measuring device. The fluorescence polarization degree P can be calculated by exposing the measuring region 23 to excitation light.

[0075] A third embodiment of this disclosure is a assay kit for measuring the concentration of a target compound contained in food using a fluorescence immunoassay method. The target compound is selected from the group consisting of mycotoxins, shellfish toxins, chemicals used to feed animals, and chemicals used to grow crops. The assay kit includes:

[0076] Measurement unit;

[0077] Dialysis fluid to be filled into the measuring unit;

[0078] Antibodies that specifically bind to the target compound; and

[0079] Fluorescent labeling of target compound derivatives.

[0080] When the dialysate is placed in a measurement unit that can be used for both sample preparation and fluorescence polarization measurement, sample preparation and measurement operations can be easily performed. Furthermore, in fluorescence immunoassay methods, based on the principle of competitive binding immunoassay, FPIA indispensablely uses antibodies that specifically bind to the target compound and fluorescently labeled derivatives of the target compound. Therefore, measurement kits that include antibodies corresponding to the target compound and fluorescently labeled derivatives of the target compound are extremely useful.

[0081] When such an antibody is commercially available, the commercially available product can be used. Alternatively, even if such an antibody is not commercially available, antibodies can be produced using an immunogen, wherein the immunogenic carrier material binds to the target compound via an amide bond or other groups. The immunogenic carrier material can be selected from conventionally known substances. The immunogenic carrier material can be any of the following: immunogenic protein, polypeptide, carbohydrate, polysaccharide, lipopolysaccharide, nucleic acid, etc. The immunogenic carrier material is preferably a protein or polypeptide, more preferably bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or thyroglobulin. This immunogen can be used to prepare polyclonal and monoclonal antibodies using well-known methods. Generally, the immunogen (preferably a mixture of immunogen and adjuvant) is injected into one or more different sites in a host animal (such as a rabbit, goat, mouse, guinea pig, or horse). Further injections are performed at regular or irregular intervals at the same or different sites. The titer is assessed as needed to obtain the desired antibody. The antibody can be recovered by collecting blood from the host animal, etc. It should be noted that the antibodies in this disclosure may be fragments of immunoglobulins (such as Fab, F(ab')2, or Fv), rather than complete immunoglobulins. It is sufficient if at least one epitope of the antibody binds to the target compound. Furthermore, commercially available products may be used for either antibodies or fragments.

[0082] Derivatives of the target compound can be prepared by binding a fluorescently labeled compound to any of the functional groups of the target compound. Examples of fluorescently labeled compounds include chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-hydroxyfluorescein, 5-carboxyfluorescein, 5-aminofluorescein and 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein. Fluorescently labeled compounds can be bound to the target compound using known methods to prepare derivatives of the target compound.

[0083] In this disclosure, the measurement kit includes a measurement unit in which a dialysis membrane, preferably for a target compound, is disposed; a target compound derivative corresponding to the target compound; and an antibody specifically binding to the target compound. The dialysis solution can be provided as a group of multiple liquids, allowing selection of a suitable liquid based on the presence of the target compound. Furthermore, the target compound derivative or antibody can be prepared as a liquid reagent by pre-dissolving the target compound derivative or antibody in a suitable solvent. Additionally, the measurement kit may include other reagents, standards, buffer solutions, diluents, etc., required for the measurement.

[0084] The fourth embodiment of this disclosure is a sample solution preparation apparatus for an immunoassay method using fluorescence, and the sample solution preparation apparatus includes:

[0085] A container that holds a solution containing the target compound; and

[0086] One or more dialysis cartridges are placed in a container.

[0087] The ratio of the total capacity of the dialysis cartridges to the capacity of the container (total capacity of the dialysis cartridges / capacity of the container) is 1 / (1×10⁻⁶). 4 The range is from 1 / 10 to 1 / 10. For example, the device is one in which the dialysis cartridge 5 is disposed in the container 1, such as... Figure 1 As shown. Utilizing such a simple structure, this device is preferably used for in-situ sample preparation.

[0088] The container is used to prepare a solution containing the target compound or to contain a solution containing the target compound for dialysis. The capacity of the container refers to the total capacity it can hold when placed under normal operating conditions. Furthermore, the capacity of the dialysis cartridges refers to the total capacity the dialysis cartridges can hold when arranged under normal operating conditions. Multiple dialysis cartridges can also be arranged in the container. When multiple dialysis cartridges are arranged, the capacity refers to the total capacity of the dialysis cartridges. The sample solution preparation apparatus of this disclosure may have a stirring device installed below the container. Figure 8 An example of a sample solution preparation apparatus is shown in which dialysis cartridge 5 is housed in container 1 and stirrer 17 is arranged below container 1 as a stirring device. The ratio of the total capacity of dialysis cartridge 5 to the capacity of container 1 (total capacity of dialysis cartridges / capacity of container) is 1 / (1×10⁻⁶). 4 The range is from 1 / 10 to 1 / 10, preferably in the range of 1 / 1 × 10. 2 To 1 / 1×10 4 More preferably within the range of 1 / 1×10 2 To 1 / 1×10 3Within the range. Even considering operability, assuming the fluid is filled up to 80% of the capacity of container 1 or dialysis cartridge 5, when the total capacity of dialysis cartridge 5 / container capacity is within 1 / (1×10⁻⁶). 4 When the concentration is within the range of 1 / 10, the dialysate recovered after dialysis by dialysis cartridge 5 can have a concentration of a specific component that is substantially the same as the concentration of the specific component contained in the solution filled in container 1.

[0089] although Figure 8 The illustration shows a configuration in which a single dialysis cartridge 5 is arranged within a container 1, but alternatively, multiple dialysis cartridges 5 can be arranged. Furthermore, each dialysis cartridge may have a plug for anchoring the cartridge to the container 1 and a cap for preventing leakage of dialysate through the inlet. The cap may be, for example, screw-on or assembled. Figure 9A The diagram shows a configuration in which three dialysis cartridges 5 are arranged, and dialysis cartridges 5 with stoppers 5a and lids 5b are housed within a container 1. For example, a dialysis cartridge 5 with a lid 5b is filled with dialysis fluid and covered with the lid 5b, then placed together with a solution containing the target compound in a lidded container. By vibrating the lidded container from side to side, upwards and downwards, etc., the distribution between the solution containing the target compound and the dialysis fluid can be ensured without the use of a stirring device.

[0090] In addition, multiple dialysis cartridges can be connected to each other via connecting components. Figure 9B The diagram shows a configuration in which three dialysis cassettes 5 are connected by connecting member 5c and housed in container 1.

[0091] Furthermore, the dialysis box can be in the shape of a long tube, along which dialysis membranes can be arranged or multiple membranes can be connected intermittently. Figure 10 The diagram illustrates a long tubular dialysis cartridge 5 comprising a dialysis-capable component 5d and a non-dialysis-capable component 5', with the dialysis-capable component 5d arranged in the liquid-containing portion of the container 1, and the non-dialysis-capable component 5' joined to the upper portion of the container 1. A dialysis membrane can be used as the dialysis-capable component. The molecular weight cutoff is 2 × 10⁻⁶. 2 Up to 2×10 5 Within the range, preferably within 2×10 3 Up to 2×10 5 Within the range, more preferably within 2×10 4 Up to 2×10 5 Within the range. Components without dialysis capability 5d can be, for example, polyethylene terephthalate, polyester resin, vinyl resin, etc.

[0092] Example

[0093] This disclosure will be described in detail below with the aid of illustrative examples, which are not intended to limit this disclosure in any way.

[0094] (Example 1)

[0095] The concentration of fluorescein sodium was adjusted to 1.06 × 10⁻⁶ by adding fluorescein sodium. -4 A few milliliters of milk containing M were prepared and used as a solution containing the target compound. A 500 μL capacity microdialysis cartridge (including a molecular weight cutoff of 4 × 10⁻⁶) was used. 2 Up to 1.4×10 5 A dialysis membrane was filled with 300 μL of pure water and immersed in a solution containing the target compound for 2 hours. After immersion, the fluorescence spectra of the dialysate and the solution containing the target compound were observed. The results were... Figure 11 As shown in the figure, this indicates that the solution containing the target compound has peaks at 470 nm and 520 nm, while the dialysate has a main peak at 520 nm. Therefore, the dialysis membrane can separate the peaks of sodium fluorescein.

[0096] (Example 2)

[0097] The extract was prepared as follows: sodium fluorescein and 70 ml of pure water were added to 7 g of brown wheat that had been ground using an electric grinder to adjust the sodium fluorescein concentration to 1.06 × 10⁻⁶. -4 M, which is used as a solution containing the target compound. A 300 μL microdialysis cartridge (including a molecular weight cutoff of 4 × 10⁻⁶) 2 Up to 1.4×10 5 A dialysis membrane was filled with 300 μL of pure water and immersed in a solution containing the target compound for 2 hours. The fluorescence spectra of the dialysate after 30 minutes, 1 hour, and 2 hours of immersion, and the fluorescence spectrum of the solution containing the target compound after 2 hours of immersion, were observed. Results were obtained in... Figure 12 As shown in the figure, the peak height of the fluorescence spectrum of the dialysate increases with immersion time, indicating that the target compound is transferred to the dialysate side.

[0098] (Example 3)

[0099] Grind 10 grams each of salmon, pork, and carrot in a small grinder and place them separately into 50 mL filter membranes with a mesh size of 100. Three containers, each with a capacity of 130 mL, each contain 100 mL of pure water and one of the filter membranes containing the ground food.

[0100] A 300 μL microdialysis cartridge (including a molecular weight cutoff of 4 × 10⁻⁶) 2 Up to 1.4×10 5 The dialysis membranes were filled with 300 μL of pure water and immersed in each container for 90 minutes. After immersion, the food extract and dialysate were recovered from the containers, and fluorescence was measured. Results were obtained in... Figure 13 As shown in the figure. In the case of any of these foods, the content of fluorescent substances (such as riboflavin) contained in the extract is reduced.

[0101] (Example 4)

[0102] A 110 mL sample solution containing chloramphenicol (CAP: molecular weight 323.1) dissolved in pure water at a concentration of 107 ng / mL was placed in a 130 mL container. Four microdialysis cartridges (each with a 300 μL capacity and composed of a molecular weight cutoff of 4 × 10⁻⁶) were prepared. 2 Up to 1.4×10 5 Each dialysis membrane (composition) was filled with 250 μL of pure water as the dialysate. After placing the stir bar into the container, the sample solution was dialyzed while stirring. The CAP concentration in the microdialysis cartridge was measured by LC / MS at the start of dialysis and after dialysis at 90, 180, 300, and 1350 minutes. The results are shown in Table 1.

[0103] Table 1

[0104]

[0105]

[0106] (Example 5)

[0107] A 110 mL sample solution containing chloramphenicol (CAP: molecular weight 323.1) dissolved in milk at a concentration of 112 ng / mL was placed in a 130 mL container. Four microdialysis cartridges (each with a 300 μL capacity and composed of a molecular weight cutoff of 4 × 10⁻⁶) were prepared. 2 Up to 1.4×10 5 Each dialysis membrane (composition) was filled with 250 μL of pure water as the dialysate. After placing the stir bar into the container, the sample solution was dialyzed while stirring. The CAP concentration in the microdialysis cartridge was measured by LC / MS at the start of dialysis and after dialysis at 90, 180, 300, and 1260 minutes. The results are shown in Table 2.

[0108] Table 2

[0109]

[0110] According to Example 5, the dialysis fluid exhibits a lower material collection rate compared to the material collection rate using pure water from the sample solution as shown in Example 4. However, even when using milk containing fat and protein as the sample solution, chloramphenicol can be recovered by dialysis.

[0111] For illustrative purposes, the foregoing has described some exemplary embodiments. Although specific embodiments have been presented in the preceding discussion, those skilled in the art will recognize that changes in form and detail may be made without departing from the broader spirit and scope of the invention. Therefore, the specification and drawings are to be viewed in an illustrative rather than restrictive sense. Consequently, this detailed description should not be considered limiting, and the scope of the invention is defined only by the included claims and the full scope of their equivalents.

Claims

1. A fluorescence-based immunoassay method for measuring the concentration of a target compound contained in food, the immunoassay method comprising: Preparation steps for preparing a sample solution include a dialysis step, wherein the dialysis step involves contacting a solution containing a food-derived target compound, which is included, through a dialysis membrane with a dialysate excluding a fluorescently labeled compound, to transfer the target compound into the dialysate. The measurement step is used to measure the concentration of a target compound contained in food by comparing the fluorescence intensity or fluorescence polarization emitted by a marker of the target compound in the sample solution with a pre-prepared calibration curve, wherein... The dialysis membrane has a molecular weight cutoff of 2 × 10⁻⁶. 2 Up to 2×10 5 Within the range of Da, and The volume of the dialysate is relatively smaller than the volume of the solution containing the target compound.

2. The immunoassay method according to claim 1, wherein in the dialysis step, the ratio of the volume of the dialysate to the volume of the solution containing the target compound (volume of dialysate / volume of solution containing the target compound) is in the range of 1 / (10000) to 1 / 10.

3. The immunoassay method according to claim 1 or 2, wherein the dialysis step comprises filling the dialysis fluid into the dialysis cartridge having the dialysis membrane on its outer periphery, and immersing the dialysis cartridge in the solution containing the target compound to transfer the target compound to the dialysis fluid.

4. The immunoassay method according to claim 1 or 2, wherein the solubility of the target compound in the dialysate is higher than the solubility of the target compound in the solution containing the target compound.

5. The immunoassay method according to claim 1 or 2, wherein the dialysis step is performed at a temperature where the temperature of the dialysate is higher than the temperature of the solution containing the target compound.

6. The immunoassay method according to claim 1, wherein the dialysis step comprises: Compare the specific gravity of the solution containing the target compound with the specific gravity of the dialysate; The dialysis membrane is placed on the surface of the one with a higher specific gravity in the solution containing the target compound and the dialysate. as well as The target compound solution and the other of the dialysate are poured onto the dialysis membrane to transfer the target compound to the dialysate.

7. The immunoassay method according to claim 1 or 2, wherein the dialysis step is performed while stirring the solution containing the target compound and / or the dialysate using a stirrer blade, the stirrer blade comprising silver-plated iron oxide particles or silver-iron oxide particles.

8. The immunoassay method according to claim 1 or 2, wherein the dialysis step is performed by placing the impurity adsorbent in the solution containing the target compound.

9. The immunoassay method according to claim 1 or 2, wherein the target compound is selected from the group consisting of mycotoxins, shellfish toxins, chemicals for animal husbandry, and chemicals for crop cultivation.

10. A measuring unit for measuring the concentration of a target compound contained in a food in an immunoassay method according to any one of claims 1 to 9 by comparing the fluorescence intensity or fluorescence polarization emitted from a label of the target compound with a pre-prepared calibration curve, and for containing a dialysate excluding the fluorescently labeled compound, said measuring unit comprising: The measurement area includes components that transmit wavelengths in the range of 300 to 800 nm; as well as Dialysis membrane, the dialysis membrane having a 2×10 2 Up to 2×10 5 The molecular weight cutoff of Da.

11. A assay kit for measuring the concentration of a target compound contained in food in any one of claims 1 to 9 by comparing the fluorescence intensity or fluorescence polarization emitted from a marker of the target compound with a pre-prepared calibration curve, said target compound being selected from the group consisting of mycotoxins, shellfish toxins, chemicals for animal husbandry, and chemicals for crop cultivation, said assay kit comprising: The measuring unit according to claim 10; The dialysate excluding fluorescently labeled compounds and to be filled into the measurement unit; Antibodies that specifically bind to the target compound; as well as Fluorescent labeling of target compound derivatives.

12. A sample solution preparation apparatus for an immunoassay method according to any one of claims 1 to 9, the sample solution preparation apparatus comprising: A container that holds a solution containing the target compound; as well as One or more dialysis cartridges, which are placed in a container and receive dialysate that does not contain a fluorescently labeled compound. The ratio of the total capacity of the dialysis box to the capacity of the container (total capacity of the dialysis box / capacity of the container) is in the range of 1 / (10000) to 1 / 10.

13. The sample solution preparation apparatus according to claim 12, The dialysis box is equipped with a lid at the dialysate inlet of the dialysis box.

14. The sample solution preparation apparatus according to claim 12 or 13 further includes a stirring device for stirring the solution contained in the container.

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