KR102021176B1 - Monoclonal antibody and inspection Kit. - Google Patents

Abstract

Provided are monoclonal antibodies, test tools, and test kits capable of specifically detecting Kudoa mucospores directly from fish such as flounder. The monoclonal antibody which destroys the spore shell and the cells inside the spores by freezing and thawing the spores of the genus Mycoccus mucosa and acquiring the protein which comprises the said cell, and produced it as an antigen is Kudo septum puntata Since it binds to the proteins constituting the internal structure of the genus Kudoa spp., Specific from the Kudoa spp. Such as Kudoa septum punta directly from fish meat such as halibut where spores also have unformed Kudoa spp. Can be detected. Since the test kit using the monoclonal antibody is provided with a sampling tool made of a circumferential grooved bar such as a screw, a large amount of fish such as flounder can be tested.

Description

Monoclonal antibody for testing and test kit using same {Monoclonal antibody and inspection Kit.}

The present invention relates to a monoclonal antibody capable of detecting a Kudosa mucosa spore, which is a parasite of fish, and a test kit (immune chromat test specimen, ELISA test tool) using the monoclonal antibody, and a test kit equipped with the test tool. will be.

There is the pathogenicity to human in Kudoza mucospores parasitic to fish meat and may cause food poisoning. For example, it is known that food poisoning caused by flounder reproduction is caused by Kudoa septempunctata, a kind of Kudoa mucospores parasitic on flounder. These Kudoa slime spores form spores in fish meat, but there are also Kudoa slime spores that do not form spores in fish meat. In addition, in the case of flounder meat, Kudoa thyrsites and Kudoa lateolabracis, which are Kudoa mucospores, other than Kudoa septum puntata, and which are not pathogenic to humans, are parasitic. Known.

Therefore, in the examination of the genus Kudoa mucospores, the corresponding spores of spore-unformation can also be detected, and in the case of the flounder meat, Kudoa tyrcites and Kudoa laterolabrasis are distinguished from each other. It is necessary to be able to selectively detect to prevent food poisoning.

(Prior art)

As a test for the detection of food poisoning by Kudoza mucosporosis, a test for detecting Kudoa septum puntata, which is parasitic in flounder meat, is known, and quantitative reverse transcription (RT) using convention PCR (Polymerase Chain Reaction) and RNA-by PCR Methods (see Non-Patent Document 1), methods by real-time PCR (see Non-Patent Document 2), and methods using monoclonal antibodies as test reagents (see Patent Document 1) are known.

However, the method according to the PCR method (Non Patent Literature 1 and Non Patent Literature 2) requires an amplifier of DNA, and a long time over several hours is required for determining the degree of inspection.

Patent Document 1 discloses a method of using a monoclonal antibody as a test reagent for the test of Kudoa septum punta using an immunological measurement method. The method of patent document 1 adopts the immunochromat graph method among the immunological measurement methods, and can test for a short time, and the apparatus is also called an immunochromat graph test tool (hereinafter referred to as an "immune chromat test piece.") Simple to use. Since the inspection method of patent document 1 is a method of detecting Kudoa septa punta spores, the presence of Kudoa septa punta spores is required. In Kudoa Septempunta infected with flounder, as described above, not only the spores, that is, the spores are covered with the spores, but also many of the spore shells are unformed. In order to detect the tempunt tarta, the spore shell must also detect an unformed one. However, the test method according to Patent Document 1 and the monoclonal antibody used in the same should be tested in the state of the spores after extracting and purifying Kudoa septum punta spores from the flounder meat. Therefore, it is not possible to directly detect Kudoa septum puntata in flounder meat which has spores of unformed Kudoa septum punta. In addition, the monoclonal antibody not only responds to Kudoa septum punta, but also paradoxizes the muscles of the flounder and exhibits responsiveness to Kudoa tyrcites without pathogenicity to humans. Different from test, kudoa septum punta can not be detected selectively (hereinafter referred to as "specific").

When we perform examination of Kudoa septum puntata for a large quantity of flounder in a short time, it is necessary to collect flounder meat easily and quickly from flounder and easily detect Kudoa septum puntata directly from the flounder Patent Document No. 1 does not describe an inspection tool and an inspection kit relating thereto, and has a problem to provide an inspection instrument or an inspection kit for inspecting a large quantity of flounder.

(Task 1)

The monoclonal antibody used for the test of the Kudoa mucospores such as the conventional Kudoa septum punta can detect the spores of the Kudoa mucospores easily and specifically. However, it was difficult for the monoclonal antibody to directly detect the Kudoa mucosa in the fish meat parasitic of the spore-forming Kudoa mucosa. In the case of the flounder, it was also difficult to specifically detect Kudoa septum puntata, which causes food poisoning by distinguishing it from Kudoa tyrcetes which is not pathogenic to humans. Therefore, the problem that needs to be solved in the prior art is that (I) monoclonal antibody used for the examination of Kudoa mucospores, such as Kudoa septem puntata, does not respond to Kudoa mucospores without spores. The spores cannot detect Kudoa mucosa directly from fish meat such as flounder infected with Kudoa mucus spores, including unformed Kudoza mucosa, and (II) in the case of flounder, it is also reactive to Kudoa tyrcites It does not specifically react to Kudoa Septem puntata.

(Task 2)

For the examination of Kudoa mucospores such as the conventional Kudoa septum puntata, the test instrument or the test kit using the conventional monoclonal antibody described above is used, so that the spores of the Kudoa mucospores from fish meat are not purified. Because fish meat cannot be used for inspection directly, a large quantity of fish could not be inspected. In addition, the collection tool of inspection was not suitable for a large amount of inspection. Therefore, the problem to be solved in the prior art is that the test kit used for the test of Kudoza mucospores cannot easily and quickly collect fish meat from fish infected with this Kudoa mucospores, and Kudoa mucospores parasitic Due to the inability to specifically detect the fish directly, it is impossible to detect the Kudoa mucosa in a short time from a large amount of fish.

MEANS TO SOLVE THE PROBLEM This inventor discovered the method by which the monoclonal antibody produced by the method including the process of the following (1)-(5) can solve the said subject 1.

(1) (antigen protein adjustment step) The Kudoza mucosa is isolated from fish meat, and the spore suspension solution containing the spores of the Kudoza mucosa is frozen and thawed, thereby processing the spore shell and the cells inside the spores. Destroying and adjusting the suspension solution in which the protein constituting the cell is eluted;

(2) (antibody production cell acquisition process) We inoculate protein obtained by step (1) into animal of mammal or bird one or more times, and in antibody body produce the antibody producing cell which produces antibody responsive to the protein Extracting the antibody-producing cells from the animal,

(3) (Cell fusion step) A step of fusing the antibody-producing cells obtained in step (2) and myeloma cells of the same animal species as the animal to produce inactivated antibody-producing cell A;

(4) (screening step) a step of selecting inactivated antibody-producing cell B in response to the protein from the inactivated antibody-producing cell A obtained in step (3), using a protein derived from the Kudoa mucosporecus;

(5) (cloning step) A step of separating the inactivated antibody-producing cell B selected in step (4) as a single cell (hereinafter referred to as "cloning"), and then generating a monoclonal antibody in the cell.

In step (1), the spores of the Kudoa genus mycoccal spores (spore suspension solution suspended in phosphate buffered physiological saline (hereinafter referred to as "PBS")) are frozen and thawed to freeze and melt the spore shell and the cells inside the spores. It can be destroyed. By destroying the spore shell, the internal structure of the Kudosa mucosa spores covered with the spore shell is exposed in the suspension solution, and the cells contained in the internal structure are destroyed by the freezing and thawing, thereby destroying the cells. The protein comprised can be eluted in the said suspension solution, and the suspension solution containing the said protein (henceforth an "antigen protein") used as an antigen for inoculation (immunity) to the animal in the process (2) can be adjusted. The antigenic protein obtained at the process (1) is a protein derived from the internal structure of the spore of the Kudoa genus mucosa.

Since the monoclonal antibody according to the present invention is an antibody produced by inactivating antibody-producing cells obtained by inactivating and cloning antibody-producing cells obtained by inoculating the animal in the step (2) with the protein as an antigen, The monoclonal antibody responds to the antigenic protein of interest. Therefore, the monoclonal antibody according to the present invention responds to the Kudoa mucospores, whose spores (shells) are unformed, that is, the Kudoa mucospores exposed to their internal structure. Therefore, the monoclonal antibody produced by the said process is applicable directly without extracting or refining spores from the fish meat contaminated with the said Kudoa slime spores including the said Kudoa slime spores which have no spores (shells). Can be detected.

In step (1), the spores of the Kudosa mucosa spores are frozen and thawed, and then a surfactant is added to the suspension solution and solubilized, so that the protein constituting the spore shell and cells inside the cells. And a surfactant combine to form micelles in the solution, and obtain the protein from the micelle that becomes an antigen for inoculation (immunization) in the animal in step (2).

In step (1), after spore freezing and thawing spores of the Kudo subsp. Mucus spores, the spore suspension solution containing the spores is irradiated with ultrasonic waves to further destroy the cells in the spores. By eluting the protein constituting the in the solution, it is possible to obtain the antigen protein of the antigen. By adding the solubilization treatment and the ultrasonic irradiation in the step (1), the protein remaining in the cells in the spores is further eluted in the solution, thereby increasing the amount of the antigenic protein contained in the solution.

Therefore, when the animal obtained by the method obtained by adding the treatment or the like to the step (1) is inoculated into the animal, it reacts to the protein constituting the cells of the internal structure of the spores of the genus Kudoza mucosa rather than the freeze-thaw treatment alone. It is possible to easily obtain antibody-producing cells that produce antibodies, and to obtain monoclonal antibodies that respond to Kudoa genus mucosa.

In the step (1), when the fish meat is flounder meat, the Kudoa septum punta spores parasitic on the flounder meat are frozen and thawed, and the cells inside the spores are destroyed to obtain the antigenic protein of the step (2).

In step (4), inactivated antibody-producing cells in response to an antigenic protein derived from the corresponding Kudoa mucosporosis among the inactivated cells A inactivated in step (3) are obtained. By selecting B, the monoclonal antibody produced by the inactivated cell B selected and cloned in step (5) becomes an antibody that specifically reacts with a specific Kudoza mucosa, which is a cause of food poisoning. .

In the case of the flounder meat, in step (4), in the inactivated antibody-producing cell A obtained in step (3), in response to the antigenic protein derived from Kudoa septum puntata, Kudoa tyrcites and kudoa The monoclonal antibody which the inactivated antibody producing cell C which does not respond to the antigenic protein originating in a latteorabrasis is selected, and the inactivated antibody producing cell C which was selected and cloned by the process (5) produces is Kudoa Since it does not react with Tyrcites or Kudoa laterolabrasis, but only with Kudoa septum punta, it becomes an antibody suitable for the test which specifically detects Kudoa septum punta from flounder meat.

As a test tool using the monoclonal antibody according to the present invention, a test tool that can specifically and simply detect the Kudoa mucospores such as Kudoa septem puntata is suitable. As such a test tool, the immunochromat test piece which uses a well-known immunochromat method, or the ELISA method test tool which uses a well-known ELISA method is mentioned. Any test tool employs two types of monoclonal antibodies related to the present invention, which does not require any special apparatus, and is suitable for the simple and specific detection of the Kudoa mucosa.

In the immunochromat test specimen according to the present invention, the monoclonal antibody according to the present invention is used as the first and second monoclonal antibodies to the Kudoa genus mucosa. The immunochromat test specimen is a complex in which a sample pad onto which a sample is loaded and a second monoclonal antibody (labeled antibody) labeled on the downstream side of the sample are applied in advance and mixed with a sample spilled from the sample pad and the corresponding labeled antibody ( on the membrane and downstream thereof, where the complex flows out from the complex pad, and the downstream side thereof is provided with a complex pad on which the conjugate is formed and developed, and a detection portion on which the first monoclonal antibody (solidifying antibody) is fixed. It consists of an absorbent pad that absorbs excess sample or composite, and a backing sheet made by supporting the sample pad, membrane, composite pad, and absorbent pad. In the detection unit of the membrane, the solidified antibody reacts with the antigenic protein derived from the Kudoa genus mucosa in the sample constituting the complex to detect the Kudoa mucosa.

In addition, since the immunochromat test piece is in the form of a rectangular sheet in its overall shape, and the supply side (sample pad side) and the downstream end (absorption pad side) of the sample are the same shape, when the sample is added to the sample pad, the sample pad It may be difficult to determine which side the side is on. In that case, the shape of the edge part of a sample pad side and an absorption pad side can also be changed into another shape.

The ELISA method test tool which concerns on this invention uses the monoclonal antibody which concerns on this invention, and this monoclonal antibody is a 1st and 2nd monoclonal antibody with respect to said Kudoa mucospores, A monoclonal antibody is an ELISA test tool, characterized in that it comprises an immobilized plate and a solution containing a corresponding second monoclonal antibody labeled by an enzyme.

In order to solve the above-mentioned problem 2, the test kit according to the present invention, by inserting a sample picking tool to the fish, and extracting, the sample picking tool which can collect the fish meat pieces from the fish and the fish A sample solution in which the sample collection tool was placed in a container (hereinafter referred to as a "tube") containing a buffer solution, vibrated the tube, peeled off the fish meat from the sample collection tool, and then fragmented to elute Kudoza spores in a buffer solution. A sample solution adjusting unit for homogenizing, a sample solution supplying tool for supplying the sample solution to the test tool, and a test tool for detecting Kudox mucosa from the sample solution using the monoclonal antibody according to the present invention.

The sample collection tool is inserted into the fish meat section of the sample and is extracted, and it becomes a sampling tool that can directly collect fish meat (sample) from the living body of the fish. Can be harvested to inspect large quantities of fish.

The sample solution adjusting unit inserts the sample tool with the fish meat collected with the sample collecting tool into a container containing a buffer solution, and adjusts the sample solution. In other words, the container is shaken up and down, and the fish meat attached to the sample collecting tool is peeled off in the buffer solution, and the separated fish meat is crushed and broken into small pieces by colliding with the specimen sampling device or the wall of the tube by the vertical vibration. The fish meat is further dispersed uniformly in the buffer by vibration, homogenizing the sample solution. Thereby, the sample solution which accelerated the elution to the buffer solution of the Kudoa genus mucospores (antigen) in fish meat can be obtained. As a result, the monoclonal antibody used for the said test | inspection becomes easy to recognize the said antigen in the test tool in which the said sample solution was dripped.

The test tool can detect the Kudoa mucospore from the sample solution using the monoclonal antibody according to the present invention. The monoclonal antibody according to the present invention directly and specifically detects the Kudoa mucosa from the fish meat contaminated with Kudoa mucosa, including the spore-free Kudoa mucosa. Therefore, the said test tool using this can perform the test which detects Kudoa mucospores directly and specifically from fish meat.

As described above, the test kit according to the present invention including the sample collecting tool, the sample solution adjusting unit, and the test tool can directly and specifically detect the Kudoa mucosa from the fish infected with Kudoza mucosa. Large quantities of fish can be tested.

As the test tool included in the test kit according to the present invention, an immunochromat test piece or an ELISA method test tool can be used. All are well-known test | inspection tools, and can detect the Kudoa genus mucospores, such as Kudoa septem puntata specifically.

If the sample collection tool included in the test kit according to the present invention is a circumferential grooved bar-shaped sampling tool, the amount of the sample (fish meat) required for the inspection in the groove portion of the sampling tool is simply inserted and extracted into the fish meat. Because fish can be collected, fish meat can be collected directly, quickly and easily, so that a sample can be collected in a short time even when a large amount of fish is inspected.

In the sample solution adjustment unit included in the test kit according to the present invention, when the fish meat is crushed and further fragmentation is promoted, a homogenizer may be used instead of a tube. By using the homogenizer, it is possible to further reduce the fish meat collected by the sampling tool.

The monoclonal antibody produced by the method including the steps (1) to (5) above reacts to the Kudoa genus mycoid spores with spores and spore shells, and thus Kudoza mucosa is suitable for the test which directly detects Kudoza mucosa in the fish meat without extracting and purifying spores from fish such as halibut infected with the included Kudoza mucosa. In the case of flounder meat, it does not respond to Kudoa Tyrcites or Kudoa latheorabrasis, but specifically to Kudoa mucospores (Kudoa septum puntata in the case of flounder meat) It can be tested to specifically detect the Kudoa mucospore from fish meat.

By using the test kit according to the present invention, it is possible to directly and specifically collect fish meat directly from the living body of the fish, and adjust the sample solution suitable for the use of tests such as immunochromat test specimens from the collected fish meat. As a test tool using Kudoa mucospores, it can be detected specifically and conveniently. According to the present invention, in the case of the flounder, the fish species are distinguished from Kudoa tyrcites and Kudoa latheorabrasis, which are parasitic on the flounder, and react only specifically to Kudoa septum puntata from the flounder. Since the Kudoa septum puntata can be detected quickly, easily and directly, it is possible to provide a test kit that can rapidly inspect a living body of fish such as a large flounder.

Fig. 1 shows a photograph (phase contrast microscopy and fluorescein isothiocyanate (FITC) fluorescence staining) in which a monoclonal antibody according to the present invention binds to a protein constituting cells inside the spores of Kudoa septum punta.
Fig. 2 is an image showing the results of Western blots in which monoclonal antibodies (two kinds) according to the present invention recognize proteins near molecular weights of 25,000 to 37,000.
3 is a schematic view relating to the configuration of an immunochromat test piece according to the present invention.
Fig. 4 is a schematic diagram relating to the construction of a test kit using an immunochromat test piece as a test tool in accordance with the present invention.
Fig. 5 is a schematic diagram relating to the configuration of a test kit using an ELISA method test tool as a test tool in accordance with the present invention.

(Explanation of process (1)-(5))

The monoclonal antibody which concerns on this invention is produced through the method made including the process of process (1)-(5). Step (1) is to separate the Kudosahic spores from fish meat, and to freeze and thaw the spore suspension solution including the spores of the Kudoa spores to destroy the spore shell and the cells inside the spores. The process of adjusting the suspension solution which elutes the protein which comprises the said cell, The process (2) inoculates the antigenic protein obtained by the process (1) to the animal of a mammal or a bird more than once, and in the body of the said animal, In the step (3), the antibody-producing cells obtained by step (2), the animal-producing cells obtained in step (2), and the antibody-producing cells that produce antibodies that respond to the protein are produced. Myeloma cells of the same animal species are fused to prepare inactivated antibody-producing cell A. Step (4) is in the inactivated antibody-producing cell A genus obtained in step (3). On, a process for the Kudo subgenus using the antigenic protein derived from the mucous gondii screening inactivation antibody-producing cells B in response to the protein. Step (5) is a step of separating the inactivated antibody-producing cell B selected in step (4) as a single cell, and then producing a monoclonal antibody in the cell.

In the step (1) (antigen protein adjusting step), the mucosporosis of the genus Kudoa is isolated from the fish meat by a method such as a percholl density gradient centrifugation, and purified, and then suspended in PBS and suspended in PBS. Cryopreserve at ° C. The following three methods are used to destroy the cells of the spores (spore shell and spores) of the Kudosa mucosa in the preserved suspension, and elute the proteins constituting the cells in the suspension solution. The suspension solution containing the protein which is an antigen for inoculating (immunizing) the animal of () can be adjusted.

(a) Freezing and thawing treatment

The stored spore suspension solution is returned to room temperature, diluted with PBS, repeatedly frozen one or more times, and then thawed. The freezing temperature is -5 ° C or lower and -196 ° C, and the melting temperature is preferably 100 ° C or lower and 0 ° C or higher. Since the cells of the spores are physically damaged and destroyed by this treatment, the proteins constituting the cells of the spores (and the inside thereof) are eluted in the suspension solution, and the inoculation (immune) to the animals in the step (2) is performed. The suspension solution containing the protein of interest as the antigen can be adjusted.

(b) freezing, thawing and solubilizing

The spore suspension solution is diluted with PBS, subjected to freeze-thawing twice or more, and the surfactant is added to the solution and left at room temperature for one day. As the surfactant, sodium dodecyl sulfate (SDS), octyl phenoxy polyethoxy ethanol or monolauric acid polyoxy ethylene sorbitan is suitably used. In this solubilization treatment, a surfactant binds to a protein constituting the cells inside the spores, forms micelles in the solution, and the antigen for inoculation (immune) from the micelle to the animal in step (2). This protein can be obtained.

(c) freeze thawing and sonication

After diluting the spore suspension solution with PBS, performing freeze-thawing twice or more, and cooling with ice, the spore suspension solution containing the spores is irradiated with ultrasonic waves, and the cells inside the spores are destroyed to form the cells. The present protein can be eluted in the solution to obtain the protein as an antigen for inoculation (immunization) to the animal in step (2).

In step (2) (antibody producing cell acquisition step), the protein obtained in step (1) is inoculated (immunized) with the antigen of an animal of mammal or bird one or more times. The animal is preferably a mouse, a rat, a rabbit or a chicken, and a mouse is more preferable for the good handling and accumulation of past immunity-related data. The site to be inoculated is preferably intraperitoneal, intradermal, subcutaneous, muscle or vein. In the immunization process to the said animal, the antibody potency in the serum of the said animal is measured by ELISA method, and the animal with high antibody potency of serum is selected. The selected animal can be inoculated with the antigenic protein, and in the body of the animal, antibody-producing cells that produce antibodies that respond to the protein can be produced, and the antibody-producing cells (spleen) can be extracted from the animal.

In step (3) (cell fusion step), the cell suspension containing the antibody-producing cells obtained in step (2) and myeloma cells of the same animal species as the animal are placed in one container, and the polyethylene glycol ( Cell fusion agents such as hereinafter, "PEG") are added to the cell-fusion of antibody-producing cells and spleen cells to prepare inactivated antibody-producing cell A.

In step (4) (screening step), inactivated antibody-producing cell A obtained in step (3) is selected from inactivated antibody-producing cell B which produces an antibody that specifically reacts with the Kudo subspecies mucosa. Screening ”is used. The protein derived from the Kudoa genus mucosporosis is used as an antigen, and it selects by ELISA method. That is, inactivated antibody-producing cell B with high antibody potency is selected. In the case of the flounder, an inactivated antibody which produces the antibody which does not react with the protein derived from Kudoa tyrcites and Kudoa latheorabrasis, but responds specifically to the protein derived from Kudoa septum puntata. Produce cell C is screened. The above screening is carried out by measuring the antibody potency of inactivated antibody-producing cells by ELISA and selecting the cells having high antibody potency, that is, those cells having high reactivity to the antigen.

In step (5) (cloning step), the inactivated antibody-producing cells B or C selected in step (4) are cloned, and monoclonal antibodies are generated in the cells. In steps (4) and (5), the steps of screening and cloning the inactivated antibody-producing cells B or C are repeated two or more times.

In step (5), for the halibut fish species, positive wells (inactivated antibody-producing cell B) that showed reactivity with a protein derived from Kudoa septum puntata were cloned to produce clones of inactivated antibody-producing cell B. do. The monoclonal antibodies produced by the clones exhibit high reactivity to proteins derived from Kudoa septum punta.

In addition, in this screening process, not only Kudoa septempunta but Kudoa Tyrcites and Kudoa latheorabrasis are subjected to cross-reaction tests. This screening (step (4)) and cloning (step (5)) are carried out several times to produce clones of inactivated antibody-producing cell C. The monoclonal antibody produced by this clone does not react to Kudoa tyrcetes and Kudoa laterolabrasis, but specifically to Kudoa septum puntata.

Among the monoclonal antibodies related to the present invention produced by the method produced by the step (1) to (5), an antibody to be used for a test tool (immune chromat test piece) using the immunochromat method is selected. . For the immunochromat test specimen using the immunochromat method, the monoclonal antibody used to detect the Kudoa septum puntata infected with the flounder meat is produced by a method produced by including the steps (1) to (5). From these monoclonal antibodies, three types of antibodies (Ks-3 C5, Ks-6 F9, Ks-1 B11) selected by repeating the process described in paragraph 0039 are used.

In order to construct the immunochromat method, since it is necessary to sandwich an antigenic protein with two types of monoclonal antibodies, a combination of antibodies suitable for sandwiching the antigenic protein is selected by the sandwich ELISA method. As a result of the selection by the sandwich ELISA method, it is narrowed down to the combination of Ks-3 C5 and Ks-6 F9 and the combination of Ks-1 B11 and Ks-1 B11. Next, the cross-reactivity of Kudoa Tyrcites with Kudoa Latteorabrasis is examined. As a result, since the combination of Ks-3 C5 and Ks-6 F9 had low crossover, the antibody used for the immunochromat method is determined by this formation.

Figure 1 shows a photograph (phase contrast microscopy and FITC fluorescence staining) in which the Ks-3 C5 antibody and Ks-6 F9 antibody bind to the protein inside the spores of Kudoa septum punta. Ks-3 C5 antibody and Ks-6 F9 antibody do not react to Kudoa Tyrcites and Kudoa latheorabrasis, but specifically to Kudoa septum puntata, and the subclass is IgG1, and the light chain It is the κ chain and recognizes proteins in the range of 25,000 to 37,000 molecular weights of the internal structure of Kudoa septum punta.

The image of the result of the western blot method of the antigen (Kudoa septem puntata) which the antibody recognizes is shown in FIG. M in the image is a protein molecular weight marker, lane 1 and lane 2 each represent the corresponding antigen recognized by two kinds of the corresponding antibodies, lane 1 uses Ks-6 F9 antibody, and lane 2 is Ks-3. C5 antibody is used. Figure 2 shows that the antibody recognizes proteins in the range of 25,000 to 37,000 molecular weights of the internal structure of Kudoa septum punta.

The two types of monoclonal antibodies were clones of inactivated antibody-producing cell C deposited as an accession number "NITEP-02181" as of January 15, 2016 to a product evaluation technology-based mechanism patent microorganism deposit center of an independent administrative corporation ("hybrid"). From hybrid horse deposited with antibody (Ks-3C5 antibody) produced from) and product evaluation technology base mechanism patent microorganism deposit center from January 15, 2016 as accession number "NITEP-02182" It is an antibody (Ks-6 F9 antibody) produced.

Ks-3 C5 antibody and Ks-6 F9 antibody are produced from the hybrid horse deposited under accession number "NITEP-02181" and the hybrid horse deposited with "NITEP-02182" made by the well-known method. The monoclonal antibody in the present invention specifically reacts to the Kudoa septum punta spores.

(Immunochromat Specimen Used in Test Tool)

The immunochromat test piece uses two kinds of antibodies (monoclonal antibodies according to the present invention) obtained in the monoclonal antibody preparation step, and the membrane to which the first antibody is immobilized and the labeled second antibody are immobilized. A composite pad and a sample pad (a sample pad which is a supply part of a sample suspension containing fish pieces such as flounder, an absorbent pad that absorbs an excess of a sample, and a backing sheet serving as such a support) can be laminated. The material constituting the absorbent pad is preferably made of a fibrous material, a porous material, or other material having the corresponding function in order to have a function of developing and retaining the sample or composite.

In the performance of this test piece, the detection sensitivity in the case of Kudoa septem puntata in which the antigen parasitic in the flounder meat is around 5000 spores, and the crossover in the case of the flounder meat is Kudoa Tyrcithes and Kudoa laterolabrasis. It does not react, but reacts specifically to Kudoa Septem puntata. In addition, the use of this test piece is not limited only to the flounder meat, but can also be used for the detection of the Kudoa mucospores parasitic in other fish meat.

 (ELISA method test tool used for test tool)

ELISA test tool is a test tool using a known ELISA method using two types of monoclonal antibodies related to the present invention, 96 web micro titanate, micro plate solidifying antibody (using Ks-6 F9 antibody), It consists of a perocytase-labeled antibody (using Ks-3 C5 antibody), a washing solution, a blocking solution, a substrate, and a reaction stopper.

The performance of this test tool is about 40 spores in the case of Kudoa septum puntata whose antigens are parasitic on the flounder meat, and the crossover in the flounder meat is Kudoa Tyrcites and Kudoa latheorabrasis. It does not react, but reacts specifically to Kudoa Septem puntata. In addition, the use of the ELISA test tool is not limited to the flounder but also can be used in Kudoa mucospores parasitic to other fish meat.

(Inspection kit)

The test kit is buffered by a sample picking tool which can obtain a fish piece (sample) of the quantity required for the test by inserting it into the fish meat of the sample and extracting the sample picking tool with the fish meat collected from the living body of the fish. The sample solution adjusting unit for adjusting the sample solution by eluting the Kudosa mucosa spores in the buffer, the sample solution supplying tool for supplying the sample solution to the test tool, and the monoclonal antibody related to the present invention. The sample solution consists of a test tool for detecting the Kudoop mucosa.

As specimen collection tool which can collect fish meat of quantity (preferably 10 mg-20 mg, but quantity out of this range.) Necessary for examination by inserting specimen collection tool into fish meat of sample and extracting from fish Use a circumferential grooved rod-shaped tool. As the rod-type sampling tool, a screw-type sampling tool using commercially available screws (pieces or mini-pieces) in which the grooves are screw-shaped, or a rod-shaped sampling tool or the like in which the grooves are formed at least one in a non-helix shape in the circumferential direction of the rod Can be mentioned.

In particular, a screw type sampling tool using a commercially available piece can be inserted into the fish meat and pulled out to extract the amount of fish meat required for the inspection by inserting it into the fish groove. In the state, it puts into the tube of a sample solution adjustment part, and can adjust a homogenized sample solution by adding a vibration. In addition, the screw type sampling tool can be easily obtained at low cost, and can be directly and easily collected from a large amount of sample (a living body of a fish), and an inspection can be performed to detect a Kudo subspecies mucosa from the fish.

Although the screw (cross screw; JISB11121995) or a mini piece can be used suitably, this screw is not restrict | limited if a groove | channel is a spiral shape. The material preferably has a strength that can be repeatedly used for inserting and extracting iron-based materials such as steel and stainless steel, nonferrous metals such as copper, copper alloys, aluminum and aluminum alloys, inorganic materials such as ceramics, and fish such as flounder. If so, it may be an organic material such as plastic or wood material, or a composite material of an organic material and an inorganic material. As for the dimension of a screw, Preferably, a nominal diameter (outer diameter of a screw) is 1-5 mm, and the thing of 20-80 mm in length is mentioned.

There is no restriction in particular as long as the quantity required for the inspection can be obtained by simply inserting it into the fish meat of the sample and extracting it, and the screw strength can withstand the insertion and withdrawal.

In the tube of the sample solution adjusting unit (see FIGS. 4 and 5), samples such as fish meat can be crushed, broken into pieces, and the homogenized sample solution can be adjusted as described above. In order to adjust the sample solution, a biomasher, a finger masher or other homogenizer may be used instead of the tube.

The sample solution adjusted by the said sample solution adjustment part is taken out from the said tube, and is supplied to a test tool. As this sample solution supply tool, an appropriate amount of sample solution (sample) can be supplied to the test tool, and a simple and easy thing is preferable. For example, a dropper, a pipette, etc. may be mentioned, but there is no particular limitation.

As the test tool, an immunochromat test piece using a monoclonal antibody according to the present invention and an ELISA method test tool can be used. By using the antibody, the test tool can detect Kudoa mucospores such as Kudoa septum puntata directly and specifically and easily from fish meat such as flounder.

(Performance of the corresponding test kit using the immunochromat specimen)

Screws were used as specimen collection tools. Pinch the screw directly to the flounder, pull out the flounder meat, adjust the suspension of the flounder meat (sample solution) with PBS with the sample solution adjusting tool, and drop the sample solution onto the sample pad of the immunochromat specimen with a dropper. Checked. As a result, the specimen responds only to flounder meat infected with Kudoa septum punta. In addition, the test kit using this test piece is not limited only to the test of a flounder, but can also be used for the test of other fish species.

(Performance of the test kit using the ELISA method)

Screws were used as specimen collection tools. The screw was stuck directly to the flounder, and the flounder meat was collected, the suspension of the flounder meat was adjusted with PBS at the sample solution adjusting unit, and the suspension was injected into the dropper into each web of the 96 web microplate. As a result, according to the test tool, the absorbance of flounder meat infected with Kudoa septum punta becomes high, and the test kit can specifically detect Kudoa septum punta from flounder meat. In addition, the test kit using this test tool is not limited to the test of the flounder, but can be used for the test of other fish species.

Although the example which implemented this invention is demonstrated to Examples 1-6, this invention is not limited to these Examples.

(Production of Monoclonal Antibody)

Monoclonal antibodies related to the present invention were produced by the following method.

Process (1) (adjustment of antigen protein)

After separating and purifying the Kudoa septum punta spores from the percol density gradient centrifuge than the flounder meat infected with Kudoa septa puntata, the spores were suspended in PBS to prepare a spore suspension solution, and then the solution was -80. Cryopreservation was carried out at ℃. Proteins (antigen proteins) which are antigens for inoculation into mice were obtained through the following three methods.

(a) Lyophilized Antigens

The solution was diluted with PBS, the solution after the dilution was frozen at -80 ° C, and the treatment of melting at 25 ° C was repeated twice.

(b) freeze thawing and solubilizer treatment

After diluting the solution with PBS and freezing and thawing the solution after the dilution under the above conditions twice, octyl phenoxy polyethoxy ethanol (0.1% TritonX-100) as a surfactant (solubilizer). , 0.5% dodecyl sulfate (SDS) or monolauric acid polyoxyethylene sol butane (0.1% Tween20) was added and solubilized for one day at room temperature.

(c) freeze thawing and sonication

The solution was diluted with PBS, and the treatment after freezing and thawing the solution after the dilution under the above conditions was repeated twice, followed by ultrasonic treatment while cooling with ice.

Process (2) (acquisition of antibody producing cell)

The antigen protein obtained by the step (1) was inoculated to mouse. 1 Prepare of three BALB / cmouse per bunch, the first doses (immunity) is 1 meeting immunization, mouse 1 maridang, 1 × 10 ⁶ of Kudo Oh counts the amount of the protein can be obtained from tempun Tata spores ( Antigenic protein) was emulsified in Freund's complete adjuvant (DifcoLaboratories), and after the second immunization, emulsified in Freund's incomplete adjuvant (DifcoLaboratories) and inoculated subcutaneously, subcutaneously, intramuscularly, intraperitoneally or intravenously three times. . One week after the third immunization, serum collected from the vein of the tail of the mouse is obtained, and the antibody potency in the serum is measured by ELISA method (serum antibody potency measurement, details described in paragraph 0056), and the mouse with high antibody potency Was selected (see Table 1 below). They inoculated the amount of the protein can be obtained from without the use of adjuvant into the abdominal cavity of the selected mouse 1 × 10 ⁶ spores of Kudo Oh forceps tempun tartar, and after three days the harvested spleen (antibody producing cells). The antibody producing cells were crushed using a stainless steel mesh on a chalet containing RPMI1640 culture medium to obtain a cell suspension containing the antibody producing cells.

(Measurement of antibody potency in serum by ELISA method)

Purify with percol density gradient centrifuge and prepare Kudoa Septem puntata, cryopreserved at -80 ° C. Freeze and thaw the spore suspension solution adjusted to the concentration of 1 × 1 × ¹⁰⁶ (spouse number of Kudoa septum puntata) / mL under the same conditions as in step (1), and then dissolve the solution ( The solubilized spore solution subjected to the same solubilization treatment as in 1) was injected into the respective webs of the 96 web microplates so as to be 1 × 10 ³ / web and placed at 4 ° C. overnight. Thereafter, the solubilized spore solution was removed, and then washed three times in PBS containing 0.05% Tween20. Thereafter, 0.5% gelatin-containing PBS was injected into 300 µL of each web as a blocking solution, and placed at room temperature for 1 hour. After the standing solution, the blocking liquid was removed and washed three times in 0.05% Tween20-containing PBS. Subsequently, the serum obtained from the mouse inoculated with Kudoa septum puntata was diluted 500-fold, and diluted five-fold there, and 100 µL of the mouse serum-diluted solution was added to each web and placed at room temperature for 1 hour. After placing at room temperature, the mouse serum diluted solution was removed, and washed three times in PBS containing 0.05% Tween20. Subsequently, perokisidase-labeled anti-mouse IgG antibody (Kirkegaard & PerryLaboratories) was diluted 2500-fold with 0.5% gelatin-containing PBS, added to each web, and left for 1 hour at room temperature. After diluting the perokisidase-labeled anti-mouse IgG antibody dilutions, the cells were washed three times in PBS containing 0.05% Tween20. Then, 0.5 mg / mL o-phenylene diamine dibasic acid salt (OPD) substrate solution (Sigma-Aldrich) was added to 100 µL of each web, placed at room temperature for 10 minutes, and 50 µL of 2N sulfuric acid was added to stop the reaction. In addition, the absorbance at 490 nm was measured with an absorbance measuring device. Through the absorbance, the antibody titer of the antigenic protein by three types of treatments of step (1) was compared.

Antibody potency of antibody-producing cells in serum collected from mouse immunized with three kinds of treatments for Kudoa Septem puntata 0.5% SDS solubilizing antigen (data only when serum dilution ratio 500 times) Is shown in Table 1.

K. septempunctata 0.5% SDS solubilizing antigen 0.5% Gelatin (Background) (a) Freeze-thawing treatment antigen inoculated mouse 0.756 0.044 (b) Freeze thawing and 0.5% SDS solubilizing agent inoculated mice 1.598 0.059 (c) Freeze thawing and sonicated antigen inoculated mice 1.146 0.047

The figure is an absorbance value of 490 nm. According to Table 1, both (a) freeze-thawed antigen inoculated mouse, (b) freeze-thawed and 0.5% SDS solubilized antigen-inoculated mouse, and (c) freeze-thawed and sonicated antigen-inoculated mouse were absorbed to the absorbance of 0.5% gelatin in the background. In comparison, the absorbance of the Kudoa Septem puntata antigen is greatly increased, and it reacts strongly to the antigen, and three kinds of antigens ((a) freeze-thawing antigen, (b) freeze-thawing and 0.5% SDS solubilizing antigen, ( c) Antibody potency rises about mouse inoculated with freeze thawing and sonication antigen, and antibody produced based on three kinds of antigens is 0.5% SDS solubilizing antigen (cell of spore inside of Kudoa septum punta) It includes a protein derived from. Thereby, in the monoclonal antibody produced from the antigenic protein obtained by the freezing and thawing treatment of step (1), there is an antibody that reacts with the antigenic protein derived from the cells in the spores.

Therefore, the monoclonal antibody which concerns on this invention can detect the Kudoa genus mucosporosis which does not form the target spore. In particular, the antigens subjected to solubilization in addition to the freezing and thawing treatment (Table 1 (b)) had the largest increase in the antibody potency, and then the antigens treated with ultrasound after freezing and thawing treatment (see Table 1 ( c)), and then, the antigen (Table 1 (a)) of only the freeze-E fusion treatment is used. Therefore, in the step (1), in addition to the freezing and thawing treatment of the Kudoa septum punta spores, the solubilization treatment or the irradiation with ultrasonic waves can be used to further increase the antibody potency of the mouse.

Process (3) (cell fusion process)

The cell suspension containing the antibody-producing cells obtained using the antigen of (a) of the step (1) and myeloma cells (P3X63Ag8u. 1) are each fetal calf serum (hereinafter referred to as "FBS"). RPMI1640 in a culture medium (Gibco) Two centrifugal washings (1200 rpm, 5 minutes, room temperature) were carried out, and finally, a cell suspension containing a plurality of antibody-producing cells and a plurality of myeloma cells were arranged in one round bottom tube. Centrifuge the mixture of the cell suspension of the antibody-producing cells and the bone marrow cells (1200 rpm, 5 minutes, room temperature), remove the supernatant, release the cell mass, and remove 50% polyethylene glycol (50%) of the cell fusion reagent ( 2 ml of culture medium containing PEG) (RPMI1640 culture medium (non-FBS)) was placed in a tube containing cells for 1 minute.The cells were fused with a plurality of antibody-producing cells and a plurality of myeloma cells. A was obtained. Thereafter, 10 mL of RPMI 1640 culture paper (without FBS) was added to the tube for 3 minutes.

Process (4) (selection (screening) process)

Then, 10% FBS-containing baeyangji HAT (Sigma-Aldrich) to the injection into 100 mL added to each 200μL 96 Web culture plates, were incubated one week under 37 ℃, 5% CO ₂ present. Three days after the start of the culture, the medium was exchanged in a 10% FBS-containing HAT medium. After culturing the culture medium (inactivated antibody-producing cell A) subjected to the culture medium exchange for one week, 100 μL of the culture supernatant was recovered from each web of the culture plate (hereinafter referred to as the "web"), and the ELISA method (for details, paragraph 0062) Was selected by the following two methods, and the web (inactivated antibody producing cell B or C) which advances to the next process (cloning process) was selected.

(A) Screening process by comparing only Kudoa Septem puntata

(B) Screening process in contrast to Kudoa Septempuntata, Kudoa Tyrcites, and Kudoa Latheorabrasis

As a result of two kinds of screening (data shown in Tables 2 and 3 below), Table 2 shows a highly reactive web against Kudoa septum puntata (antibodies of antibodies produced by inactivated antibody-producing cells in the web). Web with high stamina) and Table 3 were selected for a web that is highly reactive to Kudoa septum puntata and a web that is less reactive to Kudoa tyrcetes and Kudoa laterolabrasis.

Data of the screening process in contrast to Kudoa Septem puntata only Web (Inactivated Antibody Producing Cell B) K.septempunctata 0.5% SDS Solubilizing Antigen Ks-1A1 0.609 Ks-2D12 0.414 Ks-4A11 0.438 Ks-5H5 0.746 Ks-5E7 0.900

The figure is an absorbance value of 490 nm. Among about 600 webs of the culture plate, cells of 5 webs (inactivated antibody-producing cell B) described in Table 2 above, which showed high reactivity to Kudoa septum puntata were selected. The five kinds of inactivated antibody-producing cells B obtained by this step through Table 2 show high reactivity to proteins derived from Kudoa septum punta spores, including proteins derived from the internal structure of Kudoa septum punta spores. have.

Data from the screening process in contrast to Kudoa Septempuntata, Kudoa Tyrcites, and Kudoa Latheorabrasis Web (Inactivated Antibody Producing Cell C) K.septempunctata 0,5% SDS solubilizing antigen K. thyrsites 0,5% SDS solubilizing antigen K.lateolabracis 05% SDS solubilizing antigen Ks-1B11 0.859 0.132 0.142 Ks-2A2 0.659 0.152 0.112 Ks-3C5 0.793 0.050 0.046 Ks-4B1 0.632 0.044 0.049 Ks-6F9 0.326 0.043 0.050

The figure is an absorbance value of 490 nm. In the highly reactive web to Kudoa septum puntata, and also the cells of the web (inactivated antibody-producing cells C) with low reactivity to Kudoa tyrcetes and Kudoa laterolabrasis, Cells were selected.

 (Screening of Inactivated Antibody Producing Cells by ELISA Method)

Purified with a percol density gradient centrifuge, -80 ° C and cryopreserved Kudoa septum puntata were prepared. Solubilized spore solution in which the spore suspension solution adjusted to the concentration of 1 × ¹⁰⁶ / mL was frozen twice and thawed under the same conditions as in step (1), and the solution was subjected to the same solubilization treatment as in step (1). Was injected into each web of 96 web microplates so as to be 1 × 10 / web, and left overnight at 4 ° C. After removing the solubilized spore solution, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, 0.5% gelatin-containing PBS was injected into 300 µL of each web as a blocking solution, and placed at room temperature for 1 hour. After the blocking solution was removed, washing was performed three times in PBS containing 0.05% Tween20.

Thereafter, 100 µL was collected from the culture medium (culture supernatant) of the cell culture plate, and added to each web and placed at room temperature for 1 hour. After the standing, the culture supernatant was removed, and then washed three times in PBS containing 0.05% Tween20. Subsequently, perokisidase-labeled anti-mouse IgG antibody (Kirkegaard & PerryLaboratories) was diluted 2500-fold with 0.5% gelatin-containing PBS and added to each web and left for 1 hour at room temperature. After the dilution of the perokisidase-labeled anti-mouse IgG antibody was performed, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, 0.5 mg / mL OPD substrate solution (Sigma-Aldrich) was added to each 100 μL web and placed at room temperature for 10 minutes, and 2 N sulfuric acid was added to 50 μL each web to stop the reaction. Absorbance was measured. The results are shown in Tables 2 and 3.

Inactivated antibody-producing cell B (Ks-1 A1 strain, Ks-2 D12 strain, Ks-) exhibiting high reactivity to Kudoa septempunta in about 600 webs (about 600 inactivated antibody-producing cell B) of the culture plate. 4 A11 strain, Ks-5 H5 strain, Ks-5 E7 strain) were selected. Five types of inactivated antibody-producing cells B obtained in this step in Table 2 show high reactivity with respect to proteins derived from the internal structure of Kudoa septum punta spores and spores. In Table 3, five kinds of cells were expressed as inactivated antibody-producing cells C with high reactivity to Kudoa septum puntata, and as inactivated antibody-producing cells C with low reactivity to Kudoa tyrcetes and Kudoa laterolabrasis. (Ks-1 B11 strain, Ks-2 A2 strain, Ks-3 C5 strain, Ks-4 B1 strain, Ks-4 B1 strain, Ks-6 F9 strain) were selected.

For example, in the case of Ks-3 C5 strain, according to Table 3, the absorbance value of 490 nm for the 0.5% SDS solubilizing antigen of Kudoa septum punta is 0.793, whereas that of the corresponding antigen of Kudoa tyrcites is 0.793. The value is 0.050, the corresponding value of the corresponding antigen of Kudoa lateorabrasis is 0.046, the reactivity of the absorbance value to the high antigen is high, specific, the reactivity of the absorbance value to the low antigen is low and inactivated. Antibody-producing cell C (Ks-3 C5 strain) has low reactivity to Kudoa Tyrcitest and Kudoa laterolabrasis and specifically reacts to Kudoa septum puntata. Therefore, by the screening process described above, inactivated antibody-producing cell C capable of specifically detecting only Kudoa septum puntata that causes food poisoning in flounder meat was selected.

Process (5) (cloning process)

The inactivated antibody-producing cells B or C grown in the web selected in the step (4) were collected using a pipette, put into PBS, suspended, and the cell count was measured by a hemocytometer. Thereafter, the mixture was diluted in a 10% FBS-containing RPMI1640 medium so as to be 1/100 μL, and 100 μL was added to each new cell culture plate. The cells were incubated for about two weeks in the presence of 37 ° C and 0.5% CO ₂ , and then subjected to ELISA test using the culture supernatant. This operation was repeated four times, inactivating antibody-producing cells B and C were single-celled, and 5 weeks of hybrid lines were obtained for the cell B. Moreover, about the said cell C, the hybrid line of 3 weeks (Ks-3 C5 strain, Ks-6 F9 strain, Ks-1B11 strain) was finally established. Table 4 shows that the monoclonal antibodies produced by the hybrid viscera specifically react to Kudoa septempuntata and do not respond to Kudoa tyrcetes and Kudoa laterolabrasis.

Table 4 shows the antibody potency of the monoclonal antibodies produced in the hybrid strains (clone (clone of inactivated antibody-producing cell C)) obtained after screening and cloning.

Clone name (monoclonal antibody name) K.septempunctata 0.5% SDS Solubilizing Antigen K.thyrsites 0.5% SDS Solubilization K.lateolabracis 05% SDS Solubilizing Antigen 0.5% Gelatin (Background) Ks-3D5 0.602 0.046 0.042 0.040 Ks-6F9 0.377 0.043 0.040 0.040 Ks-1B11 0.764 0.103 0.110 0.042

The figure is an absorbance value of 490 nm. Table 4 shows that the antibodies produced by the three-week hybrid vicin (clone (clone of inactivated antibody-producing cell C)) are highly responsive to Kudoa septum puntata causing food poisoning, parasitic on flounder, and not pathogenic to humans. It has been shown to have low reactivity to either Kudoa tyrcetes or Kudoa laterolabrasis.

Among the monoclonal antibodies related to the present invention, a combination of monoclonal antibodies (combination of a solidified antibody and a labeled antibody) to be used as an immunochromat method (immune chromat test specimen) is selected by the sandwich ELISA method, and the selected antibody is selected. Confirmed that the protein from the interior of the Kudoa Septem punta spores is bound to.

(Adjustment of Purified Monoclonal Antibodies)

In order to produce a labeled antibody, the monoclonal antibody related to the present invention was purified by the following treatments (α) to (γ).

(α) Collection of plural

The hybrid strain thus established was cultured in a 10% FBS-containing I1640 culture medium to prepare a cell suspension adjusted to 1 × 10 ⁶ cells / mL. Three cells were administered to each hybrid vis, BALB / cmouse inoculated with 0.5 mL of pristane (wako pure drug) intraperitoneally two weeks ago, and 1 mL of the adjusted cells were inoculated intraperitoneally. Between 1 week after inoculation and 2 weeks later, ascites was collected with a syringe regularly stored in the abdomen. Per mouse, about 5 mL of ascites could be harvested. The collected ascites was centrifuged (3000 rpm, 15 minutes, 4 ° C), and the supernatant was cryopreserved at -80 ° C.

(β) a plurality of tablets

The above-obtained ascites was diluted twice with PBS, and the monoclonal antibody was purified by affinity column chromatography using ProteinG. ProteinG column (GE Healthcare) was washed with PBS 10 times the volume of the bat volume, and the diluted ascites was added. Thereafter, the column was washed with PBS 20 times the volume of the bat volume, and then 0.1 M glycine (pH 2.5) 5 times the volume of the bat was added to elute the IgG adsorbed on the column. After elution, 1 M Tris buffer (pH9.0) was added to neutralize the eluate. Then, dialysis was performed in PBS, and the solvent was substituted by PBS.

(γ) Enrichment of Monoclonal Antibodies

The purified solution was concentrated to 5 mg / mL by ultrafiltration apparatus (Mermyripoa) with a molecular weight of 50,000, and then cryopreserved at -80 ° C.

 (Perokidase Label of Purified Monoclonal Antibody)

The labeling antibody used for sandwich ELISA was produced using the monoclonal antibody refine | purified by the said (alpha)-(gamma) process. The peroxidase labeling of the purified monoclonal antibody was performed using Peroxidase Labeling Kit-NH ₂ (Dongin Chemical). Finally, 1 mg / mL of perokidase-labeled monoclonal antibody was obtained and cryopreserved at -80 ° C.

 (Selection of Monoclonal Antibody Used by Immunochromat Method (Immunochromat Specimen))

In order to establish the immunochromat method, it is necessary to sandwich the antigenic protein with two kinds of monoclonal antibodies. Therefore, a combination of antibodies suitable for sandwiching the antigenic protein among antibodies generated by inactivated antibody-producing cell C is sandwiched by the ELISA method. (Details are given in paragraph 0072) (see Table 5). As a result, it was narrowed down to the combination of Ks-3 C5 and Ks-6 F9 and to the combination of Ks-1 B11 and Ks-1 B11. Therefore, the cross reactivity between tyrcetes and laterolabrasis was examined next (see Table 6). As a result, since the combination of Ks-3 C5 and Ks-6 F9 had low crossover, the antibody used for the immunochromat method was determined by this formation.

Table 5 shows the results of the formation of clones suitable for the immunochromat method according to the sandwich ELISA method (antigen is kudoa septum punta).

Clone name (monoclonal antibody name) Plate Solidification Antibodies Ks-3C5 Ks-6F9 Ks-1B11 Perocysidase Antibody Ks-3C5 0.179 3.873 0.09 Ks-6F9 3.903 0.116 0.305 Ks-1B11 0.601 0.105 3.614

The figure is an absorbance value of 490 nm. Table 5 shows the absorbance values of the Ks-3 C5 and Ks-6 F9 combinations of 3.873 and 3.903 among the three-week clones (clones of inactivated antibody-producing cell C producing monoclonal antibodies). The combination of different combinations (e.g., Ks-1 B11 and Ks) with different values for the absorbance of the combination of -1 B11 and Ks-1 B11 is 3.614; The absorbance of -3C5 is very high compared to 0.601). Therefore, the combination of such antibodies can be used for the specific detection of antigen by the immunochromat method (immunogrommat test piece).

The detection sensitivity of the sandwich ELISA method in combination of Ks-3 C5 and Ks-6 F9 is shown in Table 6 (the antigen is Kudoa septum punta, etc.).

Plate Solidification Antibodies Ks-6P9 Perocysidase Antibody Ks-3C5 Spore count K. Septempunctata 0.5% SDS Solubilizing Antigen K. Septempunctata formalin K. Thyrsites 0.5% SDS Solubilizing Antigen K. Thyrsites Formalin K. Lateolabracis 05% SDS Solubilizing Antigen K. Lateolabracis Formalin 5 × 103 3.608 3.452 0.134 0.235 0.13 0.125 1 × 103 3.176 2.496 0.13 0.144 0.124 0.124 2 × 102 1.58 0.882 0.125 0.124 0.12 0.12 4 × 10 0.561 0.316 0.124 0.121 0.125 0.124 8.0 0.265 0.182 0.123 0.123 0.124 0.125 1.6 0.183 0.137 0.126 0.127 0.125 0.126 0.3 0.154 0.139 0.126 0.127 0.127 0.127 0.5% gelatin 0.148 0.132 0.131 0.131 0.133 0.133

The figure is an absorbance value of 490 nm. Through the results of Table 6, the detection sensitivity of the sandwich ELISA method in the combination of Ks-3 C5 and Ks-6 F9 was specific for Kudoa septum punta with 8 or more spores if solubilized spore solution of Kudoa septa punta Can be detected. Formalin spore fluid can specifically detect Kudoa septum punta in more than 40 spores.

 (Extraction of Antibodies Appropriate to the Immunochromat Method by Sandwich ELISA Method)

5 mg / mL purified monoclonal antibody was diluted to 5 μg / mL in PBS, 100 μL each was injected into each web of 96 web microplates, and placed overnight at 4 ° C. After removing the antibody solution, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, 0.5% gelatin-containing PBS was injected into 300 µmL of each web as a blocking solution, and the mixture was placed at room temperature for 1 hour. After the blocking solution was removed, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, the spore solution adjusted to the concentration of 1 × 10 6 / mL was lyophilized twice, and the solubilized spore solution subjected to the above solubilization treatment, or a formalin spore solution only suspended in formalin-containing PBS, was used. Each web of 96 web microplate was inject | poured so that it might become a web, and it was left at room temperature for 1 hour.

After removing the spore solution, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, the peroxysidase-labeled purified monoclonal antibody was diluted 200-fold with 0.55 gelatin-containing PBS, and added to each web and placed at room temperature for 1 hour. After removal of the peroxysidase-labeled purified monoclonal antibody dilution, three washes were performed in PBS containing 0.05% Tween20. Thereafter, 0.5 mg / mL OPD substrate solution (Sigma-Aldrich) was added to each 100 μL web and placed at room temperature for 10 minutes, and 2 N sulfuric acid was added to 50 μL each web to stop the reaction. Absorbance was measured.

(Confirmation of the antigen protein to which the antibody used for the immunochromat method binds)

In order to determine the antigen recognition sites of the monoclonal antibodies K-3 C5 and Ks-6 F9, immunostaining and Western blot were performed (for details, paragraphs 0074 and 0075, respectively). From these results, both K-3 C5 and Ks-6 F9 bind to proteins inside the spores of Kudoa septum punta (see Figure 1), and recognize proteins with a molecular weight in the range of 25,000 to 37,000 (near 28,000). It confirmed that it was an antibody (refer FIG. 2).

(Observation of binding state of antigen by antibody by immunostaining method)

The binding state of the monoclonal antibodies K-3 C5 and Ks-6 F9 to the protein inside the spores of Kudoa septum punta was observed by the following immunostaining method (color development method for visualizing the antigen antibody response (the corresponding binding state)). did. 10 µL of 1 × 10 ⁶ / mL formalin-containing kudoa septum puntata was used. Centrifuge twice with PBS (6,000 rpm, 3 min, room temperature) and remove the supernatant. The screening was suspended with 100 µL of purified monoclonal antibody diluted at 10 µg / mL, and left at room temperature for 30 minutes. Thereafter, the cells were centrifuged twice with PBS (6,000 rpm, 3 minutes, room temperature), and the FITC-labeled antimouse IgG antibody (SantaCruzBiotechnology, Inc) was diluted 50-fold with PBS and left at room temperature for 30 minutes. Thereafter, the resultant was centrifuged twice with PBS (6,000 rpm, 3 minutes, room temperature), and the suspension was suspended in 20 µL of PBS, and the suspension was observed under a fluorescence microscope. As shown in the phase contrast microscopy and FITC fluorescence staining of Fig. 1, it was found that the monoclonal antibodies K-3 C5 and Ks-6 F9 bind to proteins inside the spores of Kudoa septum punta.

 (Classification of antigenic protein molecular weight recognized by antibody according to western blot method)

The molecular weights of the proteins inside the spores of Kudoa septempunta recognized by the monoclonal antibodies K-3 C5 and Ks-6 F9 were classified into Western blots. 10 µL of 1 × 10 ⁶ / mL formalin-containing kudoa septum puntata was used. The equivalent amount of 10% 2-mercapto ethanol-containing SDS-PAGE sample buffer was added to the spore solution, and then heated at 100 ° C for 10 minutes. The heated spore solution was electrophoresed using SuperSep 10-20% (Wako Pure Chemical Co., Ltd.), which is a polyacrylamide gel, and then transferred to a polyvinylidene fluoride (hereinafter referred to as "PVDF") film. After the transfer, the PVDF film was blocked with 1% skim milk-containing PBS. After blocking, the resultant was washed three times with PBS containing 0.05% Tween20, and a perocysidase-labeled monoclonal antibody diluted at 10 µg / mL with a blocking solution was added and left at room temperature for 60 minutes. Thereafter, the resultant was washed three times with 0.05% Tween20-containing PBS, and 2 mL of EzWestBlue (Ato Corporation), which is a color developing substrate, was added and allowed to react for 1 minute. Then, the band which appeared after wash | cleaning with a large amount of distilled water was confirmed. As a result of classifying the protein molecular weight of the marker from the position of the appearing band, as shown in Fig. 2, the monoclonal antibodies K-3 C5 and Ks-6 F9 both recognized proteins having a molecular weight in the range of 25,000 to 37,000 (near 28,000). did.

 (Confirmation of subclasses of Ks-3 C5, Ks-6 F9)

To examine the subclasses characteristic of the monoclonal antibodies K-3 C5 and Ks-6 F9, they were tested using IsoStripMouseMonoclonalAntibodyIsotypingKit (RocheLifeScience). As a result, both antibodies belonged to the subclass of IgG1, and the light chain was classified into κ chains.

 (Deposit of hybrids)

The hybrid horse which produces the monoclonal antibody Ks-3C5 was deposited as accession number "NITEP-02181" to the product evaluation technology base apparatus patent microorganism deposit center as of January 15, 2016. The hybrid horse which produces the monoclonal antibody Ks-6 F9 was deposited with the accession number "NITEP-02182" to the patent evaluation microorganism deposit center based on the product evaluation technology base apparatus of an independent administrative corporation on January 15, 2016.

The performance of the test tools and the test tools that make up the test kit were evaluated.

 (Confirmation of production of immunochromatin test piece and detection of Kudoa septum punta)

(A) Preparation of Antibody Solidification Membrane

The concentration of Ks-6 F9 monoclonal antibody Ks-6 F9 used for application was diluted in 5 mM phosphate buffer (pH7) at 1 mg / mL, and the coating was applied to a Hi-FlowPlusHF135 nitrocellulose cellulose membrane (Merck myripoa). It applied on the conditions of 1 microliters / cm. Then, it put for 30 minutes at 60 degreeC, and dried. After drying, the solution was immersed in 0.5% 50 mM boric acid buffer (pH 5.5) and dried at room temperature for one day. After the drying, the membrane was stored in a low humidity storage chamber.

 (B) Preparation of the Composite Pad

Monoclonal antibody Ks-3 C5 bound to gold colloid was diluted with 100 μg / mL with pure water and added to 10 mL of 40 nm gold colloidal solution (BBISolutions) for 30 minutes at room temperature. Subsequently, after centrifuging twice in 20 mL 1% bovine serum albumin-containing Tris buffer (pH8) (10,000 rpm, 4 ° C. for 15 minutes), the supernatant was removed, and the screening was performed with 2 mL of 1% bovine serum albumin-containing tris. It was suspended in buffer (pH8) and refrigerated at 4 degreeC. After cold preservation, 1 mL of antibody-coated gold colloidal solution was applied to a 10 mm x 150 mm Glassfiber conjugate pad (Merck myripoa), and the glassfiber conjugatepad was dried at 60 ° C for 30 minutes using a vacuum low temperature dryer. The composite pads were stored in low humidity storage.

 (C) Lamination (gluing) process of each member

Cellulosefiber samplepad (Merck Myrpoa) and backing sheet (Lohmann) were prepared as an absorption pad as an antibody solidification membrane, a composite pad, and a sample pad which were produced above. At the pasting position of each member of the backing sheet, each member was pasted so that 2 mm overlapped in order of an antibody solidification membrane, an absorption pad, a composite pad, and a sample pad.

 (D) cutting process

The backing sheet which adhered the member was cut | disconnected to width 5mm with a cutting machine, and was stored in the low humidity storage. In addition, in order to make the shape of the edge part of the sample pad side of an immunochromat test piece and the shape of the edge part of a liquid absorption pad side different, the liquid absorption side edge part can be cut to be fine in the flow direction of a sample.

 (Quality Evaluation of Detection Sensitivity)

A Kudoa Septempunta formalin spore solution only suspended in formalin-containing PBS was diluted with PBS to a concentration of 5 × 10 ⁴ / mL, and a test sample subjected to 5-fold dilution was obtained. Thereafter, 100 µL of diluted spore solution was added dropwise to the immunochromat test specimen, and qualitative evaluation of the detection sensitivity of the immunochromat test specimen was performed.

Table 7 shows the detection sensitivity of Kudoa septum puntata of immunochromat test specimens.

K.septempunctata 5 x 104 pcs / mL 1 × 104 pcs / mL 0.2 × 104 pcs / mL PBS + - - -

From the results in Table 7, it was found that about 5,000 spores were the detection sensitivity.

 Qualitative evaluation of intersects

The formalin spore solution of Kudoa septa punta spores suspended only in formalin-containing PBS was diluted with PBS to a concentration of 5 × 10 ⁴ / mL, and 100 μL of diluted spore solution was added dropwise to the prepared immunochromat specimen. , Ks-6 F9 antibody and Ks-3 C5 antibody coated qualitatively the cross-linking of immunochromat specimens. As a target, 1 g of flounder meat was added to 1 mL of PBS, homogenized with a finger mash (Sarstedt), and the presence or absence of reactivity was evaluated in the same manner.

Table 8 shows the crossover by the immunochromat test specimens coated with Ks-6 F9 antibody and Ks-3 C5 antibody.

K septempunctata formalin K thyrsites formalin K lateolabracis formalin Flounder PBS + - - - -

In Table 8 above, when the test line on the membrane of the immunochromat test specimen in which the suspension of each sample described in Table 8 was loaded was developed (shows that the antibody reacted with the antigen.), "+" When we did not develop color, we said "-". Through the results in Table 8, the combination of Ks-6 F9 antibody and Ks-3 C5 antibody used for the test piece does not respond to Kudoa Tyrcites or Kudoa laterabrasis parasitic on the flounder, It has been recognized that it reacts specifically to septum puntata. This confirmed that the Kudoa septum punta can be detected specifically by using an immunochromat test specimen using the monoclonal antibody. Moreover, there is no restriction | limiting in particular also using this immunochromat test piece for the detection of the Kudoop mucosa of fish other than a flounder.

(Confirmation of kudoa septa puntata detection by ELISA method inspection tool)

The composition of the ELISA test tool using the monoclonal antibody related to the present invention and the performance of the test tool were confirmed.

(Constituent absence of ELISA method inspection tool)

96 web microplates for the ELISA method used Maxithorp (Thermo Scientific, Inc.). Ks-6 F9 strain was used for the microplate solidification antibody for ELISA method, and Ks-3 C5 strain was used for the perokidase-labeled antibody. The washing solution was PBS containing 0.05% Tween20. As the blocking solution, 0.5% gelatin containing PBS was used. As the substrate, 0.5 mg / mL of OPD substrate solution (Sigma-Aldrich) was used. 2 N sulfuric acid was used as the reaction terminating liquid.

 (Preparation of Peroxysidase Enzyme Labeled Antibody)

To prepare an antibody labeled with peroxysidase for use in the sandwich ELISA method, a purified monoclonal antibody (Ks-3 C5 strain) was used for Peroxidase Labeling Kit-NH2 (Dongin Chemical) and the antibody was labeled with peroxysidase. did. Finally, 1 mg / mL of perokidase-labeled monoclonal antibody (Ks-3 C5 strain) was obtained and then cryopreserved at -80 ° C.

 (Quantitative evaluation of detection sensitivity)

The formalin spore solution only of suspending Kudoa septum punta spores with formalin-containing PBS was diluted with PBS to a concentration of 5 × 10 ⁴ , followed by 5-fold dilution to obtain a test sample. Then, ELISA method test was performed using each 100 microliters of diluted spore liquid, and the reactivity was quantitatively evaluated. 5 mg / mL purified monoclonal antibody (Ks-6 F9) was diluted to 5 μg / mL in PBS, injected into 100 μL of each web of 96 web microplates, and placed overnight at 4 ° C. After removing the antibody solution, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, 0.5% gelatin-containing PBS was injected into 300 µL of each web as a blocking solution, and placed at room temperature for 1 hour. After the blocking solution was removed, washing was performed three times in PBS containing 0.05% Tween20.

Subsequently, 100 µL of the diluted spore solution was added to each web and placed at room temperature for 1 hour. After removing the diluted spore solution, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, the perokidase-labeled purified monoclonal antibody (Ks-3 C5) was diluted 2000-fold with 0.5% gelatin-containing PBS, and each web was added and left at room temperature for 1 hour. After removal of the peroxysidase-labeled purified monoclonal antibody dilution, three washes were performed in PBS containing 0.05% Tween20. Thereafter, 0.5 mg / mL OPD substrate solution (Sigma-Aldrich) was added to each 100 μL web and left for 10 minutes at room temperature, and 2 N sulfuric acid was added to 50 μL each web to stop the reaction. Was measured. Table 9 shows the detection sensitivity of the ELISA assay tool against Kudoa septum puntata.

Spore count K. septempumnctata formalin 5 × 103 2.504 1 × 103 0.974 2 × 103 0.291 4 × 10 0.108 8.0 0.058 0.5% gelatin 0.052

The figure is an absorbance value of 490 nm. From the above result, it turned out that about 40 spores are detection sensitivity.

 (Quantitative evaluation of crossover)

A formalin spore solution only suspended in formalin-containing PBS was diluted with PBS to a concentration of 5 × 10 ⁴ / mL, and 100 μL of diluted spore solution was added dropwise to the prepared immunochromat test specimens to confirm reactivity. 5 mg / mL purified monoclonal antibody (Ks-6 F9) was diluted to 5 μg / mL in PBS, injected into 100 μL of each web of 96 web microplates, and placed overnight at 4 ° C. After removing the antibody solution, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, 0.5% gelatin-containing PBS was injected into each 100 µL web as a blocking solution, and placed at room temperature for 1 hour. After the blocking solution was removed, washing was performed three times in PBS containing 0.05% Tween20. Subsequently, 100 µL of the diluted spore solution was added to each web and placed at room temperature for 1 hour. After removing the dilute spore solution, washing was performed three times in PBS containing 0.05% Tween20.

Subsequently, the perokisidase-labeled purified monoclonal antibody (Ks-3 C5) was diluted 2000-fold with 0.5% gelatin-containing PBS and added to each web and left for 1 hour at room temperature. After removal of the peroxysidase-labeled purified monoclonal antibody dilution, three washes were performed in PBS containing 0.05% Tween20. Thereafter, 0.5 mg / mL OPD substrate solution (Sigma-Aldrich) was added to 100 μL of each blood and placed at room temperature for 10 minutes, and 2N sulfuric acid was added to 50 μL of each web to stop the reaction. Was measured. Table 10 shows the evaluation results of the crosslinking of monoclonal antibodies (Ks-6 F9, Ks-3 C5) by ELISA test tool.

Table 10 shows the evaluation results of the quantitative evaluation of the crosslinking of monoclonal antibodies (Ks-6 F9, Ks-3 C5) by the ELISA test tool.

Spore count K. septempunctata formalin K. thyrsites formalin K. lateolabracis formalin 5 × 103 2.504 0.058 0.047 1 × 103 0.974 0.047 0.047 2 × 102 0.291 0.047 0.048 4 × 10 0.108 0.044 0.046 8.0 0.058 0.046 0.046 0.5% gelatin 0.052 0.049 0.05

The figure is an absorbance value of 490 nm. By comparing the absorbances of Kudoa septempunta and the absorbances of Kudoa Tyrcites and Kudoa latheorabrasis through Table 10 above, Kudoa Tyrcites and Kudoa latheorabrasis are compared Because the levels of Aceptemuntata are significantly higher (Kudoa Tyrcites, etc., are equivalent to 0.5% gelatin, the background against which the antibody does not respond), the test tool using the monoclonal antibody is Kudoa Septempun It was found that tata can be detected specifically.

(Evaluation of Test Kits)

(I) Test Kits Using Immunochromat Specimens

As a sample picking tool, a screw outer diameter 2.1 mm and a screw 38 mm in length (Yatha Screw Co., Ltd. "Mini Piece" MN-38 Cu) were used. The test piece of Example 3 was used as this immunochromat test piece. Monoclonal antibody Ks-6 F9 was applied to the antibody solidification membrane of the test piece, and monoclonal antibody Ks-3 C5 labeled with a gold colloidal solution was applied to the composite pad. Flounder meat and uninfected flounder infected with three kinds (1.23 × 10 ⁶ pieces / g, 4.5 × 10 ⁵ pieces / g, 7.5 × 10 ⁴ / g) with different infected numbers per 1 g of flounder flesh Four kinds of meat (we confirmed uninfected by microscopic examination) were made into samples in total. The screw was stuck directly to the flounder, and the flounder meat was directly collected from the livestock of the flounder. After that, the weight of the screw including the flounder meat was weighed, and the weight of the flounder meat was subtracted by subtracting the weight of the screw body that was weighed in advance.

As a result, the weight of the flounder meat collected was 15 mg. PBS was added to the tube so that the flounder meat and the flounder meat in PBS had a concentration of 10 mg / mL, and the mixture was shaken up and down sufficiently to obtain a suspension of the flounder meat. In addition, when the sample was further fragmented, a homogenizer such as a finger masher was used instead of the tube. Then, the flounder suspension in a tube was extract | collected using the dropper (for 0.2 mL, AZONE Co., Ltd.), and 3 drops were dripped at the immunochromat test piece. According to the presence or absence of the color development of the test line in the measurement site of the determination result of the immunochromat test piece, the reactivity with the flounder meat infected with Kudoa septem puntata (reactivity with Kudoa septem puntata) was confirmed. Table 11 shows the results of evaluating the performance of the test kit using the immunochromat test specimen.

K. septempunctata infected flounder Ref. 1.23 × 106 pcs / g , 4.5 × 105 pcs / g, 7.5 × 104 / g Uninfected Flounder PBS + + + - -

In Table 11, when the test line of the test piece was developed (when the monoclonal antibody (combination of Ks-6 F9 and Ks-3 C5) used in the immunochromat test piece reacted with the antigen), "+", When not developing (when not responding) was called "-". In Table 11, it does not respond to the suspension prepared from uninfected flounder meat, but reacts only with the suspension prepared from flounder meat infected with Kudoa septum puntata. By this, the test kit can collect the flounder meat quickly and easily directly from the flounder with the grooved bar-shaped sampling tool, and the presence or absence of color development of the test line of the immunochromat test piece which comprises the test kit. In addition, since the reactivity can be confirmed, it was confirmed that the reactivity to the Kudoa septum puntata can be determined quickly and easily directly from the flounder meat.

(II) Test kit using ELISA test tool

As the sample collecting tool, the same screw as in Example 4 was used. The same combination of Ks-6 F9 and Ks-3 C5 as in Example 3 was used for the ELISA microplate solidification antibody and the perokidase-labeled antibody. Flounder meat and uninfected flounder infected with three kinds (1.23 × 10 ⁶ pieces / g, 4.5 × 10 ⁵ pieces / g, 7.5 × 10 ⁴ / g) with different infected numbers per 1 g of flounder flesh Four kinds of meat (we confirmed uninfected by microscopic examination) were made into samples in total. The screw was stuck directly to the flounder and the flounder meat was collected. After that, the weight of the screw including the flounder meat was weighed, and the weight of the flounder meat was subtracted by subtracting the weight of the screw body previously measured. As a result, the weight of the flounder meat collected was 15 mg. PBS was shaken up and down in a tube sufficiently so that the flounder meat and the flounder meat in PBS became 10 mg / mL, and the suspension of flounder meat was obtained. In addition, when the sample was further fragmented, a homogenizer such as a finger masher was used instead of the tube. 5 mg / mL purified monoclonal antibody (Ks-6 F9) was diluted to 5 μg / mL in PBS, injected into 100 μL of each web of 96 web microplates, and placed overnight at 4 ° C. After removing the antibody solution, washing was performed three times in PBS containing 0.05% Tween20.

Thereafter, 0.5% gelatin-containing PBS was injected into 300 µL of each web as a blocking solution, and the mixture was placed at room temperature for 1 hour. After the blocking solution was removed, washing was performed three times in PBS containing 0.05% Tween20. Thereafter, the halibut suspension was collected into a tube using a dropper (for 0.2 mL, AZONE Co., Ltd.), and dropped into each web of three drops of 96 web microplates, and left at room temperature for 1 hour. After removing the spore solution, washing was performed three times in PBS containing 0.05% Tween20. Subsequently, the peroxysidase-labeled purified monoclonal antibody (Ks-3 C5) was diluted 2000-fold with 0.5% gelatin-containing PBS, and added to each life preserver for 1 hour at room temperature. After removal of the peroxysidase-labeled purified monoclonal antibody dilution, three washes were performed in PBS containing 0.05% Tween20. Thereafter, 0.5 mg / mL OPD substrate solution (Sigma-Aldrich) was added to each 100 μL web and left for 10 minutes at room temperature, and 2 N sulfuric acid was added to 50 μL each web to stop the reaction. Was measured. Table 12 shows the results of the performance evaluation of the test kit using the tool.

Table 12 shows the results of evaluating the performance of the test kit using the ELISA test tool.

K. septempunctata infected flounder Ref. 1.23 × 106 pcs / g 4.5 × 105 pcs / g, 7.5 × 104 / g Uninfected Flounder 0.5% gelatin 1.339 1.173 0.847 0.232 0.049

The figure is an absorbance value of 490 nm. In Table 12, the absorbance of the suspension (three types) produced from flounder meat infected with Kudoa septum punta shows a significantly higher value than the absorbance of the suspension produced from uninfected flounder meat. From this result, even in the main test kit by the ELISA test tool using the same monoclonal antibody as the immunochromat test specimen, the sample flounder meat can be collected quickly and easily from the flounder with a circumferential groove-shaped sampling tool. It was found that Kudoa septum punta can be specifically detected directly from flounder meat (including suspension).

The present invention can be directly detected from the fish meat Kudua mucin spores that are infected by fish such as flounder causing food poisoning, test tools, test kits and monoclonal antibodies used in these Can be provided.

1: sample pad 2: composite pad
3: membrane 4: absorbent pad
5: backing sheet 6: screw
7: dropper 8: tube
9: Immunochromat Specimen 10: Microplate for ELISA

Claims (25)

delete delete (1) Separating Kudoa septum punta from flounder meat, freezing and thawing spore suspension solution containing spores of the separated Kudoa septa punta to destroy the spore shell and the cells inside the spores. Adjusting the suspension solution in which the protein constituting the cell is eluted,
(2) Inoculating the protein obtained in step (1) into an animal of mammal or bird one or more times to produce antibody-producing cells in which the antibody-producing antibody is produced in the body of the animal, thereby producing the antibody-producing cells into the animal. Extraction process from
(3) a step of fusing the antibody-producing cells obtained in step (2) and myeloma cells of the same animal species as the animals to produce inactivated antibody-producing cell A;
(4) Inactivated antibody-producing cell A obtained in step (3) was selected from inactivated antibody-producing cell B in response to the protein derived from Kudoa septempunta, and Kudoa Tyrcites and Kudoa laterolabra. Selecting the inactivated antibody-producing cell C that does not react to the protein derived from the cis,
(5) A method for producing a monoclonal antibody comprising the step of isolating the inactivated antibody-producing cell B and the inactivated antibody-producing cell C selected in step (4) into a single cell, and then generating a monoclonal antibody in the cell. To;
The monoclonal antibody belongs to a subclass of IgG1, has a κ light chain, recognizes a protein in the range of 25,000 to 37,000 in Kudoa septum punta internal structure, and is deposited with accession number "NITE P-02181". A monoclonal antibody that specifically reacts to Kudoa septum punta, characterized in that it is a monoclonal antibody produced from a hybridoma or a hybridoma deposited with accession number "NITE P-02182".
delete delete delete delete delete delete delete Monoclonal antibody that specifically reacts to Kudoa septem puntata prepared by the method of claim 3
Specificity of Kudoa septum puntata in halibut meat samples was obtained using a monoclonal antibody produced from a hybridoma deposited with accession number "NITE P-02181" or a hybridoma deposited with accession number "NITE P-02182". Detection method of Kudoa septum punta
Hybridoma deposited with accession number "NITE P-02181" or accession number "NITE P-02182" delete delete delete delete delete delete delete delete delete delete delete delete

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