JP2007169207A - Antibody, method of detecting tricholoma matsutake-derived component and kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means to simply and quantitatively detect a Tricholoma matsutake-derived component present in food and drink, a drug and the like with accuracy. <P>SOLUTION: The method of detecting a Tricholoma matsutake-derived component comprises detecting the Tricholoma matsutake-derived component with the use of an antibody which specifically binds to an α-glucan-protein complex derived from Tricholoma matsutake. For example, by performing the ELISA with the use of an antibody to MPG-1 (anti-MPG-1 antibody) of an α-glucan-protein complex inherent in Tricholoma matsutake, the Tricholoma matsutake derived component can be quantitatively detected with high accuracy. Accordingly, the content of a specific α-glucan-protein complex in, for example, food and drink and the like can be quantitatively detected, and further whether or not the food and drink actually contain a Tricholoma matsutake-derived component can be easily distinguished with high accuracy. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an antibody, a matsutake-derived component detection method, a kit, and the like. More specifically, an antibody (such as an anti-MPG-1 antibody) that specifically binds to an α-glucan-protein complex derived from matsutake, a matsutake-derived component detection method in which the antibody is applied to an ELISA method, and the like The present invention relates to a matsutake-derived component detection kit in which an antibody, a reagent, or the like is made into a kit.

  Matsutake (scientific name “Tricholoma matsutake”) is a mushroom belonging to the family Agaricidae and is distributed in the temperate region. The mycelium of matsutake grows by spreading mycelium on the roots of red pine and the like, and in the fall, gives rise to fruiting bodies.

  Matsutake is loved as a luxury food in Japan. As foods and drinks using matsutake, cooked rice, soup, etc. are particularly preferred. In addition, instant foods with a matsutake flavor (rice cooked rice, soup powder, etc.) are also widely distributed. In addition, matsutake (or its dry powder) may be added to miso soup, tea, yogurt, or the like.

  In addition, in recent years, various physiological activities of matsutake-derived components have been clarified, and pharmaceuticals and health foods and drinks containing matsutake-derived components are also increasing. For example, Patent Document 1 discloses an antihypertensive agent containing matsutake or an extract thereof, Patent Document 2 discloses a diabetes therapeutic agent, and Patent Document 3 discloses a cancer preventive agent. .

As other prior art related to the present invention, for example, Patent Document 4 discloses a method for specifically detecting glucan in a test sample using a monoclonal antibody against alpha 2-macroglobulin. In addition, methods for quantifying useful substances and allergens contained in foods by using the degree of aggregation caused by an antigen-antibody reaction as an index are disclosed.
JP 2005-225803 A JP 2005-225801 A Japanese Patent Laid-Open No. 2004-210695 JP-T-2001-519183 JP 2004-333171 A

  It has been difficult to quantitatively and accurately detect components derived from matsutake contained in foods and drinks and pharmaceuticals. In particular, since there are many high molecular weight substances such as glucan in the physiologically active substances derived from matsutake, almost no effective quantitative means have been known. Therefore, the main object of the present invention is to provide a means for simply, quantitatively and highly accurately detecting matsutake-derived components contained in foods and drinks and pharmaceuticals.

  The present invention provides a matsutake-derived component detection method for detecting a matsutake-derived component using an antibody that specifically binds to the matsutake-derived α-glucan-protein complex.

  For example, an antibody (anti-MPG-1 antibody) that specifically binds to MPG-1, which is an α-glucan-protein complex unique to matsutake, is used as the antibody. By using these antibodies as capture antibodies and / or detection antibodies in the ELISA method, components derived from matsutake can be detected quantitatively and with high accuracy.

  As a specific procedure, a known ELISA method or the like can be applied. In this case, for example, a capture antibody immobilization procedure for immobilizing the antibody on a predetermined surface of the reaction field, an antigen supply procedure for supplying a target sample to the reaction field, and an antibody modified so that it can bind to a detection enzyme Including a detection antibody supply procedure for supplying the reaction field to the reaction field, a chromogenic substrate supply procedure for supplying a chromogenic substrate for the detection enzyme to the reaction field, a chromogenic reaction detection procedure for detecting a reaction between the detection enzyme and the chromogenic substrate, and the like.

  By using the matsutake-derived component detection method according to the present invention, it is possible to quantitatively detect the content of a specific α-glucan-protein complex in foods and drinks and pharmaceuticals. Therefore, since the intake (dose) of a specific physiologically active substance can be grasped, it is useful from the viewpoint of daily health care.

  For example, when using an anti-MPG-1 antibody, since MPG-1 is a component intrinsic to matsutake, it is possible to easily and accurately determine whether matsutake-derived components are actually contained in the food or drink. Therefore, the quality of the product can be evaluated easily and with high accuracy.

  In addition, the matsutake-derived component detection method according to the present invention can be used, for example, when quantitatively detecting the concentration of a specific α-glucan-protein complex in blood. Thereby, when a specific physiologically active substance is ingested, its pharmacokinetics can be grasped.

  Note that the matsutake-derived component detection method according to the present invention can be made into a kit. In that case, the kit is modified, for example, so that the antibody is bound to a predetermined surface as a capture antibody (microplate, microtube, etc.), and the detection antibody is bound to a detection enzyme. An antibody, a detection enzyme and its substrate (chromogenic substrate), other ELISA reagents, and the like are appropriately combined.

  According to the present invention, matsutake-derived components can be detected quantitatively and with high accuracy. The present invention is useful for detecting and quantifying components derived from matsutake, for example, contained in foods, drinks, pharmaceuticals, and other samples.

  In Example 1, an anti-MPG-1 antibody was produced as an example of an antibody that specifically binds to an α-glucan-protein complex derived from matsutake.

  The antibody was prepared in the order of (1) preparation of antigen, (2) preparation of antiserum (immune serum), (3) measurement of antibody titer of antiserum, and (4) purification of antiserum. Hereinafter, the outline of the procedure will be described in order.

(1) Preparation of antigen:
The antigen was prepared by the following procedure (see FIG. 1).

  First, 5 g of 0.2 M sodium hydroxide aqueous solution was added to 100 g of matsutake mycelium preparation CM6271 (manufactured by Kureha Co., Ltd., hereinafter the same), and the components were extracted into the solution by stirring at 25 ° C. for 1 hour. The supernatant (“Extract (E)”) was recovered after separation (15,000 rpm, 20 minutes, 4 ° C.).

  Next, this supernatant (“Extract (E)”) is neutralized with hydrochloric acid, and an equal volume of chloroform / methanol mixed solvent (2: 1) is added to this solution, followed by shaking and mixing. The layer was separated into a layer (“Water Phase (EW)”) and an organic solvent layer (“Ch · Me Phase”), and each was separated. Then, the aqueous layer (“Water Phase (EW)”) was dialyzed against pure water for 3 days using a cellulose dialysis membrane (molecular weight: 8,000 Da), and then freeze-dried.

  The aqueous layer (“Water Phase (EW)”) was then purified by DEAE ion exchange chromatography. The lyophilized powder obtained from the aqueous layer (“Water Phase (EW)”) was dissolved in 50 mM Tris-HCl buffer (pH 7.5, hereinafter referred to as “Tris-HCl”), and then DEAE TOYOPEARL PAK 650M. The solution was applied to a packed column (pore diameter: 22 mm, length: 20 cm), and linear gradient elution was performed using eluent A (Tris-HCl) and eluent B (1M sodium chloride-containing Tris-HCl), and the absorbance at 280 nm. And the elution pattern shown in (A) on the right side of FIG. 1 was obtained. The flow rate of the eluate was set to 5 mL / min.

  Then, based on the obtained elution pattern, the aqueous layer (“Water Phase (EW)”) is fractionated into five fractions (“EW-1” to “EW-5”), of which the EW-2 fraction Was dialyzed against distilled water for 20 days using a dialysis membrane (molecular weight: 13,000 Da) and then freeze-dried.

  Next, gel filtration of the EW-2 fraction was performed. The lyophilized powder obtained from the EW-2 fraction was dissolved in Tris-HCl and then applied to a Sephacryl S-100 (Amersham Biosciences) packed column (diameter 16 mm, length 30 cm), and the absorbance at 280 nm was measured. The elution pattern shown in (B) on the right side of FIG. 1 was obtained by measurement. Tris-HCl was used for column equilibration.

  Then, based on the obtained elution pattern, the EW-2 fraction was further fractionated into three fractions (“EW-21” to “EW-23”).

  Next, the EW-21 fraction was subjected to gel filtration with higher accuracy. The solution was applied to a Sephacryl S-500 (Amersham Biosciences) packed column (diameter 16 mm, length 60 cm), the absorbance at 280 nm was measured, and the elution pattern shown in (C) on the right side of FIG. 1 was obtained. . For equilibration of the column, Tris-HCl was used as described above.

  Then, based on the obtained elution pattern, the EW-21 fraction was further fractionated into three fractions (“EW-211” to “EW-213”). Among them, the EW212 fraction (hereinafter referred to as “MPG-1”) was used as an antigen.

(2) Preparation of antiserum:
Antiserum was prepared by the following procedure.

  First, the first immunization was performed. An equal amount of a phosphate buffered saline (hereinafter referred to as “PBS”) solution of MPG-1 that is the target antigen and Freund's Complete Adjuvant (manufactured by Sigma-Aldrich) is mixed. A highly viscous emulsion liquid was prepared using an ultrasonic generator. The emulsion solution was subcutaneously injected into the back of each of two 4-week-old female New Zealand white rabbits (purchased from Oriental Yeast Co., Ltd.) so that the amount of MPG-1 was 0.5 mg / feather. .

  Next, the second to fifth immunizations were performed. A PBS solution of the antigen MPG-1 and Freund's Incomplete Adjuvant (manufactured by Sigma-Aldrich) were mixed to prepare an emulsion. Then, this emulsion solution was subcutaneously applied to the back of each New Zealand white rabbit 4 times every 2 weeks from 2 weeks after the first injection so that the amount of MPG-1 was 0.5 mg / feather. Injected.

  Next, the sixth to ninth immunizations were performed. A PBS solution of the antigen MPG-1 and TiterMax gold adjuvant ("TITERMAX" is a registered trademark of Titamax, Inc., purchased from UPTIMA INTERCHIM) were prepared to prepare an emulsion solution. Then, this emulsion solution was subcutaneously applied to the back of each New Zealand white rabbit 4 times every 2 weeks from 2 weeks after the fifth injection so that the amount of MPG-1 was 0.5 mg / feather. Injected.

  In the third to eighth immunizations, 0.5 to 1.0 mL of blood was collected from the ear artery one week after each emulsion injection. In the ninth immunization, one week after the emulsion injection, whole blood was collected from the carotid artery under ketamine / xylazine anesthesia. Each collected blood was allowed to stand at room temperature for 1 hour and stored overnight at 4 ° C., followed by centrifugation (3,000 rpm, 10 minutes) to obtain serum.

(3) Measurement of antibody titer of antiserum:
The antibody titer of each serum (antiserum) obtained from two rabbits was measured by ELISA method and dot blot method.

  First, the outline of the procedure for antibody titer measurement by the ELISA method is shown below.

  First, an MPG-1 solution was dispensed into a 96-well plate at 50 ng / well, and each well was coated with MPG-1. Next, 100 μL of a 100 to 24,300-fold diluted solution of the obtained serum was dispensed to each well and reacted at 25 ° C. for 3 hours.

  Next, each well was washed three times with PBS containing 0.05% Tween 80 (hereinafter referred to as “PBS-Tween”), and then an alkaline phosphatase-labeled anti-rabbit IgG antibody solution (“Protein Detector ELISA kit, Rabbit IgG”). , AP ", purchased from Funakoshi Co., Ltd., the same applies hereinafter) was dispensed into each well and reacted at 25 ° C for 1 hour.

  Next, each well was washed three times with PBS-Tween, and then the substrate attached to the kit was added and allowed to stand for 15 minutes to develop color. And the light absorbency in 405 nm was measured using the plate reader.

  The results are shown in FIG.

  FIG. 2 is a graph showing the results of antibody titer measurement by ELISA. In the graph, the horizontal axis represents the serum dilution rate, and the vertical axis represents the absorbance. In the graph, the broken line indicated by “1-0” indicates the absorbance when the serum obtained from the first individual before immunization was used, and the broken line indicated by “1-7” indicates the seventh from the first individual. Absorbance when using the serum obtained at the time of the first immunization, the broken line indicated by “1-8” is obtained when the serum obtained at the time of the eighth immunization from the first individual is used. The broken line indicating the absorbance as “1-9” represents the absorbance when the serum obtained from the first individual during the ninth immunization is used. Similarly, the broken line indicated by “2-0” indicates the absorbance when serum obtained from the second individual before immunization was used, and the broken line indicated by “2-8” indicates the eighth from the second individual. Absorbance when using the serum obtained at the time of the first immunization, the broken line indicated by “2-9” is the case of using the serum obtained at the time of the ninth immunization from the second individual. Absorbance is expressed respectively.

  As shown in FIG. 2, in both individuals, when the serum obtained at the time of the 7th and subsequent immunizations was used, the absorbance (antibody titer) was higher than when the serum obtained before the immunization was used. Increased significantly. For example, the absorbance at the time of 100-fold dilution when using serum obtained at the time of the seventh and subsequent immunizations is changed from a value of 0.5 or less before immunization to a value of 2.0 to 2.5. Rose to. This result shows that the serum obtained during the seventh and subsequent immunizations has a high serum antibody titer.

  Then, the outline | summary is shown below about the case where an antibody titer measurement is performed by the dot blot method.

  First, a solution of antigen MPG-1 (1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, respectively) was dropped and fixed on a PVDF (“polyvinylidene difried”, hereinafter the same) membrane. Next, a 1,000-fold dilution of serum was added dropwise to the fixed MPG-1, and reacted at 25 ° C. for 1 hour.

  Next, after thoroughly washing with PBS-Tween, an alkaline phosphatase-labeled anti-rabbit IgG antibody solution was dropped and reacted at 25 ° C. for 1 hour. Next, after washing 3 times with PBS-Tween, the substrate attached to the kit was added to cause color development.

  The results are shown in FIG.

  FIG. 3 is an immunoblot photograph showing the results of antibody titer measurement by the dot blot method. In the figure, “MPG-1 concentration (ng)” represents the amount of MPG-1 dripped onto the PVDF membrane. “Individual 1” and “Individual 2” represent the two rabbits used in this example. “Before immunization” is the case of using serum obtained before immunization, and “5th” to “9th” are the cases of using serum obtained after the number of immunizations. Represents each.

  As shown in FIG. 3, in both individuals, when serum before immunization was used, spots were hardly detected, whereas when serum obtained after immunization was used, anti-IgG antibody A spot was detected. Further, as the number of times of immunization increased, the spots due to the anti-IgG antibody became clear, and the spots could be detected even when the MPG-1 concentration was low. These results indicate that the serum obtained by immunization has a high serum antibody titer, and that the serum antibody titer increased as the number of immunizations increased.

(4) Antiserum purification:
The serum (antiserum) obtained by immunization was purified by the following procedure to prepare an anti-MPG-1 antibody solution.

  First, 5 mL of the obtained antiserum was passed through a 0.45 μm filter, and an equal amount of 0.02 M sodium phosphate solution (pH 7.0) was added to the antiserum. Next, this antiserum solution was passed through a 1 mL volume of HiTrapr Protein A FF Column (antibody purification column, manufactured by Amersham Biosciences). This column was previously equilibrated with 0.02 M sodium phosphate solution (pH 7.0).

  Next, 10 mL of 0.2 M sodium phosphate solution was applied to the column, and the column was washed. Then, 0.1 M sodium citrate solution (pH 3.0) was applied as an eluent, and the antibody-containing solution was recovered. .

  Next, a sodium hydroxide solution was added to the obtained antibody-containing solution to neutralize it, and then the solvent was replaced with PBS using Sephadex G-25 Superfine Column (manufactured by Amersham Biosciences), and anti-MPG-1 antibody A liquid was obtained.

  Hereinafter, in Example 2 to Example 4, it was examined whether the anti-MPG-1 antibody prepared in Example 1 has an action of suppressing the physiological activity of MPG-1.

  In previous studies by the present inventors, it was found that MPG-1 prepared in Example 1 has a physiological activity that binds to mouse CD11c positive cells and promotes cytokine production in vitro. Therefore, in Example 2, the anti-MPG-1 antibody prepared in Example 1 was examined to determine whether MPG-1 has an action of suppressing binding to CD11c positive cells.

  First, mouse CD11c positive cells were separated by the following procedure.

  Eight-week-old male C57BL / 6 mice (purchased from Nihon Charles River Co., Ltd.) are killed, mesenteric lymph nodes are removed, tissues are loosened with tweezers, passed through a 200 mesh sieve, and single cell suspension A suspension was prepared. Next, the single cell suspension was washed three times with RPMI 1640 medium by centrifugation, and then suspended in RPMI 1640 medium supplemented with 10% fat calf serum.

  Next, using a mouse anti-CD11c antibody-attached bead (“CD11c micro beads”, manufactured by Miltenyi Biotec GmbH, Germany) and a magnetic cell sorter (“MiniMACS Separator”, manufactured by Miltenyi Biotec GmbH, Germany), a positive suspension from CD11c. Was separated and recovered. The procedure followed the attached protocol. As a result of the purity test by flow cytometry, about 80% of the collected cell suspension was CD11c positive cells.

Subsequently, ¹²⁵ I labeling of MPG-1 was performed by the following procedure.

The ¹²⁵ I labeling of MPG-1 was performed using the Bolton-Hunter reagent (N-succinimidyl 3- (4-hydroxy-5- [ ¹²⁵ I] iodophenyl) propionate hydride succinimide ester, manufactured by Amersham Biosciences, Inc.) The protocol was followed.

  First, the benzene solution of the Bolton-Hunter reagent placed in a small glass tube was dried in a nitrogen stream. Next, under ice cooling, 100 μg / 50 μL of MPG-1 solution was added to the small glass tube and reacted for 10 minutes, and then 0.5 mL of 1M glycine aqueous solution was added to stop the reaction.

Next, the volume of the reaction solution was increased to 2.5 mL, and then applied to a gel filtration column (product name “PD-10”, manufactured by Amersham Biosciences) filled with Sephadex G25, and the fraction passed through ( ¹²⁵ I labeling). MPG-1) was recovered. The radiometric value of the obtained fraction was 1.99 × 10 ⁹ cpm / 100 μg.

  Subsequently, the assay was performed.

First, a CD11c positive cell suspension is put into an Eppendorf tube (manufactured by Eppendorf Co., Ltd.), and ¹²⁵ I-labeled MPG-1 and the anti-MPG-1 antibody solution prepared in Example 1 are added to the Eppendorf tube (Eppendorf Co., Ltd.) in predetermined amounts. Mix and react for 1 hour at 4 ° C.

Next, each reaction solution is centrifuged, the supernatant is removed, the cell pellet is washed three times with PBS, and then a radiometric value of ¹²⁵ I-labeled MPG-1 bound to the cells is obtained using a γ counter. It was.

The results are shown in Table 1.

In the table, sample 3 represents the result when ¹²⁵ I-labeled MPG-1 and the anti-MPG-1 antibody solution prepared in Example 1 were added to the CD11c positive cell suspension.

Sample 1, sample 2, and sample 4 are controls. In the table, Sample 1 shows the result when only ¹²⁵ I-labeled MPG-1 was added to the CD11c positive cell suspension, and Sample 2 also showed ¹²⁵ I-labeled MPG-1 and an excess amount (50 times). Sample 4 represents the result when the standard antibody was used in place of the anti-MPG-1 antibody prepared in Example 1, respectively. Each experimental procedure is the same as in the case of Sample 3.

  As shown in Table 1, when the anti-MPG-1 antibody solution prepared in Example 1 was added (Sample 3), the amount of MPG-1 bound to the cells was decreased as compared with the case where it was not added (Sample 1). . This result shows that the antibody according to the present invention suppresses the binding of MPG-1 to CD11c positive cells.

In Sample 2, the amount of MPG-1 bound to the cells was greatly reduced by adding an excessive amount of unlabeled MPG-1. This result shows that the binding of the CD11c positive cell suspension and ¹²⁵ I-labeled MPG-1 in sample 3 is specific rather than non-specific.

In previous studies by the present inventors, the MPG-1 prepared in Example 1, coupled directly with TGF-beta _1, was found to be inhibiting physiological activity the activity of TGF-beta _1. In a third embodiment, it was examined whether the anti-MPG-1 antibody prepared in Example 1, there is the effect of suppressing the binding of MPG-1 and TGF-beta _1.

First, (1) ¹²⁵ I-labeled rhTGF-β ₁ (hereinafter referred to as “ ¹²⁵ I-labeled TGF”) solution (manufactured by Amersham Biosciences) 2 μL (corresponding to 0.24 μg / mL), (2) MPG -1 50 mM Tris-HCl solution 200 μL (50 μg) and (3) 10 μg or 50 μg of the antibody solution prepared in Example 1 were mixed and reacted at 37 ° C. for 3 hours.

Next, ¹²⁵ I-labeled TGF and MPG-1 conjugates and non-conjugates were recovered from the reaction solution by DEAE ion exchange chromatography. AKTA Prime (manufactured by Amersham Biosciences) was used as the HPLC apparatus, and DEAE Sepharose FF (1 mL) was used as the ion exchange resin column. The detection wavelength was set at 280 nm.

The specific procedure for chromatography is as follows. First, 200 μL of the reaction solution was applied to this column and washed with 10 mL of 50 mM Tris-HCl buffer to recover a non-adsorbed fraction (a conjugate of ¹²⁵ I-labeled TGF and MPG-1). Next, linear gradient elution was performed using eluent A (Tris-HCl) and eluent B (Tris-HCl containing 1M sodium chloride) (elution amount: 20 mL), and the adsorbed fraction (unbound ¹²⁵ I-labeled TGF). ) Was recovered. The eluate was collected every 1 mL with a fraction collector.

Next, for each of the recovered non-adsorbed fraction (a conjugate of ¹²⁵ I-labeled TGF and MPG-1) and the adsorbed fraction (non-bound ¹²⁵ I-labeled TGF), a radiometric measurement value was obtained using a γ counter. It was.

The results are shown in Table 2.

  In the table, sample 3 represents the result when 10 μg of the antibody prepared in Example 1 was added, and sample 4 also represents the result when 50 μg was added.

  Sample 1 and sample 2 are controls. In the table, sample 1 shows the result when MPG-1 and the antibody prepared in Example 1 are not added, and sample 2 shows the result when MPG-1 is added (the antibody prepared in Example 1 is not added). Case) results. Each experimental procedure is the same as in the case of Sample 3 or Sample 4.

As shown in Table 2, compared to the case where MPG-1 was added (Sample 2) and the case where MPG-1 was not added (Sample 1), the radiation measurement value of the conjugate of ¹²⁵ I-labeled TGF and MPG-1 was greatly increased. Increasing and the ratio of conjugates to total radiation measurements also increased significantly.

  On the other hand, when 10 μg of the antibody prepared in Example 1 was added (sample 3), the ratio of the conjugate to the total radiation measurement value was 67.2%, and when 50 μg was added (sample 4), the ratio was 40 Decrease to 8% respectively.

  These results suggest that the antibody according to the present invention suppresses the binding between MPG-1 and TGF-β.

  In previous studies by the present inventors, it was found that MPG-1 prepared in Example 1 has a physiological activity that suppresses the growth of sarcoma 180 cells (solid cancer cells). Therefore, in Example 4, it was examined whether the anti-MPG-1 antibody prepared in Example 1 has an action of suppressing the growth inhibition of sarcoma 180 cells by MPG-1.

First, 1 × 10 ⁶ sarcoma 180 cells were transplanted subcutaneously into the lower heel of 5-week-old female ICR mice (purchased from Clea Japan Co., Ltd.). As the sarcoma 180 cells, those maintained by passage in the peritoneal cavity of female ICR mice at Kureha Biomedical Research Institute, Inc. were used. In this experiment, 8 mice were used for each group.

  Next, from the next day after sarcoma 180 cell transplantation, MPG-1 was intraperitoneally administered every other day at 100 mg / kg / dose. Further, the anti-MPG-1 antibody solution prepared in Example 1 was intravenously injected twice a week for 2 weeks after transplantation at 50 μg / animal.

  On the 25th day after transplantation, the mice were killed to remove tumor nodules, and their weights were measured.

The results are shown in Table 3.

  In the table, sample 3 represents the results when both MPG-1 and anti-MPG-1 antibody were administered.

  Sample 1, sample 2, and sample 4 are controls. In the table, sample 1 shows the results when physiological saline is administered instead of MPG-1, sample 2 shows the results when MPG-1 is administered, and sample 4 shows the anti-tumor produced in Example 1. The results when using the standard antibody instead of the MPG-1 antibody are shown. Each experimental procedure is the same as in the case of Sample 3.

  In the table, “inhibition rate” indicates a tumor cell growth inhibition rate, and the value obtained by subtracting the average tumor weight of each sample from the average tumor weight of sample 1 is divided by the average tumor weight of sample 1 to be 100. Calculated by multiplying.

  As shown in Table 3, tumor cell growth inhibition rate decreased from 96% to 84% when anti-MPG-1 antibody was administered (sample 3) and when only MPG-1 was administered (sample 2) did. This result suggests that the antibody according to the present invention has an action of suppressing the growth inhibition of sarcoma 180 cells by MPG-1.

  As described above, the results of Example 2 to Example 4 indicate that the antibody prepared in Example 1 has an action of suppressing the physiological activity of MPG-1.

  In Example 5, an attempt was made to construct an ELISA system using the anti-MPG-1 antibody prepared in Example 1.

  First, the capture antibody was immobilized by the following procedure. First, the anti-MPG-1 antibody solution prepared in Example 1 was diluted with 0.05 M carbonate buffer (pH 9.6) so as to be 10 μg / L. Next, 200 μL of the prepared antibody solution is dispensed into each well of a micro ELISA plate (product name “Coster EIA / RIA 8well STIRIP”, Corning, USA) for 3 hours in a 4 ° C. wet box. The antibody was immobilized by allowing to stand. Then, after the antibody was immobilized, the antibody solution was removed, and each well was washed three times with PBS-Tween. Washing was performed by putting PBS-Tween into each well, permeating on a micromixer at 25 ° C. for 3 minutes, and then removing PBS-Tween.

  Subsequently, the detection antibody was labeled with biotin. Biotinylation Kit (Sulfo-OSu, manufactured by Dojindo Laboratories) was used for biotin labeling. The procedure followed the attached protocol.

  First, put the antibody solution prepared in the above procedure into a sample tube, add a sodium bicarbonate buffer solution, and adjust the salt concentration to 50 mM and the protein concentration to 5.0 mg / 0.5 mL. Mixed well. Next, Biotin- (AC5) 2Sulfo-OSu was prepared to 10 mg / 750 μL, 17.5 μL of the solution was added to the antibody solution, and mixed well using a vortex mixer, and then reacted at 25 ° C. for 2 hours. . Next, the reaction solution was applied to a gel filtration column, and a biotin-labeled antibody solution was recovered. The gel filtration column was equilibrated with PBS.

  Subsequently, an assay by ELISA was performed.

  First, 300 μL of a BSA (bovine serum albumin, hereinafter the same) solution was dispensed into each well of a plate on which a capture antibody was immobilized, thereby blocking nonspecific reaction of the antigen protein (blocking treatment). Next, after removing the blocking solution, 100 μL of an MPG-1 solution having a predetermined concentration (10 ng / mL to 10 μg / L) was dispensed and allowed to stand at room temperature for 1 hour (binding of capture antibody and antigen).

  Next, 100 μL of a biotin-labeled antibody solution having a predetermined concentration was dispensed into each well of the plate and allowed to stand at room temperature for 1 hour (binding antibody-antigen-detection antibody binding). Next, the biotin-labeled antibody solution was removed, and each well was washed 5 times with PBS-Tween.

  Next, 100 μL of HRP-labeled streptavidin (HRP-Streptavidin Conjugate, manufactured by ZYMED LABORATORIES) was dispensed into each well of the plate and allowed to stand at room temperature for 1 hour (supplementary antibody-antigen-detection antibody-HRP Combined). Next, HRP-labeled streptavidin was removed and each well was washed 3 times with PBS-Tween. “HRP (horseradish peroxidase)” is an enzyme (peroxidase) used for detecting an antigen by ELISA or the like, and catalyzes a color reaction when a chromogenic substrate is added.

  Next, MPG-1 was detected by a color reaction with HRP. The detection of the chromogenic reaction was performed using Protein Detector ELISA Kit (this kit contains “ABTS (peroxidase chromogenic substrate)” manufactured by KPL).

  Equal amounts of the reagents “ABTS Substrate Solution” and “Peroxidase Solution B” included in the kit were mixed, 200 μL of the solution was dispensed into each well on the plate, and allowed to stand for 5 to 15 minutes for reaction. . Next, 100 μL of the reagent “Peroxidase Stop Solution” included in the kit was dispensed to stop color development. Next, the absorbance at 405 nm was measured using a plate reader, and a standard line was created based on the measured value.

  The results are shown in FIG. In FIG. 4, the horizontal axis represents MPG-1 concentration (logarithm), and the vertical axis represents absorbance (logarithm).

  As a result, as shown in FIG. 4, a linear relationship was obtained between the concentration of the MPG-1 solution (10 ng / mL to 10 μg / L) and the absorbance. This indicates that MPG-1 can be detected by the ELISA system according to the present invention, and quantitative detection of MPG-1 is possible.

  In Example 6, using the ELISA system constructed in Example 5, it was examined whether or not MPG-1 contained in the fruit bodies of various mushrooms.

  First, various mushroom bodies shown in Table 4 below were purchased from mail order / department stores. For the identification and confirmation of purchased mushrooms, refer to “Primary Color Japanese New Fungi Encyclopedia (I)” and “Primary Color Japanese New Fungus Encyclopedia (II)” (both issued by Hoikusha, 1987). This was done by observing the morphology of

  Next, the purchased fruit body was freeze-dried in a raw state and pulverized to obtain a mushroom powder. Next, 1.0 L of pure water was added to 20 g of each mushroom powder, followed by stirring and extraction at 98 ° C. for 1 hour, centrifugation (15,000 rpm, 20 minutes, 4 ° C.), and the supernatant was collected. Further, 1.0 L of pure water was added to the residue remaining after the supernatant was collected, and the same operation as described above was performed, and the supernatant was again collected and combined with the previously collected supernatant. The whole supernatant was dialyzed against pure water for 3 days using a dialysis membrane (“Spectra / Por3 membrane”, fractional molecular weight 3,500 D, manufactured by Spectrum), then freeze-dried, and “mushroom hot water” An “extraction fraction” was prepared.

  Next, after dissolving the prepared mushroom hot water extraction fraction in pure water to a concentration of 10 mg / mL, using the ELISA system constructed in Example 5, each mushroom contains MPG-1 I checked. The content of MPG-1 was obtained from the measured absorbance using the standard line in FIG.

The results are shown in Table 4. The detection limit was 1 μg / g.

  As shown in Table 4, MPG-1 could be detected quantitatively by using the ELISA system constructed in Example 5. Moreover, it was confirmed that MPG-1 is a component specific to matsutake and is hardly detected from other mushrooms. In addition, it has been found that MPG-1 is contained most in matsutake from Kyoto.

  In Example 7, the ELISA system constructed in Example 5 was used to examine whether or not MPG-1 contained in the matsutake mycelium.

  In this example, eight Matsutake strains (IFO06915, IFO06925, IFO06935, IFO06935, IFO30605, IFO30605, IFO30606, IFO460038) that were transferred from the Fermentation Research Institute and maintained by Kureha Biomedical Research Institute, Inc., and , Using Suehirotake strain (Kureha storage number CM556), which has been isolated and maintained by Kureha Biomedical Research Laboratories, with isolated hyphae isolated from fruit bodies of Suehirotake collected in Tochigi Prefecture in 1966 It was.

  First, a sterilized medium (containing 3% glucose and 0.3% yeast extract, pH 6.0) is put into a 500 mL Erlenmeyer flask, and each of these strains is inoculated therein, and cultured at 24 ° C. for 10 weeks. did.

  Next, after culturing for 10 weeks, the culture (medium and cell mass) was dispersed and crushed using a homogenizer, placed in a water bath at 93 to 98 ° C., and stirred for 3 hours using a stirrer. And after cooling to room temperature, it centrifuged (12,000 rpm, 20 minutes, 4 degreeC), and collect | recovered supernatants. The residue remaining after the supernatant was collected was subjected to the same operation as described above, and the supernatant was again collected. This operation was repeated 3 times in total, and the collected whole supernatants were combined. Then, using a dialysis membrane (“Spectra / Por3 membrane”, molecular weight cut-off 3,500D, manufactured by Spectrum), the whole supernatant is dialyzed against pure water for 3 days, and the dialysis membrane solution is concentrated with a rotary evaporator. After that, it was freeze-dried to prepare “mycelium hot water extraction fraction”.

  Next, after the prepared mycelium hot water extraction fraction was dissolved in pure water to a concentration of 10 mg / mL, MPG-1 contained in each mycelium using the ELISA system constructed in Example 5. Investigate whether it is. The content of MPG-1 was obtained from the measured absorbance using the standard line in FIG.

The results are shown in Table 5. The detection limit was 1 μg / g.

  As shown in Table 5, MPG-1 could also be detected from the matsutake mycelium. That is, this experimental result shows that by using the ELISA system according to the present invention, it is possible to detect not only the content of components derived from Matsutake fruiting bodies but also the content of components derived from Matsutake mycelium.

  In Example 8, the reactivity between the ELISA system constructed in Example 5 and the polysaccharide preparation was examined.

  Among the polysaccharides used in this example, glycogen II, glycogen IX, dextrin, corn starch, rice starch, amylose, amylopectin, pullulan, laminarin, yeast glucan, barley glucan and inulin were purchased from Sigma-Aldrich. Lentinan was purchased from Ajinomoto Co., Inc., and Sonifilan was purchased from Kaken Pharmaceutical Co., Ltd.

  The preparation derived from Agaricus coconut body (scientific name “Agaricus blazei Murill”) is made from commercially available Brazilian dried Agaricus product (purchased from Uchida Shoji) as a raw material, and the method of Mizuno et al. (Takashi Mizuno et al, “Antitumor fruiting body of Agaricus blazei Murill ”Agric Biol Chem, 54: 2889-2896, 1990).

  The specific procedure is as follows.

  Agaricus koji dried fruit bodies were finely pulverized, 1 L of 80% ethanol was added to 100 g, and stirring and extraction operation was performed at 85 ° C. for 3 hours, and the operation was repeated three times to obtain an extraction residue. Next, 1 L of pure water was added to this extraction residue, and a stirring extraction operation was performed at 98 ° C. for 3 hours, and this operation was repeated three times to obtain an extract. Next, after concentrating the extract under reduced pressure to 500 mL using a rotary evaporator, 4 times volume of ethanol was added and allowed to stand for 3 hours, followed by centrifugation (12,000 rpm, 30 minutes, 4 ° C.), The precipitated fraction was collected.

  The precipitated fraction was then purified by DEAE ion exchange chromatography. First, the precipitate fraction was dissolved in 50 mM Tris-HCl buffer (pH 7.5), and then the solution was applied to a DEAE ion exchange column, and eluent A (Tris-HCl) and eluent B ( 1M sodium chloride-containing Tris-HCl) was used for linear gradient elution, and the FA-1 and FA-2 fractions were collected. The column was equilibrated with 50 mM Tris-HCl buffer (pH 7.5).

  Next, gel filtration of those fractions was performed. The FA-1 fraction and the FA-2 fraction were respectively applied to Sephacryl S-100 (Pharmacia) packed columns (diameter: 16 mm, length: 30 cm), and were applied to FA-1-a and FA-2-b. Corresponding fractions were obtained. Further, these fractions were chromatographed, and fractions corresponding to FA-1-a-β, FA-1-a-α, FA-2-a-β, and FA-2-a-α, respectively. Got the minute.

About each obtained fraction, as a result of measuring sugar: protein ratio by the colorimetric method, the ratio was 98: 2, 99: 1, 99: 1, 99: 1, respectively. As a result of analyzing the constituent sugars using HPLC, most of the fractions were glucose, and galactose, fucose, ribose, mannose, etc. were also detected. As a result of ¹ H-NMR analysis for each of the obtained fractions, it was found that the FA-1-a-β fraction was 4.6 ppm (β-1,4 or β-1,6 bond) and 5.4 ppm. In the FA-1-a-α fraction, the peak of (α-1,4 or α-1,3 bond) is expressed as FA-2 in the FA-1-a-α fraction. In the -a-β fraction, a peak of 4.6 ppm (β-1,4 or β-1,6 bond) was observed.

  Specimens derived from the dried fruit mushrooms (scientific name “Phellinus linteus”) are obtained from commercial fruit mushroom fruit bodies, and the method of Kim et al. (GY Kim et al, “Purification and characterization from the fruiting body of Phellinus liteus (Berk and MA Curtis ) Teng. ”Biosour. Technol. 89: 81-87, 2003).

  The specific procedure is as follows.

  1 g of 80% ethanol is added to 100 g of dried dried fruit body fruit (“Natural Meshimakobu 100% powder”, manufactured by Nippon Seiyaku Co., Ltd.), followed by stirring and extraction at 85 ° C. for 3 hours, and the operation is repeated three times. The extraction residue was obtained. Next, 1 L of pure water was added to this extraction residue, and a stirring extraction operation was performed at 98 ° C. for 3 hours, and this operation was repeated three times to obtain an extract. Next, after concentrating the extract under reduced pressure to 500 mL using a rotary evaporator, 4 times volume of ethanol was added and allowed to stand for 3 hours, followed by centrifugation (12,000 rpm, 30 minutes, 4 ° C.), The precipitated fraction was collected.

  The precipitated fraction was then purified by DEAE ion exchange chromatography. First, the precipitate fraction was dissolved in 50 mM Tris-HCl buffer (pH 7.5), and then the solution was applied to a DEAE ion exchange column, and eluent A (Tris-HCl) and eluent B ( 1M sodium chloride-containing Tris-HCl) was used for linear gradient elution, and fractions corresponding to PL were collected. The column was equilibrated with 50 mM Tris-HCl buffer (pH 7.5).

  As a result of measuring the sugar: protein ratio by a colorimetric method for the obtained PL fraction, the ratio was 99: 1. As a result of analyzing the constituent sugars using HPLC, most of all the fractions were glucose, and mannose, galactose, ribose and the like were also detected.

  After dissolving the above polysaccharide preparation in pure water to a concentration of 10 mg / mL, it was examined whether each preparation contains MPG-1 using the ELISA system constructed in Example 5. . The content of MPG-1 was obtained from the measured absorbance using the standard line in FIG.

The results are shown in Table 6. The detection limit was 1 μg / g.

  As shown in Table 6, all 19 types of polysaccharide preparations were negative for reaction.

  In Example 9, the mushroom health food marketed in a pharmacy or the like was examined using the ELISA system constructed in Example 5 to determine whether a matsutake-derived component was contained.

  The mushroom health foods used were Matsumax 600 ("Matsumax" is a registered trademark and sold by Sankyo Corporation), Matsumax 200 ("Matsumax" is a registered trademark and sold by Sankyo Corporation), and Sensei Russia (registered trademark, distributor: Sandley Co., Ltd.), Manjutan (distributor: Kanebo Pharmaceutical Co., Ltd.), Agaricus Blazei (distributor: FANCL Corporation), Agaricus koji concentrate (distributor: Sunwell Corporation), Hime Matsutake (Distributor: Nippon Chemifa Co., Ltd.), Meshima Cobb (Distributor: FANCL Co., Ltd.), Meshima Cell Direct (registered trademark, Distributor: Lifenics Co., Ltd.), King Meshima Cobb (Space Three Co., Ltd.), Uchida Reishi (distributor: Uchida Shoji), Maitake extract (distributor: FANCL Corporation), Rokkaku Reishi (distributor: Suntory Ken) ), Hatakeshimeji (Publisher: Corporation Yongchang source), mushroom power (registered trademark, Publisher: Takara Agri Co., Ltd.), which is more than 15 dishes.

  After adding pure water to these products and suspending them to a concentration of 1 g / 100 mL, they were dispersed and crushed using a homogenizer, placed in a water bath at 93 to 98 ° C., and stirred with a stirrer for 3 hours. . And after cooling to room temperature, it centrifuged (12,000 rpm, 20 minutes, 4 degreeC), and collect | recovered supernatants.

  And about the supernatant, it was investigated whether each product contained MPG-1 using the ELISA system constructed in Example 5. The content of MPG-1 was obtained from the measured absorbance using the standard line in FIG.

The results are shown in Table 7. The detection limit was 1 μg / g.

  As shown in Table 7, only Matsutake-derived products could be detected using the ELISA system according to the present invention.

  In Example 10, it was investigated whether the matsutake-derived component contained in food and drink can be quantitatively detected by using the ELISA system constructed in Example 5.

  As foods and drinks containing components derived from matsutake, matsutake rice, matsutake mycelium bread, matsutake flavored soup, matsutake mycelium miso soup, matsutake mycelium tea, and matsutake mycelium yogurt were prepared.

  Preparation of matsutake rice was performed according to the following procedure.

  First, after washing 3 rice (420 g), the rice was drained with a sieve, and then water was added so as to be 1.5 times the weight of the rice after draining, followed by immersion for 30 minutes. The total weight was 1,050 g. Next, 1.8 g of matsutake mycelium dry powder was added and cooked with a rice cooker (product name “SR-VTM10”, manufactured by National Corporation). And after cooking, it moved to the rice stand, applied the wet cloth, and left to cool. In the control, 1.8 g of wheat flour was used instead of matsutake mycelium.

  Separately, rice with matsutake was prepared using a retort product. Using “rice cooked matsutake mushroom rice (Ezaki Glico Co., Ltd.)”, it was heated according to the described cooking procedure to prepare matsutake rice.

  Next, for each matsutake-containing rice, 3 L of pure water was added to 100 g of rice, dispersed and crushed using a homogenizer, placed in a 98 ° C. water bath, and stirred with a stirrer for 3 hours. And after cooling to room temperature, centrifugation (8,000 rpm, 30 minutes, 4 degreeC) was performed, supernatant was collect | recovered, and the supernatant was frozen and stored with the household refrigerator.

  The production of matsutake mycelium bread was performed according to the following procedure.

  Home bakery (automatic home bakery bread club BB-HA10, manufactured by ZOJIRUSHI CORP.) Attached to bread case 210ml of drinking water, bread crumb (for bakery bread club mixed bread BB-MJ10-J1) 330g, dry yeast 3g, matsutake mycelium dried 1.8 g of powder was added and bread was prepared according to the described cooking procedure. In the control, 1.8 g of wheat flour was used instead of matsutake mycelium.

  Next, the prepared bread was divided into three parts, 3 L of pure water was added to each piece, dispersed and crushed using a homogenizer, placed in a 98 ° C. water bath, and stirred for 3 hours using a stirrer. And after cooling to room temperature, centrifugation (8,000 rpm, 30 minutes, 4 degreeC) was performed, supernatant was collect | recovered, and the supernatant was frozen and stored with the household refrigerator.

  Preparation of matsutake-flavored soup, miso soup with matsutake mycelia, and tea with matsutake mycelium were performed according to the following procedure.

  The soup was prepared by pouring hot water in accordance with the described cooking procedure for “Ryoten's soup matsutake (manufactured by Marumiya Food Industry Co., Ltd.)” and the instant product of the soup that was described as having a matsutake flavor. . Miso soup was prepared by adding 1.8 g of dried matsutake mycelium powder to commercial instant miso soup and pouring hot water. The tea was prepared by adding 1.8 g of dried matsutake mycelium powder to a predetermined amount of tea leaves and pouring hot water.

  Next, after cooling these foods and drinks to room temperature, centrifugation (8,000 rpm, 30 minutes, 4 degreeC) was performed, supernatants were collect | recovered, and the supernatants were cryopreserved with the domestic refrigerator.

  The yogurt containing matsutake mycelium was prepared according to the following procedure.

  First, 1.8 g of dried matsutake mycelium powder and 60 mL of commercially available plain yogurt (starter) were added to 400 mL of commercially available milk in a paper pack container, and the mixture was well stirred to close the opening of the paper pack container. In the control, 1.8 g of wheat flour was used instead of matsutake mycelium.

  Next, hot water was poured into a yogurt maker (product name “MM-AV05D”, manufactured by ZOJIRUSHI CORP.), A paper pack container was immersed therein, the lid was closed, and fermentation was performed for about 8 hours. And after fermentation, the whole paper pack container was put into a household refrigerator and stored refrigerated.

  Next, 50 g of yogurt was taken out from the paper pack container, 200 mL of pure water was added, dispersed and crushed using a homogenizer, placed in a 98 ° C. water bath, and stirred using a stirrer for 3 hours. And after cooling to room temperature, it centrifuged (8,000 rpm, 30 minutes, 4 degreeC), the supernatant was collect | recovered, and the supernatant was frozen and preserve | saved again with the household refrigerator.

  As mentioned above, about the supernatant collect | recovered from each foodstuff, using the ELISA system constructed | assembled in Example 5, respectively, MPG-1 content was quantified. The content of MPG-1 was obtained from the measured absorbance using the standard curve in FIG.

The results are shown in Table 8. The detection limit was 1 μg / g.

  As shown in Table 8, by using the ELISA system constructed in Example 5, matsutake-derived components contained in food and drink could be detected quantitatively. Moreover, it turned out that the food / beverage which does not actually contain a matsutake origin component can be discriminate | determined simply and highly accurately by using the ELISA system which concerns on this invention as the result of the sample 7 shows.

  In Example 11, immunostaining was attempted using the anti-MPG-1 antibody prepared in Example 1.

  First, matsutake mycelium preparation CM6271 at 150 mg / kg or MPG-1 prepared in Example 1 was orally administered to 8-week-old male C57BL / 6 mice at 25 mg / kg. Next, the mice were killed 1 hour or 5 hours after oral administration, and Peyer's patches of intestinal immunity tissue were removed and fixed in formalin.

  Next, a preparation specimen of the tissue was prepared by a predetermined method, and immunostaining was performed using the anti-MPG-1 antibody prepared in Example 1.

  The results are shown in FIG. FIG. 5 is a photomicrograph showing the dynamics of MPG-1 in the mouse intestinal Peyer's patch after oral administration of the matsutake mycelium preparation.

  In the figure, the left photograph (A) is when matsutake mycelium preparation is not administered, the middle photograph (B) is when killed 1 hour after administration of matsutake mycelium preparation, and the right photograph (C) is The same applies when killed after 5 hours. A portion surrounded by an ellipse is a portion stained by immunostaining. The magnification of each micrograph is 10 times.

  FIG. 5 shows that MPG-1 is localized in the M cell region of the intestinal Peyer's patch 1 hour after oral administration, and locally in the parafollicular region in addition to the M cell region 5 hours after oral administration. I found out that it exists. Moreover, from this experimental result, it was found that MPG-1 reaches the intestinal tract immune tissue while maintaining its antigenic structure and binds to cells of the tissue.

  In Example 12, using the ELISA system constructed in Example 5, the course of MPG-1 concentration in the blood when matsutake mycelium preparation was administered was examined.

  First, after suspending matsutake mycelium preparation CM6271 in a 0.5% methylcellulose 400CP (manufactured by Shin-Etsu Chemical Co., Ltd.) solution at a rate of 200 mg / kg, 7-week-old SD male rats (Nippon Charles River) 10 mL / kg was orally administered to the company.

  Next, blood was collected 1, 4, 8, 24, and 168 hours after CM6271 administration. Next, the collected blood was allowed to stand at room temperature for 1 hour and then stored at 4 ° C. overnight. Then, centrifugation (3,000 rpm, 10 minutes) was performed to obtain serum.

  Next, using the ELISA system constructed in Example 5, the MPG-1 content in each obtained serum was quantified. The content of MPG-1 was obtained from the measured absorbance using the standard curve in FIG.

  The results are shown in FIG. FIG. 6 is a graph showing the course of MPG-1 concentration in serum after oral administration of the matsutake mycelium preparation.

  In the graph, the horizontal axis represents the elapsed time (hr) from oral administration of the matsutake mycelium preparation, and the vertical axis represents the MPG-1 content (ng / mL) in the serum.

  From FIG. 6, when the matsutake mycelium preparation was orally administered, the blood concentration of MPG-1 increased from about 4 hours after administration, and reached the maximum concentration about 8 to 24 hours after administration, and 168 hours passed. Later, it was found that blood levels were significantly reduced.

  According to the present invention, it is possible to accurately detect the amount of α-glucan-protein complex contained in foods and drinks and pharmaceuticals, in particular, health foods and supplements taken daily. Therefore, it is useful in that the quality assurance degree of those products can be increased. Further, according to the present invention, since the intake (dose) of a specific physiologically active substance can be accurately grasped, it is also useful from the viewpoint of health care (health management).

  In addition, according to the present invention, since the blood concentration of a specific physiologically active substance can be accurately grasped, the pharmacokinetics after intake of food and drink can be grasped, which is useful from the viewpoint of health care (health management).

The figure which shows the antigen preparation procedure and the fraction used in Example 1. FIG. The graph which shows the antibody titer measured in Example 1 by ELISA method. In Example 1, the immunoblot photograph which shows the result of the antibody titer measurement by the dot blot method. In Example 5, the graph which shows the standard line showing the relationship between MPG-1 density | concentration and a light absorbency. In Example 11, the microscope picture which shows the dynamics of MPG-1 in the mouse | mouth Peyer's board after a matsutake mycelium formulation oral administration. In Example 12, the graph which shows the progress of the density | concentration of MPG-1 in serum after oral administration of a matsutake mycelium formulation.

Claims (8)

  An antibody that specifically binds to an α-glucan-protein complex derived from matsutake.   The antibody according to claim 1, which is an anti-MPG-1 antibody that specifically binds to MPG-1, which is a component unique to matsutake.   A matsutake-derived component detection method for detecting a matsutake-derived component using the antibody according to claim 1 or 2.   The matsutake-derived component detection method according to claim 3, wherein the antibody is used as a capture antibody and / or a detection antibody in an ELISA method. Capture antibody immobilization procedure for immobilizing the antibody on a predetermined surface of a reaction field;
An antigen supply procedure for supplying a target sample to the reaction field;
A detection antibody supply procedure for supplying the antibody modified so that it can bind to a detection enzyme to the reaction field;
A chromogenic substrate supply procedure for supplying the chromogenic substrate of the detection enzyme to the reaction field;
A color reaction detection procedure for detecting a reaction between the detection enzyme and the color substrate;
The matsutake-derived component detection method according to claim 3 or 4, comprising at least   A kit for detecting an ingredient derived from matsutake, comprising at least the antibody according to claim 1.   The matsutake-derived component detection kit according to claim 6, further comprising a reaction field in which the antibody is immobilized on a predetermined surface as a capture antibody. The matsutake-derived component detection kit according to claim 6 or 7, wherein the detection antibody comprises the antibody modified so as to be capable of binding to a detection enzyme.