JP5467288B2 - Odor detection system for beverage production line - Google Patents

Description

  The present invention relates to a highly sensitive detection method for a target substance. More specifically, the present invention relates to a method for specifically detecting a very small amount of a low molecular target substance such as a scent in a beverage production line using an antibody and a surface plasmon resonance sensor in a short time. The present invention is useful in the field of manufacturing foods including beverages, pharmaceuticals, cosmetics, and the like.

Conventional technology

  In recent years, with the spread of supply chain management, beverage factories have increased opportunities to produce many types of products using the same production line.

  If the flavor component peculiar to a certain product remains in the production line (scented) and then shifts to the production of the next product, the flavor component of the previous product may shift to the next product, which may cause a problem as a strange odor. is there. The flavoring of the beverage line can be detected by a sensory test, but the sensory test requires a skilled inspector, and is considered to be easily affected by physical condition, mood and the like. Therefore, objective and high-accuracy quality control by sensory inspection is difficult. At present, flavoring should be dealt with by CIP (Claining In Place) using chemicals or excessive cleaning with heated hot water. This is one of the factors that greatly increase beverage production costs.

  On the other hand, an immunoassay utilizing an antigen-antibody reaction has become one of the main means in ultra-trace analysis of antigen substances ranging from low to high molecules. Among immunoassays, the most widely used at present is measurement by the Enzyme-Linked Immunosorbent Assay (ELISA) method. However, in the ELISA method, the reaction depends on passive diffusion, and the measurement takes time to analyze the state in which the antigen-antibody binding reaction has reached equilibrium. For detection, it is necessary to label the antibody with an isotope or an enzyme.

  Therefore, in recent years, an SPR immunosensor that combines a surface plasmon resonance (SPR) detection technique and an immunoassay has attracted attention. The SPR immunosensor can immobilize either the target substance or the antibody against it on the sensor chip surface, supply and contact the solution containing the other, and capture the fluctuation of the resonance angle generated based on the mass change due to binding. To detect the target substance. According to the SPR immunosensor, it is possible to immediately detect the binding reaction generated on the sensor chip, and it is not necessary to label the antibody.

  Up to now, high-sensitivity measurement of benzo (a) pyrene, bisphenol A, 2,4-dichlorophenol, trinitrotoluene (TNT), etc. has been studied with SPR immunosensors (Non-Patent Documents 1 to 4, Patent Document 1). Moreover, the application to the high sensitivity measurement of the flavor component used for a drink by the present inventors has also been examined (nonpatent literatures 5-7).

JP 2008-058176 A

Sakai, A. Torimaru, K. Shinahara, N. Miura, T. Imato, K. Toko and K. Matsumoto; Preparation of a Polyclonal Antibody and a Bioassay for Nitroaromatic Compounds by an Enzyme-linked Immunosorbent Assay Technique and a Surface Plasmon Resonance Biosensor; Sensors and Materials, 15, 439 (2003) R. Shankaran, K. Matsumoto, K. Toko and N. Miura; Development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance; Sensors and Actuators B, 114, 71 (2006) R. Shankaran, K. V. Gobi, T. Sakai, K. Matsumoto, K. Toko and N. Miura; Surface Plasmon Resonance Immunosensor for Highly Sensitive Detection of 2,4,6-trinitrotoluene; Biosensors and Bioelectronics, 20, 1750 (2005) R. Shankaran, KV Gobi, K. Matsumoto, T. Imato, K. Toko and N. Miura; Highly Sensitive Surface Plasmon Resonance Immunosensor for Parts-per-trillion Level Detection of 2, 4, 6-trinitrophenol; Sensors and Actuators, B 100, 450 (2004) KV Gobi, K. Matsumoto, K. Toko, H. Ikezaki, N. Miura; Enhanced Sensitivity of Self-assembled Monolayer based Surface Plasmon Resonance (SPR) Immunosensor for Detection of Benzaldehyde using Single-step Multi-sandwich Immunoassay; Anal. Bioanal. Chem., 387, 2727-2735 (2007) KV Gobi, K. Matsumoto, K. Toko, N. Miura; Highly Regenerable and Storageable All-chamical Based PEG-immunosensor Chip for SPR Detection of ppt Levels of Fragrant Compounds from Beverage Samples; Sens. & Instrumen. Food Qual, 2 ( 4), 225-233 (2008) Shintaro Ohashi, Hiroya Ishikawa, Kiyoshi Toko, Norio Miura, Kiyoshi Matsumoto; Preparation and Evaluation of Anti-Anthranilate Methyl Antibody for Fragrance Sensor, Abstracts of the 45th Chemistry-Related Branch Joint Kyushu Conference (2008)

  An object of this invention is to provide the system for the detection of the off-flavor substance in the wash water of a beverage production line.

  For the detection of off-flavor substances in the wash water of the beverage production line, even if the flavor component of the beverage is present at a concentration on the order of ppb, it can be perceived as a problem by humans and it can be a problem. It is necessary to construct a highly sensitive measurement system that can detect even at low concentrations. In addition, from the viewpoint of avoiding excessive washing of the beverage production line, quick measurement is necessary to appropriately determine the end point of washing. Therefore, for detection of off-flavor substances for this purpose, application of an SPR immunosensor that is highly sensitive and capable of rapid detection is expected.

However, according to the study by the present inventors, detection of flavor components in a beverage production line by an SPR immunosensor has the following difficulties:
That is, in general, when an immunoassay is used, since the specificity of an antibody to an antigen is high, a substance having a completely different chemical structure from a substance recognized as an antigen does not hinder detection. This is the same when used in combination with SPR. Contrary to expectations, however, the beverage water in the beverage production line contains not only flavor components but also bitterness and astringency components derived from beverages, although these components have a completely different chemical structure from the flavor components. Surprisingly, it proved to be an obstacle in immunoassays.

  In addition, the bitterness and astringency components are not usually perceived as a taste by humans at a low concentration (in the order of ppm) contained in the washing water of the beverage production line. Therefore, bitterness and astringency components have never been a problem in the beverage production line. On the other hand, as described above, the flavor component is perceived as a strange odor even when it is present at a concentration (ppb order) that is extremely lower than the bitterness / astringency component. There is currently no detection system that takes into account both substances present in the ppm order and substances present in the ppb order.

  The inventors of the present invention aimed to develop an SPR immunosensor for beverage production management, producing an antibody specific to a flavor component, and an ELISA method based on the indirect competition method, and actually measuring the flavor component with an SPR immunosensor. I have tried evaluation. In addition, as a result of examining appropriate pretreatment of the target substance, the present invention has been completed.

The present invention provides the following:
1) In a system for analyzing a target substance in a specimen using an SPR immunosensor that combines surface plasmon resonance (SPR) detection technology and immunoassay:
The specimen is wash water from a beverage production line and contains a detection hindrance substance, and the target substance is a beverage-derived flavor component;
A pretreatment unit for pretreating a sample to remove a detection hindrance substance;
Supply unit that supplies the sample from which the detection hindrance substance has been removed to the target surface or the sensor surface on which the antibody against the target substance is immobilized, and the amount of change in the SPR generation condition based on the reaction between the target substance and the antibody on the sensor surface A measuring unit for measuring
A system for analyzing the presence or concentration of a target substance in a specimen based on a measured change amount.
2) In a method for analyzing a target substance in a specimen using an SPR immunosensor that combines SPR detection technology and immunoassay:
The specimen is wash water from a beverage production line and contains a detection hindrance substance, and the target substance is a beverage-derived flavor component;
Pre-treat the specimen to remove the detection hindrance,
Specimens from which detection hindering substances have been removed are supplied to the target substance or the sensor surface on which the antibody against the target substance is immobilized, and the amount of change in the SPR generation condition based on the reaction between the target substance and the antibody on the sensor surface is measured Including the steps of:
A method of analyzing the presence or concentration of a target substance in a specimen based on a measured change amount.
3) The method according to 2), wherein the detection hindrance substance is one or more selected from a beverage-derived bitter or astringent ingredient.
4) Pre-processing
The method according to 2) or 3), wherein (a) a step of capturing a detection hindrance substance; and / or (b) a step of vaporizing and collecting the target substance.
5) The method according to any one of 2) to 4), wherein the sensor surface is a metal thin film in which a target substance-carrier protein complex is immobilized in advance.
6) The method according to 5), wherein the complex is immobilized on the surface of the metal thin film by covalently bonding to a functional group oriented by forming a self-assembled film on the surface of the metal thin film. .
7) An SPR measuring device for carrying out the method described in 2):
A device in which a target substance-carrier protein complex has a sensor unit immobilized by covalent bonding to a functional group oriented by forming a self-assembled film on the surface of a metal thin film.
8) A sensor chip for use in an SPR measuring device for carrying out the method according to claim 2:
A sensor chip, wherein a target substance-carrier protein complex is immobilized by a covalent bond to a functional group oriented by forming a self-assembled film on the surface of a metal thin film.
9) A method for producing beverage 1 comprising:
(1) sampling the wash water during washing of the beverage production line that produced the beverage 2 containing the flavor component X;
(2) Using the sampled wash water as a specimen, the flavor component X is analyzed by the method described in 2);
(3) Based on the analysis result, the washing is finished; and (4) the production method including a step of producing the beverage 1 not containing the flavor component X using the beverage production line after the washing.

  In the present invention, a surface plasmon resonance (SPR) method is used. An apparatus for this purpose usually includes a light source for irradiating light, a prism, an optical system detector, and a flow path for an object to be measured, and is used with a sensor chip having a metal thin film. In this method, when light is totally reflected at the interface between the metal thin film and the prism, if the wave number of the evanescent wave and the wave number of the surface plasmon coincide, the energy of the incident light is used to excite the surface plasmon and resonates, and the reflected light The strength of the decreases. The angle at which such dimming is observed depends on the refractive index of a medium such as a solution in contact with the outside of the metal thin film. Therefore, if the incident angle at which the intensity of the reflected light is reduced is measured, the refractive index of the solution can be found, and the amount of change reflects the variation in the amount of substance in the solution in contact with the sensor.

  In the present invention, an immunoassay is used. An immunoassay refers to a method of detecting a minute amount of an antigen substance using an antigen-antibody reaction.

  In the present invention, “analysis” refers to confirmation of presence / absence (presence) of a target substance in a sample and / or specification of the concentration of the target substance. In the system or method of the present invention, the amount of change in the measured SPR occurrence condition is included in the conversion formula for the concentration obtained by comparing with the reference data obtained in advance, if necessary. Or processed by collating with a calibration curve and specified as the presence or absence or concentration of the target substance.

  The target substance in the present invention is a flavor component derived from a beverage. In the present invention, the term “flavor ingredient” refers to an ingredient that forms a flavor of food including beverages, except in special cases. Flavors include peach flavor, grape flavor, apple flavor, blueberry flavor, cassis flavor, grapefruit flavor, strawberry flavor, cherries flavor, lychee flavor, mango flavor, sweet summer flavor, seek salmon flavor, La France flavor, Japanese pear flavor, Kyoho Flavor, Pine Flavor, Sweet Chestnut Flavor, Vanilla Flavor, Caramel Flavor, Champagne Flavor, Rum Raisin Flavor, Maple Syrup Flavor, Almond Flavor, and Earl Gray Flavor. Peach flavor, grape flavor, and apple flavor are particularly preferred examples for application to the present invention.

The flavor component is usually a relatively low-molecular substance having a molecular weight of 5,000 or less, may have a molecular weight of 2,500 or less, and may be 1,000 or less. “Flavor ingredients” include benzaldehyde, furfural, γ-undecalactone, methyl anthranilate, ethyl crotonate, hexyl acetate, and hexyl alcohol. Benzaldehyde, furfural, and methyl anthranilate are examples of particularly preferred components for applying the present invention. In the present invention, a specimen containing a flavor component contains a detection hindering substance. Therefore, the present invention includes a pretreatment step of removing the detection hindrance substance from the specimen before being subjected to the SPR immunosensor.

  In the present invention, the detection hindrance substance refers to a substance that inhibits detection of a flavor component by an SPR immunosensor, for example, a substance having a hydrophobic portion having a property of being adsorbed to a protein. Typical examples are polyphenols that are bitter or astringent components or pigment components derived from beverages. Specific examples of detection inhibitory substances include tannins (catechins) contained in tea, more specifically, catechin, gallocatechin, catechin gallate, gallocatechin gallate, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin Galate: Anthocyanins contained in grapes.

  There is a restriction that the pretreatment for removing the detection hindering substance should not affect the concentration of the target trace component. Removal can be achieved by (a) capturing the detected hindered substance and / or (b) vaporizing and collecting only the target substance without vaporizing the detected hindered substance.

  Regarding the pretreatment by capturing the detection hindrance substance, tannin can be adsorbed and removed by polyvinylpyrrolidone (PVPP) treatment, tannase treatment (free or immobilized), one or more proteins. Adsorption removal by a column with a fixed combination of these is conceivable. From the viewpoint of little influence on the target substance concentration, adsorption removal by a column on which OVA or lysozyme, preferably OVA and lysozyme is immobilized, is good. For example, it is possible to use a column packed with a gel filtration carrier having a reactive group (for example, formyl cellulose fine) carrying a protein such as OVA and / or lysozyme. For more detailed conditions for this, reference can be made to the examples of the present specification.

  More specifically, the pretreatment by vaporization collection of the detection hindrance substance does not vaporize the substance that hinders the detection of the target substance, but forcibly vaporizes only the target substance and turns the vaporized target substance into an appropriate solvent. It is for dissolving and providing for detection. As the vaporizing means, the sample liquid to be inspected is heated by a heater (for example, an electric heater, a burner, etc.), and the sample liquid to be inspected is also aerated (bubbling) by an odorless gas (for example, nitrogen, air, etc.). ), Or decompressing the container containing the sample liquid to be examined, or any combination thereof. The collection can be performed by cooling the vaporized target substance, aerating a suitable solvent with the vaporized target substance, or pressurizing the vaporized target substance, or a combination thereof. Techniques such as Japanese Patent Application Laid-Open No. 2004-93145 (odor test method and odor test apparatus) can be applied.

  In the present invention, either the target substance or the antibody against the target substance is immobilized on the surface of the sensor chip.

In general, since low molecular weight substances having a molecular weight of 5,000 or less alone do not exhibit immunogenicity, antibodies to target substances are not immunogenic, so a complex of the target substance and a carrier protein is prepared and used as an animal (eg, mouse, rabbit, rat). ) Can be obtained by immunizing. As the carrier protein, BSA (bovine serum albumin), OVA (ovalbumin), KLH (Keyhole limpet hemocyanin), CCH ( Concholehaps concholehaps hemocyanin) can be used. In the present invention, both a polyclonal antibody and a monoclonal antibody can be used. A person skilled in the art can appropriately prepare a polyclonal antibody and a monoclonal antibody. For detailed conditions, the examples in this specification can be referred to.

  In the present invention, the target substance-carrier protein complex is immobilized on the sensor surface for surface plasmon resonance measurement, and the target containing the specimen containing the target substance and the antibody against the target substance is provided to the measurement unit. It is preferable to measure the amount of change in SPR generation conditions based on the reaction between the substance and the antibody (indirect competition method). This is because the target substance has a low molecular weight, so that a large SPR response cannot be expected even when bound to the sensor surface.

  As the carrier protein here, the above-mentioned BSA, OVA, KLH and CCH can be used, but good results may be obtained by using a protein other than the one used as an immunogen.

  A method using a self-assembled monolayer (SAM) is suitable for fixing the composite to the surface of the sensor metal thin film. SAM refers to an organic thin film formed by spontaneously collecting organic molecules. When a suitable combination of substrate material and reactive organic molecules is selected and the substrate is placed in a solution or vapor of organic molecules, the organic molecules are adsorbed on the surface of the substrate. Due to the interaction, adsorbed molecules are gathered densely, and an organic monomolecular film with uniform molecular orientation is formed on the substrate surface. When SAM is formed, the surface of the substrate is covered with the functional group opposite to the adsorbed functional group, and the physical and chemical properties of the surface can be designed and controlled by using this terminal functional group. It becomes possible. For example, the surface can be covered with a reactive functional group such as a carboxyl group, an amino group, an epoxy group, or a vinyl group, and various substances can be fixed by covalent bonding. Those in which carboxyl groups are oriented are particularly suitable for immobilizing a target substance-carrier protein complex by an amide bond. Such a method is superior in terms of stability over the method of directly immobilizing an antibody.

  According to the study by the present inventors, in the case of targeting benzaldehyde, a polyclonal antibody (BZ pAb) prepared in a mouse, an alkanethiol derivative formed by a SAM method on an Au thin film, and a complex of benzaldehyde and OVA A high sensitivity measurement with a measurement limit of 50 ppt was achieved using a sensor chip immobilized with an amide bond (SAM-BZ-OVA, amine coupling); and a polyclonal antibody (BZ pAb) prepared in rabbit , A high sensitivity measurement with a measurement lower limit of 100 ppt has been achieved using a sensor chip in which a complex of benzaldehyde and OVA is immobilized by the SAM method (SAM-BZ-OVA, amine coupling); BZ mAb) and a functional group oriented by the SAM method, a complex of benzaldehyde and OVA is immobilized, and then avidin (Av) and biotin are used for amplification by SPR. (Specifically, a mixture of an antibody (primary antibody: antibody against a target compound) and a target compound is flowed to cause competitive inhibition, and free antibody is immobilized on the sensor surface. After binding to the antigen, a ternary mixture of biotinylated antibody (biotinylated anti-IgG antibody: secondary antibody), streptavidin (Av) and biotinylated BSA is passed through. Binding to the primary antibody that is bound to the sensor surface via a sensor chip (SAM-BZ-OVA, Av / Bio amplification) High sensitivity measurement of 10ppt has been achieved. In addition, when targeting furfural, a polyclonal antibody (FF pAb) prepared in rats and a sensor chip in which a complex of furfural and BSA is immobilized by physical adsorption (FF-BSA, physical adsorption) High-sensitivity measurement with a measurement lower limit of 100ppt has been achieved. With such a sensor chip, measurement within 5 minutes is possible, and one sensor chip can be used in a regeneration process (forcing the target substance and antibody to diverge and bring the sensor surface into an initial state where the target substance can be measured. Can be used repeatedly up to about 30 times.

  The target substance can be immobilized more directly using a polyethylene glycol (PEG) monomolecular layer. A sensor in which a mixture of a thiol derivative (PEG-NH2) having an amine end and PEG-OH having a shorter chain length is monolayered by the SAM method and a target substance (for example, benzaldehyde) is immobilized is suitable for surface regeneration processes. And high durability is expected.

  In the present invention, by further using an antibody against the antibody against the target substance, it is possible to increase the mass change on the sensor surface and improve the sensitivity by amplifying the signal dependent on the concentration of the target substance.

  In the present invention, the term “system” refers to a tangible object in which a plurality of functions (devices) are logically assembled regardless of whether or not functions (devices) of each configuration exist in the same housing. The system of the present invention includes at least a pretreatment device that pretreats a specimen to remove the detection hindrance substance, a supply device that supplies the specimen from which the detection hindrance substance has been removed, and an SPR detection apparatus, Further, it may include a calculation / control unit that calculates the presence or concentration of the target substance in the sample based on the data obtained from the SPR device, and controls the washing in the beverage production line based on the calculation result as necessary. .

  The system of the present invention may further include a cleaning water sampling device in the food production line. The sampling device may be equipped with two syringe pumps and two valves for supplying the specimen and antibody, respectively, so that both can be mixed and injected, and the indirect competitive measurement described above can be performed automatically It becomes.

Preparation of anti-benzaldehyde polyclonal antibody The following is an odorous ingredient that is widely used as a fragrance for soft drinks and targeted by the present inventors. First, from the viewpoint of the antigenicity of the molecule, Benzaldehyde (BZ), which is an aromatic aldehyde having a benzene ring in the molecule, was used as a target odor component.

  As immunogens, BZ-BSA and BZ-KLH were prepared by the following method.

  4-Carboxybenzaldehyde is used as a starting material, activated with N-hydroxysuccinimide (NHS) and carbodimide reagent (EDC), then reacted with bovine serum albumin (BSA) and keyhol limpet hemocyanin (KLH) to form complexes BZ-BSA, BZ -I got KLH. BZ-BSA was used to immunize BALB / c mice (5 weeks ♀) and Japanese white rabbits (13 weeks).

  [Mouse antibody] Mice were immunized with the prepared complex BZ-BSA, and affinity of BZ was evaluated by indirect competition ELISA using the obtained polyclonal antibody.

  From this, it was shown that this antibody can detect BZ at a concentration of 50 ppb or more.

  [Rabbit Antibody] The prepared complex BZ-BSA was immunized against a rabbit, and the affinity for BZ was evaluated by indirect competitive ELISA using the obtained polyclonal antibody.

  From this, it was shown that BZ can be detected at a concentration of 1 ppb or more with this antibody.

Cellulofine-OVA-Lysozyme Column Treatment According to the study by the present inventors, it was considered that it is not easy to use an untreated sample for measurement because tannin contained in black tea binds to protein. Therefore, prior to the measurement of the actual sample, a cellulofine-OVA-Lysozyme column was prepared to remove tannin in the sample.

  After washing Formylcellulofine, ovalbumin (OVA) and Lysozyme were added to Formylcellulofine, mixed by sonication, and then stirred at room temperature for 1.5 hours. After stirring, reduction treatment was performed and blocking was performed with an ethanolamine hydrochloride solution. After the reaction, reduction treatment and washing were performed to prepare a cellulofine-OVA-Lysozyme column.

First, tannin was removed from the black tea sample using a Cellulofine-OVA-Lysozyme column. BZ-added PBS solution and black tea solution (Black tea was a commercial product, boiled, returned to room temperature, diluted 5 times with PBS) were each applied to a 5 mL Cellulofine-OVA-Lysozyme column, and finally 1 mL of was collected. Further, these solutions were diluted 5-fold to adjust the BZ concentration to 2 × 10 ⁻⁵ g / mL, and each was used as a sample solution. Subsequently, BZ was measured by indirect competitive ELISA. As the measurement object, two kinds of the above-mentioned two kinds of column-treated samples and untreated column PBS prepared at the same concentration as above and black tea samples were used.

Column untreated (A), treated (B) PBS sample with BZ added by indirect competitive ELISA using rabbit-derived anti-BZ polyclonal antibody, and column untreated (C), treated (D) black tea sample BZ The affinity for was evaluated. For the evaluation of affinity, the inhibition behavior was examined using BZ at a concentration of 10 ⁻⁵ g / mL, and the results are shown together with the results of GC analysis.

From this, the absorbance (B ₀ ) obtained from the BZ-free PBS solution was taken as 100%, and the binding rate (% bound) was calculated from the absorbances of (A), (B), (C), and (D) above. However, except for the untreated black tea sample, the other samples showed the same inhibition behavior, indicating that the BZ concentration did not change substantially before and after the column treatment. From the above, it became clear that by removing tannin, it becomes possible to detect low concentrations of BZ.

Production of anti-benzaldehyde monoclonal antibody
A monoclonal antibody-producing strain having high reactivity with BZ was established.

  A monoclonal antibody was prepared using the BZ-KLH complex as an immunogen and the rat lymph node method. Rat lymph node cells and myeloma cells (SP2 / 0) were fused, and HAT selection, screening, and cloning were repeated to establish an anti-BZ monoclonal antibody-producing hybridoma strain.

  The binding of the obtained monoclonal antibody to BZ was measured by indirect competitive ELISA.

  As a result, it was considered possible to detect BZ of about 30 ppb.

Application of SPR immunosensor for sensitive measurement of benzaldehyde
Measurement of BZ with an SPR immunosensor was performed by the indirect competitive inhibition method. The sensor chip used for measurement was washed with a mixed solution of Milli-Q: 28% ammonia water: 30% hydrogen peroxide solution = 5: 1: 1, and carboxyl groups were introduced using 16-Mercaptohexadecanoic acid. After loading the SAM sensor chip and activating with NHS and EDC, BZ-OVA was injected to introduce the immobilized antigen onto the sensor surface. After blocking the surface, the obtained BZ-OVA immobilized sensor was injected with an equal amount of a mixed solution of anti-BZ polyclonal antibody at a predetermined concentration and Bz at each concentration, and Δθ caused by antibody binding Was measured. Further, an anti-rabbit IgG antibody was injected to amplify a signal dependent on the BZ concentration. After the measurement, the sensor surface was regenerated by injecting 0.1 M glycine-hydrochloric acid buffer (pH 2.4).

    The sensorgram in which the signal was amplified with the anti-rabbit IgG antibody is shown below.

Furthermore, Δθ obtained when each concentration of benzaldehyde calculated from the signal is added is expressed as a percentage with respect to Δθ ₀ obtained when Benzaldehyde is not added.

  As a result, a decrease in Benzaldehyde concentration-dependent signal was confirmed, and a significant improvement in sensitivity was shown compared to the case where normal signal amplification was not performed. From this result, it was considered that the practical sensitivity was about 0.1 ppb.

Production of anti-Furfural polyclonal antibody
For the purpose of rapid, specific and sensitive measurement of furfural (FF), a complex of FF and a carrier protein was prepared, and an animal was immunized to obtain an antibody against FF. FF-BSA was prepared as an immunogen.

  5-formyl-2-furancarboxylic acid was used as a starting material, activated with NHS and EDC, and then reacted with BSA. After the reaction, the complex FF-BSA was obtained by dialysis / centrifugation and lyophilization. Similarly, FF-KLH and FF-C8-BSA were prepared. The prepared FF-BSA, FF-KLH, and FF-C8-BSA were used to immunize WKY rats (9weeks). The affinity for FF was evaluated by indirect competition ELISA using the obtained polyclonal antibody.

  From this, it was shown that FF can be detected at a concentration of 10 ppm with an antibody derived from FF-BSA, 10 ppb with an antibody derived from FF-KLH, and 1 ppb or more with an antibody derived from FF-C8-BSA.

Production of anti-Furfural monoclonal antibodies
A monoclonal antibody-producing strain having high reactivity with FF was established.

  Monoclonal antibodies were prepared using the FF-KLH complex as an immunogen and the rat lymph node method. Rat lymph node cells and myeloma cells (SP2 / 0) were fused, and HAT selection, screening, and cloning were repeated to establish an anti-FF monoclonal antibody-producing hybridoma strain.

  The result of measuring the binding of the obtained monoclonal antibody to FF by indirect competitive ELISA is shown below.

  From this result, it was shown that this monoclonal antibody can detect FF at a concentration of 100 ppb or more.

Preparation of anti-Methyl anthranilate polyclonal and monoclonal antibodies
For the purpose of rapid, specific and sensitive measurement of Methyl anthranilate (MA), we prepared a complex of MA analog and carrier protein, and immunized animals to acquire antibodies against MA. It was. As an immunogen, was prepared 1-methyl-2-aminotelephthalate MAT -CCH complex obtained by combining the (MAT) in Concholehaps concholehaps Hemocyanin (CCH). MAT was used as a starting material, activated with NHS and EDC, and then reacted with CCH. After the reaction, dialysis / centrifugation was performed, followed by freeze-drying to obtain complex MAT-CCH. The prepared MAT-CCH was used to immunize WKY rats (9weeks ♀). Three weeks later, after confirming a sufficient increase in antibody titer, the rats were killed and the lymph nodes were removed, and at the same time, whole blood was collected. Polyclonal antibodies were obtained from whole blood. On the other hand, rat lymph node cells and myeloma cells (SP2 / 0) were fused, and HAT selection, screening, and cloning were repeated to establish an anti-MA monoclonal antibody-producing hybridoma strain.

  The results of measuring the binding of the obtained polyclonal antibody and monoclonal antibody to MA by indirect competitive ELISA are shown below.

  From this result, it was shown that MA of 2 ppm can be detected with a polyclonal antibody and 200 ppb or more with a monoclonal antibody.

Claims (7)

In an analysis system for target substances in specimens using an SPR immunosensor that combines surface plasmon resonance (SPR) detection technology and immunoassay:
The specimen is wash water from a beverage production line and contains a detection hindrance substance, and the target substance is a beverage-derived flavor component;
A pretreatment unit for pretreating a sample to remove a detection hindrance substance;
Supply unit that supplies the sample from which the detection hindrance substance has been removed to the target surface or the sensor surface on which the antibody against the target substance is immobilized, and the amount of change in the SPR generation condition based on the reaction between the target substance and the antibody on the sensor surface A measuring unit for measuring
A system for analyzing the presence or concentration of a target substance in a specimen based on a measured change amount. In a method for analyzing a target substance in a specimen using an SPR immunosensor that combines SPR detection technology and immunoassay:
The specimen is wash water from a beverage production line and contains a detection hindrance substance, and the target substance is a beverage-derived flavor component;
Pre-treat the specimen to remove the detection hindrance,
Specimens from which detection hindrance substances have been removed are supplied to the target surface or the sensor surface on which the antibody against the target substance is immobilized, and the amount of change in the SPR generation condition based on the reaction between the target substance and the antibody on the sensor surface is measured. Including steps,
A method of analyzing the presence or concentration of a target substance in a specimen based on a measured change amount.   The method according to claim 2, wherein the detection hindrance substance is one or more selected from a beverage-derived bitter or astringent ingredient. Pre-processing
The method according to claim 2 or 3, wherein (a) a step of capturing a detection hindrance substance; and / or (b) a step of vaporizing and collecting the target substance.   The method according to any one of claims 2 to 4, wherein the sensor surface is a metal thin film in which a target substance-carrier protein complex is immobilized in advance.   The method according to claim 5, wherein immobilization of the complex to the surface of the metal thin film is performed by covalently immobilizing the complex to a functional group oriented by forming a self-assembled film on the surface of the metal thin film. .   In the SPR immunosensor, the target substance-carrier protein complex has a sensor part immobilized by covalent bonding to a functional group oriented by forming a self-assembled film on the surface of the metal thin film. The system according to claim 1.