JP2015127666A - Method of quickly detecting kudoa septempunctata - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of specifically, quickly, and easily detecting Kudoa septempunctata.SOLUTION: A method includes a step that involves use of first and second monoclonal antibodies against Kudoa septempunctata spores to form a complex containing the first monoclonal antibody fixed to a solid phase, the second monoclonal antibody that has been labeled, and a Kudoa septempunctata spore-derived protein, where the first monoclonal antibody is extracted from the Kudoa septempunctata spores and recognizes a protein having an XELRE (X represents Ala or Ser) amino-acid sequence in an N-terminal and molecular weight of approximately 23,000 as an antigen.

Description

  The present invention relates to a rapid detection method for quadruple septum puntata.

  Kudoa septempuctata is a type of myxosporeworm, and is a causative agent of food poisoning caused by raw eating of Japanese flounder. Known detection methods for quadrature septum puntata include a method using real-time PCR (Non-patent Document 1) and a method using a polyclonal antibody against quadrature septum puntata (Patent Document 1).

  However, the method using real-time PCR and the method using a polyclonal antibody require not only a special apparatus, but also the time required for the examination is about 3 hours and 1.5 hours, respectively, and it takes a long time for the determination.

  Kudoa septenpuntata forms mucous spores in the infected soleus muscle. However, so far, there is not enough information about the ecology of Kudoa septenpuntata, and little is known about its genomic information and the pathogenesis of food poisoning.

JP 2013-36943 A

Notice of Director of Monitoring and Safety Division, Food Safety Department, Ministry of Health, Labor and Welfare dated July 11, 2011

  The present invention has been made in view of the above-described background art, and an object of the present invention is to provide a method for specifically, rapidly and simply detecting a quadrupole / septentater.

  The detection method according to the present invention is a method for detecting a quadrature septum puntata by an antigen-antibody reaction using a monoclonal antibody exhibiting a reactivity specific to the quadra septum puntata.

  According to the present invention, it is possible to know whether or not there is contamination by the quadrature septa pactata in a simple and reliable manner.

FIG. 1 is a schematic configuration diagram of an immunochromatographic test tool according to an embodiment of the present invention. FIG. 2 is an image showing the results of electrophoresis of the quadratic septum puntata antigen purified by affinity using the monoclonal antibody produced by the hybridoma Ks24-15G11. Lane 1 shows the marker, and Lane 2 shows the quadrature septum puntata antigen that has been affinity-purified with the same antibody. FIG. 3 is an image showing the result of immunoblotting of the antigen recognized by the monoclonal antibody produced by the hybridoma Ks24-15G11. Lane 1 shows the marker, and Lane 2 shows the quadrature septum puntata antigen that has been affinity-purified with the same antibody.

  The detection method according to the present invention is a method that utilizes an antigen-antibody reaction using a monoclonal antibody against a specific protein of Kdoa septum pactata.

  The sample to which the method according to the present invention is applied may be any sample that may be contaminated by the presence of the quadruceptor spores, that is, the quadruceptentata. The method of the present invention is a method for detecting quadrature septum pterata spores, but if the presence of spores can be detected, infection by quadrature septum puntata becomes clear.

  Kudoa septenpuntata is a parasite detected from flounder, and the specimen from which the sample is prepared is flounder or food using flounder. The sample may be, for example, flounder or a food product using flounder as it is, or a processed product thereof. Examples of a method for preparing a sample include a method of homogenizing a specimen such as Japanese flounder in phosphate buffered saline (PBS) or physiological saline to obtain a supernatant.

  The monoclonal antibody used in the present invention has a low reactivity to at least K. yasunagai, K. amamiensis, K. iwatai, and Kdo septenpactata. It is a monoclonal antibody having high reactivity. The monoclonal antibody is extracted from Kdoa septum puntata spores and recognizes a protein having a molecular weight of about 23,000 having an amino acid sequence of XELRE (where X is Ala or Ser) at the N-terminus.

  The monoclonal antibody can be produced from a hybridoma obtained by fusing a myeloma with a spleen cell of a mouse that has been inoculated as an immunogen with a protein derived from a spore of Kdoa septenpuntata, for example, a peptide having the above amino acid sequence. Hybridomas can be generated by known methods. The monoclonal antibody can be obtained from, for example, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan. It may be an antibody (hereinafter referred to as “Ks24-15G11 antibody”) produced from the deposited hybridoma (hereinafter referred to as “hybridoma Ks24-15G11”). In addition, the monoclonal antibody used in the present invention is, for example, 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan. It may be an antibody (hereinafter referred to as “Ks18-11H5 antibody”) produced from a hybridoma deposited under accession number NITE P-01767 (hereinafter referred to as “hybridoma Ks18-11H5”). In addition, a monoclonal antibody having low reactivity to at least Kudoa Yasunagai, Kudoa Amamiensis, Kudoa Iwatai, and high reactivity to Kudoa septeptuntata can be used.

Ks24-15G11 antibody and Ks18-11H5 antibody are hybridoma Ks24-15G11 and hybridoma Ks18-11H5, respectively. Hybridoma Growth Medium (Hypoxanthine / Thymidine supplemented medium: StemCell Technologies Inc., C. medium, etc.) It is obtained by culturing at 37 ° C. in the presence of 5% CO ₂ and collecting and purifying the produced monoclonal antibody by a known method. The monoclonal antibody in the present invention includes fragments thereof, modified antibodies such as chimeric antibodies and humanized antibodies, and mutated antibodies as long as they are reactive with quadruceptor spores.

  An immunological assay (immunoassay) is a method for qualitatively or quantitatively detecting a substance using an antigen-antibody reaction. Immunological measurement methods are also known, and methods that can be used in the present invention are, for example, radioimmunoassay (RIA), enzyme immunoassay (EIA), enzyme immunoassay (ELISA), homogeneous enzyme immunoassay, Fluorescence immunoassay (FIA), fluorescence polarization, chemiluminescence immunoassay (CLIA), immunochromatography.

  Further, in the present invention, among the immunological measurement methods, the first and second monoclonal antibodies against Kdoa septenpuntata spores are used, and the first monoclonal antibody labeled with the first monoclonal antibody immobilized on a solid phase is used. A method using a so-called sandwich method as a measurement principle, which includes a step of forming a complex containing the second monoclonal antibody and a spore-derived protein of quadrature septum pactata is preferable. A method using the sandwich method as a measurement principle can be, for example, ELISA, immunochromatography, or the like. By applying the sandwich method, sensitivity and specificity are improved, and detection accuracy is increased. The complex formed by the second monoclonal antibody only needs to contain at least the spore-derived protein of quadrature septum puntata recognized by the second monoclonal antibody, and may contain the entire spore or one of the spores. Whether it is a department

  In the case of the sandwich method, the first monoclonal antibody and the second monoclonal antibody may be the same antibody or different antibodies. The first monoclonal antibody is extracted from the Ks18-11H5 antibody or the quadratic septa puntata spores and has an N-terminal XELRE (where X is Ala or Ser) and has a molecular weight of about 23,000. An antibody that recognizes a protein, for example, the Ks24-15G11 antibody is preferably used. This is because the specificity to the quadratic septum puntata increases.

  The second monoclonal antibody may also be an antibody that has low reactivity with Kudo Yasunagai, Kudo Amamiensis, and Kudo Iwatai, and shows high reactivity with Kudoa septum puntata. For example, the above-mentioned Ks18-11H5 antibody, or extracted from quadrature septum pterata spores, recognizes about 23,000 proteins having the amino acid sequence of XELRE (where X is Ala or Ser) at the N-terminus. It may be an antibody, for example the Ks24-15G11 antibody. At this time, when the same antibody as the first antibody is used, for example, when the first antibody is a Ks24-15G11 antibody, a nonspecific reaction may be observed. It is preferable to combine an antibody different from that of the first antibody, such as the Ks18-11H5 antibody, with this antibody. The second antibody to be combined with the first antibody can be appropriately determined according to detection sensitivity and crossing characteristics. In addition, either the first antibody or the second antibody may be a polyclonal antibody that is reactive with quadrature septa pactata spores. However, the use of a polyclonal antibody as the second antibody is preferably applied to ELISA.

  In the sandwich method, one of the antibodies (first antibody) is immobilized on a solid phase. The material and shape of the solid phase can be appropriately determined by those skilled in the art depending on the measurement method. Examples of solid phase materials include polyvinyl chloride, polyvinylidene fluoride (PVDF), polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide, polyacrylonitrile, polypropylene. Synthetic organic polymer compounds such as dextran derivatives, polysaccharides such as agarose gel and cellulose, inorganic polymer compounds such as glass, silica gel, and silicone, and amino compounds, aminoacyl groups, carboxyl groups, and acyl groups. And compounds having a functional group such as a hydroxyl group or a nitro group introduced therein. Examples of the shape of the solid phase include a microtiter plate (ELISA plate), a flat plate shape such as a disk, a paper piece, a particle shape such as a bead, a tubular shape such as a test tube and a tube, a fibrous shape, and a membrane shape. The first antibody is immobilized on these solid phases by a known method.

The labeling substance for labeling the second antibody may be a known labeling substance used in immunological measurement methods, for example, radioisotopes ( ¹²⁵ I, ¹⁴ C, ³² P, etc.), enzymes (β-galactosyl) Illustrative examples include tase, peroxidase, alkaline phosphatase, etc.), fluorescent substances (fluorescein derivatives, rhodamine derivatives, etc.), and insoluble particulate markers. The insoluble granular marker itself is colored and is preferably a substance that can be visually recognized. Examples thereof include colloidal metal particles such as gold colloid and platinum colloid, synthetic polymer particles (colored synthetic polymer particles) such as polystyrene latex colored with pigments, and polymer dyed particles.

  In the present invention, the immunochromatogram method is preferably used because it is easy to operate and can be accurately detected in a short time without using a special apparatus. The immunochromatogram method is a method that uses the principle of chromatography, and is a method that uses a membrane-like solid phase (a membrane carrier for chromatography) to which a first antibody is immobilized. In this method, a mixture of a prepared sample and a labeled second antibody (including a complex of an antigen and a second antibody) is developed on a chromatographic membrane carrier and then immobilized on a solid phase. This is a method of causing an antigen-antibody reaction with the first antibody.

  FIG. 1 is a schematic view showing an example of an immunochromatographic test tool according to the present invention. The immunochromatographic test tool has a test piece 1 on which a first monoclonal antibody is fixed and a labeled second monoclonal antibody. The second monoclonal antibody is provided in a form such as a container 6 such as a plastic bottle, and constitutes a so-called kit product together with the test piece 1.

  The test piece 1 has a chromatographic membrane carrier 2 on which the first monoclonal antibody is fixed, and a liquid absorption part 4 that absorbs the remaining sample after the development. The liquid absorption part 4 is provided at one end of the chromatographic membrane carrier 2, and the other end on the other side is a contact part 5 for contacting a solution containing a complex formed by contact between the second monoclonal antibody and the antigen in the sample. It has become. The chromatographic membrane carrier 2 is in the form of a rectangular piece of paper, and can develop a liquid and can carry a first antibody, such as filter paper, cellulose membranes such as nitrocellulose porous bodies and cellulose acetate membranes, amino groups And / or a nylon film such as a nylon film having an amino group substituted with a carboxyl group or an alkyl group, a PVDF film, or the like. The chromatographic membrane carrier 2 may be provided on a substrate such as a glass plate or a plastic plate.

  The liquid absorbing part 4 is made of a material that can absorb and hold a liquid, for example, a filter paper, cotton cloth, porous plastic nonwoven fabric made of polyethylene, polypropylene, or the like. A determination region 3 to which the first monoclonal antibody is fixed is provided at a position slightly away from the liquid absorption portion toward the contact portion 5.

  A sample prepared from a specimen such as flounder is first contacted with the second monoclonal antibody in a container 6 containing a labeled second monoclonal antibody, and the quadrature septum puntata present in the sample is contacted. The spore-derived protein forms a complex with the second monoclonal antibody. Next, when the contact portion 5 of the test piece 1 is brought into contact with the reaction solution in the container 6, the reaction solution as a mobile phase moves through the chromatographic membrane carrier 2 and comes into contact with the first monoclonal antibody in the determination region 3. To do. Thus, the spore-derived protein of Kudoa septenpuntata present in the sample forms a complex comprising the first monoclonal antibody and the labeled second monoclonal antibody. At this time, if the labeling substance is an insoluble granular marker such as a gold colloid, the color of the determination region changes, so that the presence of the quadrature septa pactata can be immediately confirmed visually.

  As described above, the immunochromatographic test tool according to the present invention may be a test tool provided with the first monoclonal antibody and the labeled second monoclonal antibody in one test tool instead of the kit product. For example, the inspection tool includes a substrate such as a glass plate or a plastic plate, a sample supply unit that supplies a sample, a label holding unit that holds a labeled second monoclonal antibody, and a first monoclonal antibody fixed A chromatographic membrane carrier having a determined determination region and a liquid absorption part, and a sample supply part and a label holding part are arranged on the substrate from one end part to the other end part in contact with the label holding part. And the chromatographic membrane carrier are in contact with each other and disposed on the substrate, and further, in contact with the chromatographic membrane carrier, a liquid absorbing part is provided (not shown). Such an inspection tool is also known, and for example, the inspection tool disclosed in Japanese Patent No. 530200 is exemplified. In this test tool, the antigen in the sample supplied to the sample supply unit comes into contact with the label holding unit to form a complex with the labeled second antibody. In this complex, the liquid in the sample becomes a mobile phase and is developed on the chromatographic membrane carrier, and the complex comes into contact with the first monoclonal antibody fixed in the determination region, and the color of the determination region changes. Excess sample is absorbed by the liquid absorption part.

  However, the immunochromatographic test tool of the present invention is not limited to these embodiments, and various known test tools can be used, and further includes a second determination region to which another monoclonal antibody is fixed. Needless to say, the present invention includes various modes such as a test tool provided with a control region (or a reference region) that makes it possible to determine that the antigen-antibody reaction is correctly performed.

  As described above, since the detection method of the present invention uses a highly specific monoclonal antibody, there is little cross-reaction with other genus Mycospores, and the quadrature septum puntata can be quickly and easily used. It can be detected.

  Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are merely illustrative and are not limited to the following examples.

(1) Preparation of monoclonal antibody Preparation of Kudoa septempuctata antigen
After extracting K. septempuctata spores from the soleus muscle infested with the parasite, the spores were purified by Percoll density gradient centrifugation and stored frozen at -20 ° C. Using this cryopreserved spore, the K. septempuctata antigen was prepared by the following three methods.
(a) Untreated 500 μL of K. septempuctata spore solution adjusted to 2.0 × 10 ⁷ / mL with PBS (−) was used as an antigen.
(B) Ultrasonic treatment A solution obtained by ultrasonically treating 500 μL of K. septempuctata spore solution adjusted to 2.0 × 10 ⁷ / mL with PBS (−) was used as an antigen.
(C) Surfactant and Heat Treatment 500 μL of K. septempuctata spore solution adjusted to 2.0 × 10 ⁷ / mL with 2% sodium cholate solution was mixed by vortex for 1 minute. Then, it heated at 95 degreeC for 10 minute (s), and centrifuged (4 degreeC, 15,000 rpm, 5 minutes). The collected supernatant was used as an antigen.

B. Immunization of mice Each of the mice was immunized with three types of antigens prepared by the method described above.
Three BALB / c mice per group (SPF grade, female, 8 weeks old), 100 μL of K. septempuctata antigen per mouse and adjuvant (TiterMAX Gold: CytRx Co., Norcross, GA, USA), etc. Volume mixed emulsion was inoculated intraperitoneally. Further, after 3, 5, 7 and 12 weeks, 100 μL of each mouse was additionally inoculated with an equal volume mixed emulsion of the same antigen and adjuvant (TigerMAX Gold) in the abdominal cavity of each mouse. One week after the third immunization, blood was collected from the tail vein of each mouse, and the titer of serum antibody was measured by ELISA (details are described in C. below). As a result of ELISA, the mice that showed the highest serum antibody titer at a dilution of 8,000 times or more (1 out of 3 mice using untreated K. septempuctata spore solution as an antigen) were selected.

Three days before cell fusion, 100 μL of K. septempuctata spore solution adjusted to 2.0 × 10 ⁷ cells / mL with PBS (−) was inoculated into the peritoneal cavity of the selected immunized mouse to obtain the final immunization.

C. Measurement of serum antibody titer using ELISA K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.0 × 10 ⁶ / mL with PBS (−) 300 μL Was subjected to ultrasonic treatment and diluted 10-fold with PBS (−) to obtain an antigen solution. 50 μL of the prepared antigen solution was dispensed into each well of a microtiter plate and allowed to stand overnight at 4 ° C. After removing the antigen solution, a blocking solution (20% horse serum-added PBS (-)) was added at 150 μL per well and allowed to react at room temperature for 1 hour. After removing the blocking solution, the plate was washed 3 times with 300 μL of washing solution (0.05% Tween 20 added PBS (−)) per well.

  Next, the serum sample was diluted 1,000 times with 20% horse serum and 0.05% Tween 20 in PBS (-), and the 2-fold serial dilution was further added at 50 μL per well and allowed to react at room temperature for 1 hour. It was. After removing the reaction solution, the plate was washed 4 times with 300 μL of washing solution per well and then anti-mouse IgG (Fc specific) -Peroxidase: Sigma-Aldrich, Inc., St. Louis, MO, USA. ) Was diluted 2,000-fold with 20% horse serum and 0.05% Tween 20 in PBS (-), and 50 μL was added per well and allowed to react at room temperature for 1 hour.

  After removing the reaction solution, the plate was washed 4 times with 300 μL of the above washing solution per well, and then 50 μL of enzyme substrate (1-Step Ultra TMB-ELISA: Thermo Fisher scientific Inc., Madison, W1, USA) was added per well. After reacting for 10 minutes at room temperature, 50 μL of a reaction stop solution (1 M sulfuric acid) was added per well, and the absorbance at 450 nm was measured using an automatic absorbance measurement apparatus.

D. Cell fusion Spleen cells and myeloma cells of immunized mice were fused.
Three days after the final immunization of the mice, the spleens of the immunized mice were removed and washed in Pre-Fusion Medium (StemCell Technologies Inc.) in a petri dish. After washing, the spleen was transferred to another petri dish containing Pre-Fusion Medium (StemCell Technologies Inc.) and homogenized with tweezers.

  Next, the spleen homogenization solution was filtered through a nylon mesh to obtain a uniform cell suspension. Erythrocytes were removed from the cell suspension using mouse lymphocyte specific gravity separation liquid (Lymphoryte-M: Cedalane Laboratories Ltd., Ontario, Canada), and then 25 mL of Pre-Fusion Medium (StemCell Technologies Inc. 3 times). Centrifugal washing was performed (room temperature, 1,500 rpm, 5 minutes).

After washing 3 times, the suspension was suspended in 20 mL of Fusion Medium (StemCell Technologies Inc.), diluted 10-fold with PBS (−), and the number of cells was counted (1.36 × 10 ⁸ cells from one immunized mouse). Spleen cells were obtained). Spleen cell suspension (1.76 × 10 ⁸ spleen cells suspended in 20 mL Fusion Medium (StemCell Technologies Inc.)) 2.7 × 10 ⁷ myeloma cells (P3-X63-Ag8 (U1) was added (cell ratio, splenocytes: myeloma cells = 5: 1) and centrifuged (room temperature, 1,500 rpm, 5 minutes). After centrifugation, in order to completely remove the serum components, the plate was washed three times with 25 mL of Fusion medium (StemCell Technologies Inc.) that had been preliminarily kept at 37 ° C. (room temperature, 1,500 rpm, 5 minutes).

  The cell sediment was suspended in 10 mL of Fusion medium (StemCell Technologies Inc.), transferred to a 12 mL round bottom tube, and centrifuged (room temperature, 1,500 rpm, 5 minutes). After loosening the cells sufficiently by tapping, 1 mL of 40% PEG solution (40 g of PEG-4000 dissolved in 50 mL of PBS (−), 10 mL of dimethyl sulfoxide and 1 mL of poly-L-arginine hydrochloride aqueous solution) (1 mg / mL) was added, and finally 1N hydrochloric acid was added to adjust the pH to 7.8) over 1 minute while gently stirring the cells with the tip of the pipette. The cells were gently agitated with the tip of a pipette for an additional minute.

To this, 1 mL of Fusion medium (StemCell Technologies Inc.) was added over 1 minute while gently stirring the cells with the tip of a pipette, and then 1 mL of Fusion medium (StemCell Technologies Inc.) was added with the tip of the pipette. Added over 1 minute with gentle stirring. Subsequently, 7 mL of Fusion medium (StemCell Technologies Inc.) was added over 3 minutes while gently stirring the cells with the tip of the pipette (1 mL in 7 portions). After centrifugation (room temperature, 1,000 rpm, 5 minutes), the supernatant was removed. The cells were gently loosened by tapping and then suspended in 10 mL of Pre-Fusion Medium (StemCell Technologies Inc.). After centrifugation (room temperature, 1,000 rpm, 5 minutes), the supernatant was removed. The cells were gently loosened by tapping and then suspended in 10 mL of Hybridoma Recovery Medium (StemCell Technologies Inc.). 10 mL of the suspension was transferred to a medium flask containing 40 mL of Hybridoma Recovery Medium (StemCell Technologies Inc.). Then, the cells were cultured for 18 hours in the presence of 37 ° C. and 5% CO ₂ .

E. Selective culture and cloning The fused cells obtained in (1) were selectively cultured according to the following procedure and cloned.
A 50 mL fusion cell suspension cultured at 37 ° C. in the presence of 5% CO ₂ for 18 hours was centrifuged (room temperature, 1,000 rpm, 5 minutes). The cell sediment was gently loosened by tapping and then suspended in 10 mL of Hybridoma Recovery Medium (StemCell Technologies Inc.). Transfer 10 mL of suspension to Hybridoma Selection and Cloning Medium (hypoxanthine / thymidine / aminopriten-added agar medium: StemCell Technologies Inc.) and close the lid completely, and avoid bubbles as much as possible. The bottle was mixed by inversion several times. After standing at 37 ° C. for 15 minutes, 4 mL each was dispensed into 25 60 mm Petri dishes. These were cultured at 37 ° C. in the presence of 5% CO ₂ for 10 to 14 days.

F. Collection of colonies, detection and selection of positive clones E. The colonies of the fused cells obtained in the above were collected, and positive clones were detected and selected using ELISA.
Fusion cell colonies were collected from each petri dish between 10 and 14 days in culture. The fused cells were collected using a micropipette with a volume range of 20 μL and a volume range of 2 to 20 μL, and transferred to each well of a 96-well microplate to which 200 μL of Hybridoma Growth Medium (StemCell Technologies Inc.) was added. Finally, 2592 fused cell colonies were collected from 25 Petri dishes in which the fused cells were finally cultured. The fused cells transplanted into the wells were cultured at 37 ° C. in the presence of 5% CO ₂ for 4 to 6 days. After the culture, the antibody titer contained in each culture supernatant was measured by ELISA (details are described in the section G. below).

For 36 wells that showed high OD values by ELISA, each cell suspension was transferred to each well of a 24-well plate to which 1 mL of Hybridoma Growth Medium (StemCell Technologies Inc.) was added, at 37 ° C., 5% CO _2. Cultivated in the presence for 3-4 days. After culturing, the antibody contained in the culture supernatant was examined for reactivity to K. septempuctata antigen treated by a plurality of methods and reactivity to soleus muscle using ELISA (details are described in the section of H. below). (See Table 1).

  As a result of ELISA, 7 wells were selected so that antibodies exhibiting various reactivity were obtained from those that did not react with soleus muscle, and the fused cells (hybridomas) in the wells were selected as follows. It was recloned by the method shown in the section. The seven hybridomas were named Ks4-4F6, Ks14-9H12, Ks18-11H5, Ks22-15D3, Ks24-15G11, Ks30-2C10, Ks34-25B10, respectively. In addition, antibodies produced from these hybridomas are Ks4-4F6 antibody (Ks4 antibody), Ks14-9H12 antibody (Ks14 antibody), Ks18-11H5 antibody (Ks18 antibody), Ks22-15D3 antibody (Ks22 antibody), Ks24- 15G11 antibody (Ks24 antibody), Ks30-2C10 antibody (Ks30 antibody), and Ks34-25B10 antibody (Ks34 antibody) were used.

G. Measurement of antibody titer in culture supernatant by ELISA The antibody titer in the hybridoma culture supernatant obtained in the above section was measured by the following procedure. After sonicating 500 μL of K. septempuctata spores purified to Percoll density gradient centrifugation and stored frozen at −20 ° C. to 1.0 × 10 ⁷ / mL with PBS (−), PBS ( -) Was diluted 10 times to obtain an antigen solution. A necessary amount of a 96-well microtiter plate in which 50 μL of this antigen solution was dispensed per well was prepared and allowed to stand at 4 ° C. overnight.

  After removing the antigen solution, a blocking solution (PBS (−) supplemented with 20% horse serum) was added at 150 μL per well and allowed to react at room temperature for 1 hour. After removing the blocking solution, the plate was washed 3 times with 300 μL of washing solution (0.05% Tween 20 added PBS (−)) per well. The culture supernatant was diluted 6-fold with PBS (-) containing 20% horse serum and 0.05% Tween 20 and added at 50 μL per well and allowed to react at room temperature for 1 hour. After removing the reaction solution, it was washed 4 times with 300 μL of washing solution per well, and then peroxidase-labeled anti-mouse IgG (Anti-Mouse IgG (Fc specific) -Peroxidase: Sigma-Aldrich, Inc.) was added to 20% horse serum, 0 A solution diluted 2000 times with 0.05% Tween 20 in PBS (−) was added at 50 μL per well, and allowed to react at room temperature for 1 hour.

  After removing the reaction solution, the plate was washed 4 times with 300 μL of washing solution per well, and then enzyme substrate (1-Step Ultra TMB-ELISA: Thermo Fisher scientific Inc.) was added at 50 μL per well and allowed to react at room temperature for 10 minutes. Thereafter, 50 μL of a reaction stop solution (1 M sulfuric acid) was added per well, and the absorbance at 450 nm was measured using an automatic absorbance measurement apparatus.

H. Reactivity of hybridoma-producing antibody to K. septempuctata antigen after multiple treatments The reactivity of the antibody in the culture supernatant produced by the hybridoma obtained in the above section with respect to K. septempuctata antigen subjected to a plurality of different treatments was examined according to the following procedure.
i) An antigen solution prepared by treating K. septempuctata spores and soleus muscle according to the following method was dispensed at 50 μL per well of a microtiter plate and allowed to stand at 4 ° C. overnight.
a. Ultrasonic treatment K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. were sonicated with 500 μL of spore solution adjusted to 1.0 × 10 ⁷ / mL with PBS (−), and centrifuged. (4 ° C., 15,000 rpm, 5 minutes). After collecting the centrifugal supernatant, the sediment was suspended in 1.5 mL of PBS (−). Centrifugal supernatant and sediment suspension were used as antigen solutions.
b. Treatment with 0.5% sodium cholate K. septempuctata spores purified by Percoll density gradient centrifugation and stored frozen at -20 ° C. adjusted to 1.0 × 10 ⁸ / mL with 0.5% sodium cholate solution 45 μL of the solution was mixed by vortexing for 1 minute and centrifuged (4 ° C., 15,000 rpm, 5 minutes). The centrifugation supernatant was collected and diluted 100 times with PBS (−). The sediment was suspended in 4.5 mL of PBS (−). Centrifugal supernatant dilution and sediment suspension were used as antigen solutions.
c. 0.1% Triton X treatment K. septempuctata spores purified by Percoll density gradient centrifugation and frozen at -20 ° C were adjusted to 1.0 x 10 ⁸ / mL with 0.1% Triton X solution. Vortexed for 1 minute and centrifuged (4 ° C., 15,000 rpm, 5 minutes). The centrifuged supernatant was collected and diluted 100 times with PBS (−). The sediment was suspended in 4.5 mL of PBS (−). Centrifugal supernatant dilution and sediment suspension were used as antigen solutions.
d. Homogenization treatment 450 μL of K. septempuctata spore purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.0 × 10 ⁷ / mL with PBS (−) was added to Biomasher II (Nippi Inc. , Adachi-ku, Tokyo, Japan) and centrifuged (4 ° C., 15,000 rpm, 5 minutes). A centrifugal supernatant diluted 10-fold with PBS (-) was used as an antigen solution.
e. Heat treatment K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. were adjusted to 1.0 × 10 ⁷ / mL with PBS (−), and 450 μL of the spore solution was heated at 95 ° C. for 10 minutes. And centrifuged (4 ° C., 15,000 rpm, 5 minutes). The centrifuged supernatant was collected and diluted 10-fold with PBS (−) to obtain an antigen solution.
f. Untreated (-20 ° C frozen storage)
K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. were adjusted to 1.0 × 10 ⁶ / mL with PBS (−) as an antigen solution.
g. Untreated (4 ° C storage)
K. septempuctata spores purified by Percoll density gradient centrifugation and stored at 4 ° C. were adjusted to 1.0 × 10 ⁶ / mL with PBS (−) as an antigen solution.
h. Soleus muscle
100 mg of soleus muscle, which was confirmed not to be infected with K. septempuctata, K. thyrsites and K. lateolabracis, was homogenized with BioMasher II (Nippi Inc.). 900 μL of PBS (−) was added, followed by centrifugation (4 ° C., 15,000 rpm, 5 minutes). The centrifugation supernatant was recovered and used as an antigen solution.

  ii) After removing the antigen solution, a blocking solution (PBS (-) containing 20% horse serum) was added at 150 μL per well and allowed to react at room temperature for 1 hour. After removing the blocking solution, the plate was washed 3 times with 300 μL of washing solution (0.05% Tween 20 added PBS (−)) per well.

  iii) F.M. The culture supernatant was diluted 6-fold with PBS (-) containing 20% horse serum and 0.05% Tween 20 and added at 50 μL per well and allowed to react at room temperature for 1 hour. After removing the reaction solution, it was washed 4 times with 300 μL of washing solution per well, and then peroxidase-labeled anti-mouse IgG (Anti-Mouse IgG (Fc specific) -Peroxidase: Sigma-Aldrich, Inc.) was added to 20% horse serum, 0 A solution diluted 2000 times with 0.05% Tween 20 in PBS (−) was added at 50 μL per well, and allowed to react at room temperature for 1 hour.

  iv) After removing the reaction solution, the plate was washed 4 times with 300 μL of washing solution per well, and then enzyme substrate (1-Step Ultra TMB-ELISA: Thermo Fisher scientific Inc.) was added at 50 μL per well, followed by 10 at room temperature. After reacting for 5 minutes, 50 μL of a reaction stop solution (1 M sulfuric acid) was added per well, and the absorbance at 450 nm was measured using an automatic absorbance measurement apparatus.

I. Recloning The seven clones selected in section F were cultured according to the following procedure with the culture scale expanded.
For each hybridoma clone in the selected 7 wells, the number of cells was measured, and a cell suspension with a cell concentration of 300 cells / mL was prepared with Pre-Fusion Medium (StemCell Technologies Inc.). 0.5 mL of the cell suspension (150 cells) was mixed with 9.5 mL of Hybridoma Selection and Cloning Medium (StemCell Technologies Inc.) and allowed to stand at 37 ° C. in the presence of 5% CO ₂ for 15 minutes. Each clone was dispensed into 2 60 mm Petri dishes in a volume of 4 mL without foaming, and the rest was discarded. The cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 10 to 14 days.

After culturing for 10-14 days, each Petri dish colony was observed with the naked eye. Each colony was collected using a micropipette with a volume range of 2 to 20 μL and the volume range was set to 20 μL, and transferred to each well of a 96-well microplate to which 200 μL of Hybridoma Growth Medium (StemCell Technologies Inc.) was added. Twenty-four colonies were collected in order from the largest. Next, the cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 3 to 4 days. The supernatant was collected and the antibody titer was measured using ELISA according to the above G. section.

One with high antibody titer of culture supernatant and good cell growth state is selected per clone, and adjusted to a cell concentration of 1-2 × 10 ⁵ cells / mL with Hybridoma Growth Medium (StemCell Technologies Inc.). The culture scale was expanded, and the cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 2 days. After culturing, the culture scale was expanded with an appropriate amount of Hybridoma Growth Medium (StemCell Technologies Inc.) to a cell concentration of 1-2 × 10 ⁵ cells / mL, and cultured at 37 ° C. in the presence of 5% CO ₂ for 2 days. did. After culturing, the culture scale was expanded with an appropriate amount of Hybridoma Growth Medium (StemCell Technologies Inc.) to a cell concentration of 1-2 × 10 ⁵ cells / mL, and cultured at 37 ° C. in the presence of 5% CO ₂ for 2 days. did. After culturing, 50% (volume ratio) of the hybridoma pre-fusion medium (StemCell Technologies Inc.) / Hybridoma Growth Medium (StemCell Technologies Inc.) in a cell concentration / mixture solution of 10% to 2 in a volume ratio of 1 to ⁵ mL. The cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 2 days. The culture scale was successively expanded using Hybridoma Pre-fusion Medium (StemCell Technologies Inc.), and the cells were stored frozen at an appropriate time.

J. et al. Large-scale preparation of monoclonal antibody In accordance with the following procedure, 7 recloned clones were mass-cultured to obtain monoclonal antibodies.
Each of the 7 re-cloned clones was cultured in large quantities using Hybridoma Pre-fusion Medium (StemCell Technologies Inc.), washed twice with Fusion Medium (StemCell Technologies Inc.), and then FusionSolMem A cell suspension adjusted to 2 × 10 ⁷ / mL was obtained.

  Next, 16 8-week-old BALB / c mice were given pristane (2,6,10,14-tetramethylpentadecane: Funakoshi Co., Ltd., Bunkyo-ku, Tokyo, Japan) at 500 μL per mouse. Each intraperitoneal injection was performed, and 500 μL was intraperitoneally injected again one week later. Three days later, 1 mL of the prepared cell suspension was injected intraperitoneally (injected into 2 mice per clone). Ascites was collected with a syringe from a mouse in which ascites accumulated and the abdomen was enlarged between 7 and 12 days after cell suspension inoculation. Ascitic fluid of 5 to 10 mL was obtained per mouse. The obtained ascites was centrifuged (4 ° C., 3000 rpm, 10 minutes), and the supernatant was collected. An equal volume of saturated ammonium sulfate solution (adjusted to pH 7.4 with aqueous ammonia) was added dropwise to the centrifuged supernatant for salting out. After centrifugation (4 ° C., 10,000 × g, 10 minutes), the supernatant was removed, and the salted out precipitate was dissolved in an appropriate amount of PBS (−).

K. Purification of monoclonal antibodies The monoclonal antibody prepared in large quantities in the section was purified according to the following procedure.
The salted out antibody sample was purified by Protein A Agasore column chromatography. Bio-Scal ^™ Mini Affi-Prep Protein A Cartridge (5 mL: BioRad Laboratories Inc., Hercules, Calif., USA) was washed with 5 bed volumes of binding buffer (pH 9.0: BioRad Laboratories Inc.). An antibody sample having a protein concentration of 10 mg / mL was mixed with the binding buffer at an equal volume ratio, and 5 mL thereof was applied to the column.

  After washing the column with 15 times the bed volume of binding buffer, the mouse IgG fraction was eluted into the receiver with 5 bed volumes of elution buffer (pH 3.0: BioRad Laboratories Inc.). In order to neutralize the pH of the eluate to neutral, 30 μL of 1M Tris-HCl buffer (pH 9.0) per 1 mL of eluate was dispensed in advance into a receiver.

  The purified antibody fraction is an ultrafiltration device having a molecular fraction of 30,000 (Amicon Ultracentrifugal Filters Ultracel-30K: Millipore Corporation, Billerica, MA, USA: Amicon, Ultracel are registered trademarks, respectively) The solvent was replaced with PBS (-) by gel filtration using a -10 column (GE Healthcare UK Ltd., Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England). The protein concentration of the obtained purified antibody solution was adjusted to 10 mg / mL with PBS (−) and then stored frozen at −20 ° C.

L. Peroxidase Labeling of Purified Monoclonal Antibody Peroxidase Labeling Kit-SH (Cosmo Bio. Co. Ltd., Koto-ku, Tokyo, Japan) is used to label peroxidase on the purified monoclonal antibody by the procedure described in the instructions. did. K. Using 20 μL of the monoclonal antibody solution purified in the above section 10 μL, elution was performed so that the final concentration of the peroxidase-labeled monoclonal antibody was 1 mg / mL. The labeled antibody was stored frozen at -20 ° C.

(2) Selection of monoclonal antibodies useful for immunochromatography and determination of the isotype of the selected antibodies Specificity examination of monoclonal antibody against Kudoa genus mycospores Specificity for Kudoa genus mycospore was examined according to the following procedure for 7 types of monoclonal antibodies selected in (1).
K. septempuctata spores, K. thyrsites spores, K. yasunagai spores, K. neothunni spores and cryopreserved without purification by Percoll density gradient centrifugation, purified by Percoll density gradient centrifugation and cryopreserved at -20 ° C The K.lateolabracis spore, K.iwatai spore and K.amamiensis spore were adjusted to 1.0 × 10 ⁵ / mL with PBS (−), respectively. Each 400 μL of the prepared spore solution was sonicated, dispensed 50 μL per well into a microtiter plate, and allowed to stand at 4 ° C. overnight.

  After removing the antigen solution, a blocking solution (PBS (−) supplemented with 20% horse serum) was added at 150 μL per well and allowed to react at room temperature for 1 hour. After removing the blocking solution, the plate was washed 3 times with 300 μL of washing solution (0.05% Tween 20 added PBS (−)) per well.

  Next, a peroxidase-labeled monoclonal antibody diluted 1,000-fold with 20% horse serum and 0.05% Tween 20 in PBS (-) (antibody concentration 1 μg / mL) was added in an amount of 50 μL per well. For 1 hour. After removing the reaction solution, the plate was washed 4 times with 300 μL of washing solution per well, and then enzyme substrate (1-Step Ultra TMB-ELISA: Thermo Fisher scientific Inc.) was added at 50 μL per well and reacted at room temperature for 10 minutes. Then, 50 μL of a reaction stop solution (1 M sulfuric acid) was added per well, and the absorbance at 450 nm was measured using an automatic absorbance measurement apparatus. The results are shown in Table 2. As a result, other antibodies except Ks34 antibody showed high reactivity to K. septempuctata. Among antibodies that showed high reactivity to K. septempuctata, Ks14, Ks18, Ks24, and Ks30 have low reactivity to K.lateolabracis, K.yasunagai, K.iwatai, and K.amaniensis. Indicated. Three types of K. septempuctata, K. thyrsites and K. lateolabracis, which are considered to be parasitic on the soleus muscle, can be distinguished by microscopic examination.

B. Examination of Monoclonal Antibody Reactivity in Sandwich ELISA Sandwich ELISA was performed using the purified monoclonal antibody and peroxidase-labeled monoclonal antibody for the seven types of monoclonal antibodies selected in (1). All 49 combinations of monoclonal antibodies were examined for reactivity with K. septempuctata antigen treated by three different methods.

  10 mg / mL purified monoclonal antibody was diluted 200-fold with PBS (−). The antibody dilution was dispensed at 50 μL per well and allowed to stand at 4 ° C. overnight to immobilize.

K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. were treated according to the following method to obtain antigens.
a. Heat treatment After 200 μL of K. septempuctata spore solution adjusted to 1.0 × 10 ⁷ / mL with PBS (−) was heated at 100 ° C. for 10 minutes, 20% horse serum, 0.05% Tween 20 added PBS (−) Diluted 10 times.
b. 0.1% Triton X treatment 200 μL of K. septempuctata spore solution adjusted to 1.0 × 10 ⁷ / mL with 0.1% Triton X solution was homogenized with BioMusher II (Nippi Inc.), and 20% horse serum, 0% Dilute 10-fold with PBS (-) containing 0.05% Tween20.
c. Homogenization treatment 200 μL of K. septempuctata spore solution adjusted to 1.0 × 10 ⁷ / mL with PBS (−) was homogenized with BioMusher II (Nippi Inc.), and then 20% horse serum and 0.05% Tween 20 added. Dilute 10 times with PBS (-).

  After removing the antibody solution, a blocking solution (PBS (−) supplemented with 20% horse serum) was added at 150 μL per well and allowed to react at room temperature for 1 hour. After removing the blocking solution, the plate was washed 4 times with 300 μL of washing solution (0.05% Tween 20 added PBS (−)) per well.

  Next, the peroxidase-labeled monoclonal antibody was diluted 1,000-fold with 20% horse serum and 0.05% Tween 20 in PBS (−) (antibody concentration 1 μg / mL). The diluted solution and the prepared antigen solution were dispensed at 50 μL per well and reacted at room temperature for 1 hour. After removing the reaction solution, the plate was washed 4 times with 300 μL of washing solution per well, and then enzyme substrate (1-Step Ultra TMB-ELISA: Thermo Fisher Scientific Inc.) was added at 50 μL per well and reacted at room temperature for 10 minutes. Then, 50 μL of a reaction stop solution (1 M sulfuric acid) was added per well, and the absorbance at 450 nm was measured using an automatic absorbance measurement apparatus. The results are shown in Tables 3 and 4.

C. Determination of combination of monoclonal antibodies suitable for immunochromatography Based on the above results, three monoclonal antibodies, Ks14 antibody, Ks18 antibody and Ks24 antibody, were selected as antibodies having high specificity and different reactivity. The reactivity of the monoclonal antibodies selected using the immunochromatography method was examined, and a combination suitable for the immunochromatography method was determined.

  An antibody-immobilized membrane (the preparation method is described in the following D.) and a colloidal gold labeled antibody (the preparation method is described in the following E.) were prepared. Next, immunochromatography (described in detail in F.) was performed to examine the reactivity with the K. septempuctata antigen and the presence or absence of non-specific reactions. The results are shown in Table 5.

  As a result, among those in which a positive band was observed, a non-specific reaction was not recognized, and the combination with the darkest color of the positive band was a solid-phase antibody Ks24 antibody and a detection antibody Ks18 antibody. Hybridoma Ks24-15G11 that produces these monoclonal antibodies was commissioned on December 2, 2013 to the Patent Microbiology Depositary Center, National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamashi, Kisarazu, Chiba, Japan No. NITE P-01766, and the hybridoma Ks18-11H5 was deposited with the same microorganism deposit center as the deposit number NITE P-01767 on the same date. In addition to the above combinations, as shown in Tables 4 and 5, for example, Ks4 antibody-Ks4 antibody, Ks14 antibody-Ks14 antibody, Ks14 antibody-Ks18 antibody, Ks14 antibody-Ks30 antibody, Ks18 antibody-Ks4 antibody Ks18 antibody-Ks14 antibody, Ks18 antibody-Ks18 antibody, Ks18 antibody-Ks22 antibody, Ks18 antibody-Ks30 antibody, Ks22 antibody-Ks4 antibody, Ks22 antibody-Ks22 antibody, Ks24 antibody-Ks22 antibody, Ks30 antibody-Ks4 antibody, Ks30 Antibody-Ks14 antibody, Ks30 antibody-Ks22 antibody, Ks30 antibody-Ks30 antibody, Ks34 antibody-Ks34 antibody, etc. such that the OD value is 2.0 or more (all are the first antibody on the left and the second on the right Can also be used in the sandwich method.

D. Preparation of antibody-immobilized membrane A purified monoclonal antibody was immobilized on a nitrocellulose membrane according to the following procedure.
Purified Ks18 antibody using 5 mM borate buffer (pH 8.5), Ks14 antibody and Ks24 antibody using 1 mM bovine serum albumin (BSA) and 5 mM borate buffer (pH 8.5) Diluted to 2 mg / mL.

  Next, each of the prepared antibody dilutions was applied to a Hi-Flow Plus HF135 membrane (Millipore Corporation) using a Hamilton Microliter # 701 syringe. Then, after drying at 37 degreeC for 1 hour, it preserve | saved at 4 degreeC.

E. Colloidal gold labeling of monoclonal antibody Gold colloid was labeled on the purified monoclonal antibody according to the following procedure.
5 mL of 9.0 × 10 ¹⁰ / mL colloidal gold solution (Gold Colloid 40 nm: BBI International, Cardiff, UK) was adjusted to pH 9.0 with 100 mM potassium carbonate. 10 mg / mL monoclonal antibody was diluted to 0.2 mg / mL with 2 mM sodium tetraborate buffer (pH 9.0). While stirring the pH-adjusted gold colloid solution, 500 μL of the monoclonal antibody diluted solution was added dropwise with a micropipette, and then stirred at room temperature for 30 minutes. Next, with stirring, 550 μL of 2 mM sodium tetraborate buffer solution (pH 9.0) containing 10% BSA was added dropwise with a micropipette, and the mixture was further stirred at room temperature for 30 minutes. 1 mL of this solution was dispensed into six 1.5 mL microtubes, centrifuged (room temperature, 10,000 rpm, 10 minutes), and the supernatant was removed.

  400 μL each of 1 mM BSA-added 2 mM sodium tetraborate buffer (pH 9.0) was added to the centrifugal sediment and suspended by pipetting, followed by a suspension of 6 1.5 mL microtubes. 4 mL was collected in a single 15 mL centrifuge tube and mixed well, and then dispensed in an amount of 800 μL into a new 1.5 mL microtube. After centrifugation (room temperature, 10,000 rpm, 10 minutes), the supernatant was removed.

  1 mL each of 1% BSA-added 2 mM sodium tetraborate buffer (pH 9.0) was added to the centrifugal sediment and suspended by pipetting, followed by centrifugation (room temperature, 10,000 rpm, 10 minutes). After removing the supernatant, all the centrifugal sediments were suspended in 1 mL of 1% BSA, 20% glycerol-added Tris-HCl buffered saline (TBS) (TAKARA BIO INC., Otsu, Shiga, Japan) (antibody concentration 100 μg / mL) and frozen at -20 ° C.

F. Implementation of immunochromatography The immunochromatography was performed according to the following procedure.
300 μL of K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.0 × 10 ⁶ / mL with PBS (−) were added to Biomasher II (Nippi Inc.). Was used as a test solution.

  In a well of a 96-well microplate, 30 μL of test solution and gold colloid-labeled monoclonal antibody diluted solution (gold colloid-labeled antibody solution stored at −20 ° C. was added 10% with PBS (−) containing 2% BSA and 0.1% Triton X. (Diluted) 30 μL was mixed. As a negative control, a mixed solution of 30 μL of PBS (−) and 30 μL of antibody diluent was used.

The tip of the antibody-immobilized membrane was immersed in the mixed solution in the well. The density of the band was observed 15 minutes after the immersion, and the determination was made by giving a six-level display of ++, +, + ^w , + ^ww , ±, and-according to the density.

G. Determination of isotypes of Ks24 antibody and Ks18 antibody As isotypes of Ks24 antibody and Ks18 antibody, a monoclonal antibody isotyping kit (IsoStripe: Roche Diagnostics, Indianapolis, IN, USA) was used. As a result, the isotype of the Ks24 antibody was IgG1 (λ), and the isotype of the Ks18 antibody was IgG1 (κ).

(3) Identification of recognition antigen of Ks24 antibody Purification of K. septempuctata spore-derived protein recognized by Ks24 antibody According to the following procedure, the antigen recognized by Ks24 antibody was affinity purified.
5 mg of Ks24 antibody was coupled to a HiTrap NHS-activated HP column (1 mL: GE Healthcare UK Ltd.). K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.0 × 10 ⁷ / mL with PBS (−) 3 mL of spore solution in 500 μL aliquots in 1.5 mL microtubes. Dispensed and sonicated. Thereafter, the mixture was centrifuged (4 ° C., 15,000 rpm, 5 minutes), and the supernatant was collected. To the supernatant filtered through a 0.45 μm filter, 100 μL of 10% Triton X solution was added and adjusted to 10 mL with PBS (−). The prepared solution was applied to HiTrap NHS-activated HP, and the target antigen was purified according to the instructions.

B. Analysis by immunoblotting of antigen recognized by Ks24 antibody The molecular weight of the antigen recognized by Ks24 antibody was examined according to the following procedure.
A. After adding an equal volume of mercaptoethanol-containing SDS-PAGE sample buffer (Tris SDS β-ME sample treatment solution: Cosmo Bio. Co. Ltd.) to the antigen purified in (2) diluted twice with PBS (−). The sample was heated at 100 ° C. for 10 minutes to obtain an SDS-PAGE sample. SDS-PAGE was performed using two Nu PAGE 12% Bis-Tris Precast Gels (Life Technologies, Inc., Grand Island, NY, USA). One of the two gels was transferred to a PVDF membrane, and the remaining one was stained with Coomassie brilliant blue. The membrane after the transfer was washed with 0.1% Tween 20-added TBS (TAKARA BIO INC.) And then blocked using Block Ace (DS Pharma Biomedical Co., Ltd., Suita, Osaka, Japan).

  The membrane was washed 5 times with 0.1% Tween 20-added TBS (TAKARA BIO INC.) For 5 minutes, and then peroxidase-labeled Ks24 antibody was washed with Can get signal Solution 2 (TOYOBO CO., Ltd., Osaka, Japan). And diluted to 0.1 μg / mL and reacted at room temperature for 1 hour. Next, the membrane was washed 5 times with 0.1% Tween 20-added TBS (TAKARA BIO INC.) For 5 minutes, and then reacted with an enzyme substrate (1-Step TMB Blotting Solution: Thermo scientific Inc.) for 5 minutes at room temperature. It was.

  After washing the membrane with a large amount of distilled water, the position of the appearing band was compared with the gel stained with Coomassie Brilliant Blue. In addition, a calibration curve was prepared by the least square method based on the movement distance of the band of the molecular weight marker (Precision Plus Protein 2 color standard: BioRad Laboratories Inc.), and the molecular weight of the appeared band was estimated. As a result, it was shown that the Ks24 antibody recognizes a K. septempuctata spore-derived protein having a molecular weight of about 23,000 (see FIGS. 2 and 3).

  Analysis of the amino acid sequence of the protein having a molecular weight of about 23,000 (requested by Hokkaido System Science Co., Ltd.) shows that the N-terminal amino acid sequence is XELRE (where X is Ala or Ser). It was done.

C. Examination of detection sensitivity and specificity of immunochromatography using Ks24 antibody and Ks18 antibody a. Examination of detection sensitivity of immunochromatography method using Ks24 antibody and Ks18 antibody The detection sensitivity of immunochromatography method using Ks24 antibody and Ks18 antibody according to the present invention was examined according to the following procedure.
100 μL of K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.23 × 10 ⁷ / mL with PBS (−) were added to Biomasher II (Nippi Inc.). And then diluted 10-fold with PBS (-) to prepare a test solution.

  In a well of a 96-well microplate, 30 μL of test solution and gold colloid-labeled Ks18 antibody dilution (gold colloid-labeled antibody solution stored at −20 ° C. was added 10% with PBS (−) containing 2% BSA and 0.01% Triton X). (Diluted) 30 μL was mixed. As a negative control, a mixed solution of 30 μL of PBS (−) and 30 μL of antibody dilution was used.

The tip of the Ks24 antibody-immobilized membrane was immersed in the mixed solution in the well. The density of the band was observed 15 minutes after the immersion, and the determination was made by giving a six-level display of ++, +, + ^w , + ^ww , ±, and-according to the density. As a result, it was found that the spore concentration of K. septempuctata spore solution was positive when the concentration was 1.23 × 10 ⁵ / mL or more (see Table 6).

b. Examination of specificity of immunochromatography method using Ks24 antibody and Ks18 antibody Specificity of immunochromatography method using monoclonal antibody Ks24 antibody and Ks18 antibody of the present invention was examined according to the following procedure.

Kudoa genus mycospore spores and soleus muscle extract treated by the method described below were used as test solutions.
a. 100 μL of K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.23 × 10 ⁶ / mL with PBS (−) were added to BioMusher II (Nippi Inc.). Homogenized b. 100 μL of K. septempuctata spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.23 × 10 ⁵ / mL with PBS (−) were added to BioMusher II (Nippi Inc.). Homogenized c. 100 μL of K.thyrsites spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.6 × 10 ⁵ / mL with PBS (−) were added to BioMusher II (Nippi Inc.). Homogenized d. 100 μL of K.yasunagai spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 3.6 × 10 ⁶ / mL with PBS (−) were added to BioMusher II (Nippi Inc.). Homogenized e. 100 μL of K. neothunni spores purified by Percoll density gradient centrifugation and cryopreserved at −20 ° C. adjusted to 1.1 × 10 ⁷ / mL with PBS (−) were added to BioMusher II (Nippi Inc.). Homogenized f. 100 μL of K.iwatai spores that had been cryopreserved at −20 ° C. without purification by Percoll density gradient centrifugation and adjusted to 2.32 × 10 ⁷ / mL with PBS (−) were added to Biomasher II (Nippi Inc. Homogenized in.) G. 100 μL of K. amamiensis spores that had been cryopreserved at −20 ° C. without purification by Percoll density gradient centrifugation and adjusted to 7.7 × 10 ⁶ / mL with PBS (−) were added to Biomasher II (Nippi Inc.) h. Supernatant recovered by adding 250 μL of PBS (−) to 100 mg of soleus muscle confirmed to be free of K. septempuctata, K. thyrsites and K. lateolabracis and homogenizing with BioMasher II (Nippi Inc.).

  In a well of a 96-well microplate, 30 μL of test solution and gold colloid-labeled Ks18 antibody dilution (gold colloid-labeled antibody solution stored at −20 ° C. was added 10% with PBS (−) containing 2% BSA and 0.01% Triton X). (Diluted) 30 μL was mixed. As a negative control, a mixed solution of 30 μL of PBS (−) and 30 μL of antibody diluent was used.

The tip of the Ks24 antibody-immobilized membrane was immersed in the mixed solution in the well. The density of the band was observed 15 minutes after the immersion, and the determination was made by giving a six-level display of ++, +, + ^w , + ^ww , ±, and-according to the density.

  As a result, no non-specific reaction to soleus muscle was observed, but weaker reactivity to K.thyrsites, K.iwatai and K.amamiensis than K.septempuctata. (Table 7). However, K. septempuctata, K. thyrsites and K. lateolabracis, which are considered to be parasitic on the soleus muscle, can be distinguished by microscopic examination, and K. iwatai and K. amamiensis can be distinguished by microscopic examination. In addition to being able to differentiate from flounder, this rapid detection method is considered to be a detection method that is expected to be fully used for the on-site rapid detection method used as a screening method. .

  From the results of Table 5, when Ks18 antibody is used as the first antibody of immunochromatography, Ks18 antibody is used as the second antibody, and when Ks24 antibody is used as the first antibody and Ks14 antibody is used as the second antibody, This combination can also be used as a screening method for quadrature septum pactata since it showed reactivity with quadreptepteptata.

  INDUSTRIAL APPLICABILITY The present invention can be used as a method for quickly and accurately discriminating the contamination of quadrature septum pterata in flounder.

Claims (10)

Using a monoclonal antibody that has low reactivity to at least Kudo Yasunagai, Kudo Amamiensis, and Kudo Iwatai, and high reactivity to Kudo Sept. A method for detecting a quadruple septum puntata characterized by detecting a puntata. The monoclonal antibody is a monoclonal antibody having reactivity to a protein having a molecular weight of about 23,000 having an amino acid sequence of XELRE (where X is Ala or Ser) at the N-terminus, which is extracted from Kdoa septum puntata spores. The detection method according to claim 1. The detection method according to claim 1, wherein the monoclonal antibody is a hybridoma deposited under the accession number NITE P-01766 or an antibody produced from the hybridoma deposited under the accession number NITE P-01767. The immunoassay uses first and second monoclonal antibodies against Kudoa septenpuntata spores, and the second monoclonal antibody and quadrant labeled with the first monoclonal antibody immobilized on a solid phase. Including a step of forming a complex containing a spore derived protein of Septenpuntata,
The method according to claim 1, wherein at least the first monoclonal antibody is a monoclonal antibody having a low reactivity to at least Kudo Yasunagai, Kudo Amamiensis, Kudo Iwatai, and a reactivity to Kudoa septum puntata. .   The first monoclonal antibody is reactive to a protein having a molecular weight of about 23,000 having an amino acid sequence of XELRE (where X is Ala or Ser) at the N-terminus extracted from Kdoa septenpuntata spores. The detection method according to claim 4, which is a monoclonal antibody, an antibody produced from a hybridoma deposited under accession number NITE P-01766, or an antibody produced from a hybridoma deposited under accession number NITE P-01767. An immunochromatographic test tool for detecting quadruceptor spores in a sample using first and second monoclonal antibodies against quadruceptor spores,
A chromatographic membrane carrier in which a determination region to which the first monoclonal antibody is immobilized is disposed;
A labeled second monoclonal antibody,
An immunochromatographic test tool, wherein the first monoclonal antibody is a monoclonal antibody having a low reactivity to at least Kudoa Yasunagai, Kudoa Amamiensis, Kudoa Iwatai, and a reactivity to Kudoa septenpentata.   The immunochromatographic test tool according to claim 6, wherein the first monoclonal antibody and the second monoclonal antibody are heterologous monoclonal antibodies.   The first monoclonal antibody is reactive to a protein having a molecular weight of about 23,000 having an amino acid sequence of XELRE (where X is Ala or Ser) at the N-terminus extracted from Kdoa septenpuntata spores. The immunochromatograph according to claim 6 or 7, which is a monoclonal antibody, an antibody produced from a hybridoma deposited under accession number NITE P-01766, or an antibody produced from a hybridoma deposited under accession number NITE P-01767. Inspection tool. A hybridoma deposited under accession number NITE P-01766 or NITE P-01767. Monoclonal antibody against quadratic septum pterata produced from a hybridoma deposited under accession number NITE P-01766 or NITEP-01767.