JP4084808B2 - Monoclonal antibodies specific for red larvae adhering larvae - Google Patents

Description

  The present invention relates to a monoclonal antibody or a fragment thereof specific for red larvae adhering larvae, a hybridoma producing the monoclonal antibody, a reagent for detecting red larvae adhering larvae, a detector, a detection kit, and a detection method.

Discrimination and identification of barnacle species have been conventionally performed by experts based on morphological techniques. However, barnacles are very similar in morphological stage, making it difficult for specialists to identify and identify species. Therefore, a method for discriminating species of barnacles at the attachment stage by irradiating the larvae at the stage of attachment of barnacles by analyzing the fluorescence distribution pattern specific to the species has been developed (see Patent Document 1). ).
JP 2004-12467 A

  The present invention provides a detection reagent, a detector, a detection kit, a detection method, a monoclonal antibody or a fragment thereof used for the detection, a monoclonal antibody or a fragment thereof, and the monoclonal antibody capable of specifically and easily detecting red larvae adhering larvae An object of the present invention is to provide a hybridoma that produces.

  In order to solve the above-mentioned problems, the present inventors fused a spleen cell of a mouse immunized by intraperitoneally injecting an adhesion stage larvae (cypris larvae) of a red barnacle and mouse myeloma cells, and obtained hybridoma Thus, the present inventors have completed the present invention by identifying a hybridoma that produces a monoclonal antibody that does not react with the larvae that adhere to the long-horned barnacles or the larvae that adhere to the barnacles but reacts with the larvae that adhere to the red-clawed acupoint.

  That is, the hybridoma according to the present invention is characterized in that it produces a monoclonal antibody that reacts specifically with the red larvae-adherent larvae and does not react with the larvae adhering larvae.

  In addition, the monoclonal antibody or fragment thereof according to the present invention is characterized in that it specifically reacts with red larvae-adherent larvae and does not react with vertical larvae-adherent larvae or larvae-adherent larvae.

  The reagent for detecting red larvae adhering larvae according to the present invention contains, as an active ingredient, a monoclonal antibody or fragment thereof that reacts specifically with the red larvae adhering larvae and does not react with the larvae adhering larvae or larvae adhering larvae. .

  In addition, the detector of the red barnacle adhering stage larvae according to the present invention is immobilized with a monoclonal antibody or fragment thereof that reacts specifically with the red barnacle adhering stage larvae and does not react with the vertical barnacle adhering stage larvae or the larvae adhering stage larvae. It is characterized by being.

  Further, the kit for detecting red larvae adhering larvae according to the present invention comprises a monoclonal antibody or a fragment thereof that specifically reacts with red larvae adhering larvae but does not react with the larvae adhering larvae or larvae adhering larvae.

  The method for detecting red larvae adhering larvae according to the present invention comprises adding to the sample a monoclonal antibody or fragment thereof that specifically reacts with the red barnacle adhering larvae and does not react with the vertical barnacle adhering larvae or the larvae adhering larvae, A step of acting on the red larvae adhering larvae in the sample.

  Here, as a monoclonal antibody that reacts specifically with the red larvae-adherent larvae but does not react with the larvae adhering to the larvae or the larvae-adherent larvae, for example, in the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Mention may be made of monoclonal antibodies produced from hybridoma cell lines deposited under the accession numbers FERM P-20278, FERM P-20279, FERM P-20281 and the like.

  According to the present invention, a detection reagent, a detector, a detection kit, and a detection method capable of specifically and easily detecting red larvae adhesion stage larvae, and a monoclonal antibody or a fragment thereof used therein, and Hybridomas that produce monoclonal antibodies can be provided.

  An embodiment for carrying out the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

== Method for producing monoclonal antibody or fragment thereof ==
The monoclonal antibody according to the present invention is an adherent stage larva of barnacles other than red barnacles (particularly, vertical barnacles and swordflies), or a lysate (hereinafter referred to as “coarse”) treated with an ultrasonic device, a homogenizer, etc., respectively. It can be obtained from a hybridoma that does not react with the extract (hereinafter referred to as “extract”) but produces a monoclonal antibody that reacts with the red larvae adhering larva or its crude extract (hereinafter referred to as “hybridoma according to the present invention”). . Production of a monoclonal antibody using the hybridoma according to the present invention can be performed by a known method. Specifically, the above-described monoclonal antibody can be obtained by culturing the hybridoma in an appropriate culture medium and collecting the culture supernatant. However, the hybridoma can be transformed into a mammal (eg, mouse, rat, hamster, Rabbits, pigs, cows, horses, dogs, monkeys, etc.) may be administered into the abdominal cavity and the ascites collected to obtain the above-described monoclonal antibody. The purification of the monoclonal antibody is performed by using the culture supernatant of the hybridoma described above or a lysate obtained by treating the cultured hybridoma with an ultrasonic device, a homogenizer or the like, or ascites collected from a mammal administered intraperitoneally with the hybridoma described above, It can be carried out by a conventional method, for example, ammonium sulfate salting out, chromatography (for example, ion exchange chromatography, affinity chromatography, etc.), gel filtration, or a combination of these methods.

  As the hybridoma described above, for example, a cell line deposited under the accession numbers FERM P-20278, FERM P-20279, FERM P-20281, etc. at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology is used. it can.

The fragment according to the present invention is not particularly limited as long as it is an antigen-binding site comprising a part of the above-described monoclonal antibody and including a variable region. For example, Fab fragment, F (ab ′) ₂ fragment, etc. Can be used. These fragments can be obtained by a known method, for example, a method using a proteolytic enzyme. The proteolytic enzyme may be any as long as it can obtain Fab fragments and F (ab ′) ₂ fragments. For example, a protease such as pepsin or ficin should be used. Can do.

=== Production of Hybridoma ===
The hybridoma according to the present invention can be produced, for example, by the following method. First, using a red larvae adhering larva or a crude extract thereof as an antigen, an appropriate amount of antigen (adjuband may be used) is used as a mammal (eg, mouse, rat, hamster, rabbit, pig, cow, Horses, dogs, monkeys, etc.) are intravenously, subcutaneously, intraperitoneally (1 to several times) and immunized. Thereafter, antibody-producing cells are collected from the immunized animal and fused with myeloma cells (myeloma cells) to produce hybridomas. The resulting hybridoma is not reactive with the larvae of the barnacles other than the red barnacles (especially the vertical barnacles and squirrel barnacles) or their crude extracts, but does not react with the larvae of the red barnacles or their crude extracts. By selecting a hybridoma that produces a monoclonal antibody exhibiting sex, a hybridoma that produces a monoclonal antibody specific for the red larvae-adherent larvae according to the present invention can be obtained.

  Cell fusion between the antibody-producing cells and myeloma cells is performed by, for example, culturing the antibody-producing cells and myeloma cells together in a culture medium containing a fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ). Although it can be performed, it can also be performed using electrical stimulation such as electroporation. In order to increase the efficiency of cell fusion, adjuvants such as dimethyl sulfoxide and lecithin may be included in the culture medium.

  Examples of the antibody-producing cells that can be used include spleen cells, lymph node cells, thymocytes, and peripheral blood cells. These cells can be obtained by removing spleen, lymph nodes, thymus, or peripheral blood from an animal, and crushing, filtering, centrifuging, etc. the removed tissue. In addition, as the myeloma cells, cell lines derived from various animals may be used. However, cell lines that do not themselves show resistance to drugs but that are resistant to drugs when fused may be used. preferable. Thereby, after cell fusion, selection of a hybridoma obtained by cell fusion is facilitated by culturing in a culture medium (for example, HAT medium) to which a drug is added. The antibody-producing cells and myeloma cells to be fused are preferably cells of the same species, but cells derived from animals of different species may be used.

  Selection of the above-mentioned hybridoma can be performed by routine screening or cloning. For the screening of hybridomas, for example, enzyme immunoassay (EIA: Enzyme ImmunoAssay, ELISA: Enzyme-Linked Immunosorbent Assays), radioimmunoassay (RIA: Radio Immuno Assay), Western blotting, etc. can be used. For cloning, for example, limiting dilution method, soft agar method, fibrin gel method, fluorescence excitation cell sorter method and the like can be used. In selecting a hybridoma according to the present invention, it is possible to select a hybridoma that produces a monoclonal antibody highly specific for the red larvae adhering larva or its crude extract by repeating the above screening and cloning.

  In the above-described hybridoma screening, it is sufficient to examine at least the adhesion of the red barnacles, vertical barnacles, and larvae to the attached larvae or their crude extracts, but further, barnacles other than red barnacles, vertical barnacles, Attachment larvae, bivalve attachment larvae (eg clams, mussels etc.), other larvae (eg Artemia nauplius larvae) or plankton (eg crustacean plankton other than barnacles) or their More preferably, the reactivity with the crude extract is examined. Thereby, a hybridoma producing a monoclonal antibody with higher specificity to the red larvae adhering larvae or a crude extract thereof can be obtained, and various larvae or plankton can be obtained by using the monoclonal antibody produced from this hybridoma. Larvae-adherent larvae are specifically collected from samples containing seawater (eg, seawater) and detected, that is, the presence or absence of red-female-adherent-adherent larvae, identification of red-female-adherent-adherent larvae, The amount can be measured.

== Use of monoclonal antibodies or fragments thereof ==
The monoclonal antibody according to the present invention does not show reactivity to the adhesion stage larvae of barnacles other than the red barnacles (particularly, the vertical barnacles and swordflies) or their crude extracts. Since the antibody specifically shows reactivity, the monoclonal antibody according to the present invention or a fragment thereof is considered to be useful for specific detection of the red larvae attaching stage larvae.

  Further, the monoclonal antibody or fragment according to the present invention is considered to be useful for recovering red larvae adhering larvae. The recovery of red larvae-adherent larvae can be performed by utilizing the affinity with a monoclonal antibody or a fragment thereof. For example, it can be carried out by allowing the monoclonal antibody or fragment thereof according to the present invention to which a magnetic substance has been bound to act on the red larvae adhering larvae, and then recovering the monoclonal antibody or fragment thereof according to the present invention using a magnet. . In addition, as said magnetic body, iron, iron oxide, etc. can be used, for example. In addition, methods such as FACS (Fluorescence Activated Cell sort) and panning may be used for collecting red larvae adhering larvae.

  The detection of red larvae adhering larvae according to the present invention is carried out by adding the monoclonal antibody or fragment thereof according to the present invention to a sample (for example, seawater or a lysate obtained by treating it with an ultrasonic device, a homogenizer, etc.) It can be carried out by reacting with red larvae adhering larvae or lysates thereof, and analyzing the red barnacle adhering larvae or lysates thereof bound with the monoclonal antibodies or fragments thereof by immunological techniques.

  Therefore, it can be said that the reagent, kit, or instrument containing the monoclonal antibody or fragment thereof according to the present invention is useful as a reagent, detection kit, and detector for detecting red larvae-adherent larvae.

  The reagent for detecting red larvae adhering larvae according to the present invention may be any reagent as long as it contains the monoclonal antibody according to the present invention or a fragment thereof, for example, a buffer solution (for example, phosphate, carbonate, etc.). Salt, salt solution such as hydrochloride), preservatives (eg sodium azide, etc.), substances to suppress non-specific reactions (eg, block ace, gelatin, skim milk, etc.), by immunological techniques Commonly included in reagents for detecting antigens other than antibodies or fragments thereof, such as substances (eg, labeling substances) and stabilizers (eg, BSA, goat serum) necessary for detecting red larvae adhering larvae 1 or 2 or more of these substances may be further included.

  According to the present invention, the detection of the red larvae adhering larvae comprises a filter in which the monoclonal antibody or fragment thereof according to the present invention comprises a medium (for example, paper such as filter paper, glass, fiber, modified cellulose such as nitrocellulose, nylon, plastic, etc. , Membranes, plates, dishes, etc.), for example, buffers (eg, solutions of salts such as phosphates, carbonates, hydrochlorides, etc.), preservatives (For example, sodium azide), substances for suppressing non-specific reactions (for example, block ace, gelatin, skim milk, etc.), substances necessary for detecting red larvae adhering larvae by immunological techniques ( For example, labeling substances, chromogenic substrates, secondary antibodies, chromogenic enhancers, etc.), stabilizers (eg BSA, goat serum, etc.), etc. , Typical materials for inclusion in the detector of the antigen other than an antibody or fragment thereof is 1 or 2 or more may be further included.

  As an example of the detector of the red crucible adhering stage larva according to the present invention, there are a part where the sample is dropped or a first part where the sample is immersed, and a second part where the monoclonal antibody or fragment thereof according to the present invention is immobilized. A second part having a second part immobilized thereon, such as an anti-immunoglobulin antibody that specifically reacts with the immunoglobulin of the host animal species from which the monoclonal antibody according to the present invention was produced, Is provided between the first part and the third part, and the first part includes metal colloid particles (eg, gold colloid particles), heavy metals (eg, gold, platinum, etc.), fluorescent materials (eg, , FITC (fluorescein isothiocyanate), rhodamine, phalloidin, etc.), a monoclonal antibody according to the present invention or a fragment thereof labeled with a labeling substance such as colored latex particles And chromatographic media containing Examples of the chromatographic medium include filter paper made of inorganic fibers such as glass and silica, and modified cellulose such as nitrocellulose. By using such a chromatographic medium, it is possible to detect whether or not the red barnacle adhering stage larvae are present in the sample. The principle is that when a sample containing red larvae adhering larvae or a lysate thereof is dropped or immersed in the first part of the chromatographic medium, the red barnacle adhering larvae or lysates thereof are added to the first part of the chromatographic medium. The complex reacts with the labeled monoclonal antibody or fragment thereof contained in the part to form a complex, and the complex moves to the second part by utilizing the spread of the liquid in the medium. It is supplemented by the above-mentioned monoclonal antibody or fragment thereof immobilized in the second part, and the labeling substance at that position enables the detection of the red larvae adhering stage larvae. On the other hand, in a sample that does not contain the red larvae adhering stage larvae or lysates thereof, the labeled monoclonal antibody or fragment thereof contained in the first part that did not react with the antigen contained in the sample was present. It moves to the 3rd part, it supplements with the above-mentioned secondary antibody fixed in the 3rd part, and it becomes clear by the labeling | labeling of a label | marker substance that the red-female acupuncture point larvae did not exist. In this way, it is possible to detect whether or not the red barnacle attachment stage larvae are present in the sample, depending on whether the second part is labeled or only the third part is labeled.

  On the other hand, the detection kit for red larvae adhering larvae according to the present invention may be any one as long as it contains the monoclonal antibody according to the present invention or a fragment thereof, and in addition to the monoclonal antibody according to the present invention or a fragment thereof, for example, , Buffers (eg, solutions of salts such as phosphates, carbonates, hydrochlorides, etc.), preservatives (eg, sodium azide, etc.), substances for suppressing non-specific reactions (eg, block ace, Gelatin, skim milk, etc.), substances (eg, labeling substances, chromogenic substrates, secondary antibodies, chromogenic enhancers, etc.), stabilizers (eg, BSA, goats) necessary to detect red larvae adhering larvae by immunological techniques A general substance to be included in an antigen detection kit such as serum) or the like, or the monoclonal antibody according to the present invention as described above or a fragment thereof. Detector bets is immobilized on the medium, or may include a combination of two or more of these.

Examples of the labeling substance include fluorescent substances (eg, FITC, rhodamine, phalloidin, etc.), metal colloid particles (eg, gold colloid particles), heavy metals (eg, gold, platinum, etc.), chromoproteins (eg, phycoerythrin). (PE), phycocyanin (PC), etc., radioisotopes (eg, ³ H, ³² P, ³⁵ S, ¹²⁵ I, ¹³¹ I, etc.), enzymes (eg, peroxidase, alkaline phosphatase, etc.), biotin, streptavidin, etc. However, it is not limited thereto, and a known labeling substance may be used. Further, the chromogenic substrate is not particularly limited as long as it is a chromogenic substrate for the above-mentioned enzyme. For example, diaminobenzidine (DAB), o-phenylenediamine (o-Phenylenediamine), hydrogen peroxide solution, BCIP / Nitro -TB (5-Bromo-4-chloro-3-indolylphosphate / Nitrotetrazolium blue), pNPP (para-nitorophenylphosphate), etc. can be used. The color development enhancer may be any one that can enhance the color development of the above-mentioned substrate. For example, sulfuric acid or the like can be used, and examples of the secondary antibody include the present invention. Anti-immunoglobulin antibodies, anti-Ig (H + L), anti-Ig (Fc), etc. that specifically react with the immunoglobulin of the host animal species in which the monoclonal antibody is produced can be used.

  The immunological methods described above include EIA (Enzyme Immunoassay), fluorescent immunoassay, ELISA (Enzyme-Linked Immunosorbent Assay), RIA (Radioimmuno assay), Western blotting, latex agglutination, immunochromatography, sandwich method A known method such as the above can be used.

  As described above, by using the detection method, detection reagent, detector, or detection kit of red barnacle attachment stage larvae according to the present invention, confirmation of the presence or absence of red barnacle attachment stage larvae, identification of red barnacle attachment stage larvae, It is possible to separate and measure the amount of red larvae adhering larvae.

  Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention, and do not limit the scope of the present invention.

[Example 1]
In order to obtain the monoclonal antibody of the present invention, a red barnacle (Megabalanus rosa (Pilsbry)) cypris larva obtained by indoor rearing was used as an antigen.

  7 weeks old BALB / c Jc1 mice were injected intraperitoneally with 5 antigens (1st-4th injection: 50-200 larvae / 150-200 μl PBS, 5th injection (final immunization): 300 larvae / 200 μl PBS; second dose on day 22 from first dose, third dose on day 24 from second dose, fourth dose on day 60 from third dose, 4 The fifth administration was performed on the 59th day after the second administration.) The mice were immunized.

Three days after the final immunization, the spleen is removed from the mouse and lined with a sterile stainless mesh in RPMI1640 (GIBCO) medium containing 10% FBS and 1% antibiotic (Antibiotic-Antimycotic; GIBCO). The cells were dispersed and suspended to obtain floating cells (spleen cells). The spleen cells were washed and centrifuged with RPMI1640 medium (FBS ⁺ medium) containing 10% FBS, and then centrifuged and washed three times with RPMI1640 medium (FBS ^- medium) to collect spleen cells (about 10 ⁸ cells). .

Next, myeloma cells (5 × 10 ⁷ cells) and spleen cells (about 10 ⁸ cells) were mixed, and then centrifuged to remove the supernatant to prepare a cell pellet (precipitate). A solution prepared by mixing 1 g of PEG4000 (polyethylene glycol; Article No. 9727 of MERCK) and 1 ml of FBS ^- medium was slowly added thereto and stirred, and further stirred gently for 1 minute. Thereafter, 2 ml of FBS ^- medium was slowly added and stirred for 1 minute to perform cell fusion.

After cell fusion, 8 ml of FBS ^- medium (37 ° C.) was slowly added over 1 minute, followed by centrifugation (1000 rpm × 5 minutes), and the supernatant was removed by aspiration. Add 10 ml of FBS ⁺ medium and suspend it, then add FBS ⁺ HAT medium (10% FBS, 1% antibiotics, and HAT supplement medium (SIGMA; use 1 vial of powder diluted 50 times) x 2 ml each was seeded on 5 laboratory petri dishes containing 0.5 RPMI1640 medium (GIBCO) and cultured for 5-7 days at 37 ° C. and 5% CO ₂ .

  After culturing, 700 μl of 50 mM Tris-HCl (PH7.5) was added to 700 frozen Akafuji algae larvae, homogenized and ultrasonically treated (several seconds × 5 times), and centrifuged at 5000 rpm × 20 minutes. Using a crude extract of red aphid Cyprus larvae (protein concentration; 5.74 mg / ml) as an antigen, place the adsorbed nitrocellulose membrane on each petri dish (culture medium) (for 1 day), and use an HRP-labeled secondary antibody. And reacted. After the reaction, chemiluminescence was generated and baked on an X-ray film, and the antibody production of cells in each cell was confirmed.

  In this confirmation, a hybridoma of a single colony was picked up from the cell lines that showed a positive reaction against the crude extract of red aphid Cyprus larvae, and a selective medium (HT medium (100 μM hypoxanthine added with feeder cells derived from mouse thymocytes) was selected. And transplanted into a 96-well plate containing RPMI1640 medium containing 16 μM thymidine), and hybridomas were cultured. After culturing, the culture supernatant of each well was collected and antibody production was confirmed by ELISA. In addition, about the process which is not described in particular, it processed at room temperature (a reaction was included).

(1) 50 μl of the above-described antigen (Akafuji aquarius larva crude extract; 100 μg / ml) was added to each well of a 96-well Iwaki ELISA plate, and the antigen was adsorbed to the bottom of the well overnight at 4 ° C.
(2) Each well adsorbed in (1) was washed with 200 μl of TBS (0.5 M NaCl and 20 mM Tris-HCl (pH 7.5)).
(3) Each well was blocked with 100 μl of TBS containing 1% BSA for 1 hour.
(4) Each well was washed 3 times with 200 μl of TTBS (TBS containing 0.05% Tween20).
(5) 50 μl of culture supernatant (primary antibody) obtained by culturing the hybridoma was added to each well and allowed to react for 1 hour.
(6) Each well was washed 4 times with 200 μl of TTBS.
(7) 50 μl of a secondary antibody (alkaline phosphatase-labeled anti-mouse IgG (H + L) goat antibody; ZYMED laboratory) solution (1000-fold diluted with TBS) was added to each well and allowed to react for 1 hour.
(8) Each well was washed 5 times with 200 μl of TTBS.
(9) 100 μl of substrate (alkaline phosphatase substrate kit; BIO-RAD) solution was added to each well, and color was developed by shaking for 1 hour.
(10) The absorbance of the solution in each well was measured using a microplate reader (BIO-RAD Model 550; 405 nm).

  For the hybridoma whose high antibody production ability was confirmed by the ELISA method described above, the antibody titer of the culture solution diluted in stages was further measured by ELISA method, and the operation of picking up the hybridoma having high antibody production ability was repeated three times. (The second and third cultures use RPMI1640 with HT-free, 10% FBS, 1% antibiotics, and 10% conditioned medium from J774A.1 cells, HYBRIMAX, SIGMA) ), Hybridoma clones (KA4-5b6P-1 (3) a, KA4-5b (5) a, KF1-6a4 (1) 6P-) that show higher antibody-producing ability against the crude extract of red aphid Cyprus larvae 3 and 10E3-1A11-2 (3) b) were obtained.

[Example 2]
Next, the four hybridoma clones (KA4-5b6P-1 (3) a, KA4-5b (5) a, KF1-6a4 (1) 6P-3, and 10E3-1A11-2) obtained in Example 1 were used. (3) In order to investigate whether the monoclonal antibody contained in each culture supernatant in (3) specifically reacts with red larvae larvae, red larvae, Balanus amphitrite Darwin cypris larvae, (Chthamalus challengeri Hoek) Crude extract of protein (protein) Concentration: 100 μg / ml) was confirmed in the same manner as in the ELISA method described in Example 1. Each crude extract is prepared by adding each larvae or plankton to 50 mM Tris-HCl (PH7.5), homogenizing and sonicating (several seconds x 5 times), and centrifuging at 5000 rpm for 20 minutes. did. The results of ELISA (OD ₄₀₅ value) are shown in Table 1.

[Table 1]

  As shown in Table 1, the supernatants of hybridoma clones KA4-5b6P-1 (3) a, KA4-5b (5) a, and KF1-6a4 (1) 6P-3 were added to the crude extract of the red lobster cyprus larvae. It showed reactivity to the crude extract of cypris larvae of barnacles other than red barnacles such as vertebrate and barnacles, bivalves, and crude extract of crustacean plankton other than barnacles. In addition, the supernatant of hybridoma clone 10E3-1A11-2 (1) b reacts with the crude extract of cypris larvae of all barnacles, and almost with larvae other than barnacles and crude extract of plankton. It became clear that there was no reactivity.

[Example 3]
Next, in order to examine the specificity of the culture supernatant of the hybridoma clones obtained in Example 1, the culture supernatants of the hybridoma clones KF1-6a4 (1) 6P-3 and KA4-5b6P-1 (3) a ( Using the undiluted solution or the diluted undiluted solution 4, 8, 16, 32, or 64 times), the reactivity of each larva and plankton with the crude extract was confirmed in the same manner as the ELISA method described in Example 1. did. Table 2 shows the ELISA results (OD ₄₀₅ value) when KF1-6a4 (1) 6P-3 culture supernatant is used, and Table 3 shows KA4-5b6P-1 (3) a culture supernatant. Table 4 shows the ELISA results (OD ₄₀₅ value; mean value and standard error), and Table 4 shows the ELISA results (OD ₄₀₅ value) when KF1-6a4 (1) 6P-3 culture supernatant was used. .

[Table 2]
[Table 3]
[Table 4]

  As shown in Tables 2 to 4, the reaction specificity of the culture supernatants (monoclonal antibodies) of hybridoma clones KF1-6a4 (1) 6P-3 and KA4-5b6P-1 (3) a to the red lobster cyprus larvae is It was found to be at least 32 times higher than that for Cypris larvae and Iwafutsubo Cyprus larvae.

[Example 4]
Next, in order to examine whether or not the monoclonal antibody produced by the hybridoma obtained in Example 1 specifically reacts with the red larvae larvae themselves, the monoclonal antibody of the hybridoma clone KA4-5b6P-1 (3) a was used. In the inverted microscope, perform immunostaining for larvae or plankton, such as red-breasted cyprips larvae, red-breasted nauplius larvae (5th stage), vertebrate larvae puppy larvae, mussel pedy verijer larvae, outdoor plankton (mainly copepoda), etc. Each larva was observed. In addition, in the following, processes (including reactions) were performed at room temperature for processes not specifically described.

(1) Akafuji azalea Cyprus larvae, Tateji Mafuji Acupuncture larvae, Akafuji Acupuncture nauplii larvae (5th stage), mussel pediberger larvae, outdoor plankton (mainly copepoda), etc. were each placed in a 96-well plate and fixed with 5% formalin. .
(2) Each well was washed with 200 μl of PBS, and further washed with 200 μl of PBST (PBS containing 0.05% Tween20).
(3) 200 μl of PBST containing 1% BSA was injected into each well and blocked at 4 ° C. for 8 hours.
(4) 200 μl of KA4-5b6P-1 (3) a monoclonal antibody (primary antibody; solution diluted 8 times with PBST containing 1% BSA) was added to each well and reacted at 4 ° C. overnight.
(5) Each well was washed 4 times with 200 μl of PBST.
(6) A secondary antibody (alkaline phosphatase-labeled anti-mouse IgG (H + L) goat antibody; solution diluted 400 times with PBST containing 1% BSA) 200 μl was added to each well and reacted at 4 ° C. for 8 hours. .
(7) Each well was washed 4 times with 200 μl of PBST.
(8) 200 μl of substrate (alkaline phosphatase substrate kit; BIO-RAD) solution was added to each well for color development.

  As a result, it was clarified that the monoclonal antibody of the hybridoma clone KA4-5b6P-1 (3) a specifically reacts with the thoracic limb basal tissue of the Akafujitsubo Cyprus larvae but does not react with the larvae of the T. elegans.

  In addition, it did not react to blue mussel pediberger larvae and field plankton (mainly Copepoda), but it was revealed that the basal horn larvae (5th stage) showed weak reactivity around the anterior horn base. Based on this, it was considered that the monoclonal antibody produced by the hybridoma clone KA4-5b6P-1 (3) a may be somewhat reactive to the red pheasant nauplius larvae in addition to the red vaccinia cyprips larvae.

[Example 5]
Next, in order to confirm which monoclonal antibody produced from the hybridoma clone specifically reacts with the genus Akafuji apocytic larvae, and whether the antigen is expressed in the adult Akafuji acupuncture larvae, In addition, each crude extract of the adult and red barnacles was separated by SDS-PAGE and stained with CBB, and Western blotting was performed using a monoclonal antibody produced from the hybridoma clone KF1-6a4 (1) 6P-3. In addition, in order to confirm that the antigens expressed in larvae or adults other than red swordfish larvae and adult red scabbard larvae are not reactive, the crude extracts of swordfish larvae and adult swordflies are also subjected to CBB staining and Western blotting was performed. The result is shown in FIG. The left side in FIG. 1 shows the result after CBB staining, and the right side shows the result by Western blotting. In addition, “M” in FIG. 1 is a molecular weight marker (the left arrows in FIG. 1 indicate 116.2 kDa, 66.3 kDa, 42.4 kDa, and 30 kDa from the top, respectively), and “A” is a rough adult barnacle The extract (protein amount: 5 μg), “B” is the crude extract of adult red barnacles (protein amount: 5 μg), “C” is the crude extract of vertebrate aphid Cyprus larvae (protein amount: 5 μg), “D” is Each of the red larvae crude extract (protein amount: 5 μg) is shown.

  As shown in FIG. 1, the monoclonal antibody produced from the hybridoma clone KF1-6a4 (1) 6P-3 does not react with any antigens of adults of adults of red and white wings, and larvae of red and white wings, and red larvae of red and white wings. It was revealed that it reacts with an antigen of approximately 30 kDa (arrow on the right side in FIG. 1). From this, it was considered that the monoclonal antibody produced from the hybridoma cell line according to the present invention might react with an antigen of about 30 kDa in red larvae.

  From the above results, KA4-5b6P-1 (3) a, KA4-5b (5) a, and KF1-6a4 (1) 6P-3 are considered to have high reaction specificity against red larva Cyprus larvae. It was clarified that red larvae can be specifically detected and recovered by using monoclonal antibodies produced by these hybridoma cell lines.

  Therefore, these hybridoma cell lines were deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (KA4-5b6P-1 (3) a: Deposit number FERM P-20281, KA4-5b (5) a: Deposited) Number FERM P-20278, KF1-6a4 (1) 6P-3: accession number FERM P-20279).

In one embodiment of the present invention, the result of separating each adult or larvae crude extract and staining with CBB and the antigen to which the monoclonal antibody produced from the hybridoma clone KF1-6a4 (1) 6P-3 reacts are obtained by Western It is a figure which shows the result investigated by the blot method.

Claims (12)

  A hybridoma that produces a monoclonal antibody that reacts specifically with larvae that adhere to the red-clawed acupuncture point but does not react with larvae that adhere to the long-tailed barnacle or the larvae.   The hybridoma according to claim 1, wherein the hybridoma is deposited under any deposit number selected from the group consisting of FERM P-20278, FERM P-20279, and FERM P-20281.   A monoclonal antibody or a fragment thereof that specifically reacts with red larvae-adherent larvae but does not react with vertical larvae-adherent larvae or larvae-adherent larvae.   The monoclonal antibody according to claim 3, which is produced from a hybridoma deposited under any accession number selected from the group consisting of FERM P-20278, FERM P-20279, and FERM P-20281 That fragment. A reagent for detecting red larvae adhering larvae,
A detection reagent comprising, as an active ingredient, a monoclonal antibody or a fragment thereof that reacts specifically with red larvae-adherent larvae but does not react with vertical larvae-adherent larvae or larvae-adherent larvae.   The monoclonal antibody is produced from a hybridoma deposited under any accession number selected from the group consisting of FERM P-20278, FERM P-20279, and FERM P-20281. The reagent for detection as described. It is a detector of the red barnacle adhesion stage larvae,
A detector comprising a monoclonal antibody or a fragment thereof, which specifically reacts with red larvae-adherent larvae but does not react with vertical larvae-adherent larvae or staghorn acupuncture-adherent larvae.   The monoclonal antibody is produced from a hybridoma deposited under any accession number selected from the group consisting of FERM P-20278, FERM P-20279, and FERM P-20281. The detector described. A detection kit for red larva affixed larvae,
A detection kit comprising a monoclonal antibody or a fragment thereof that specifically reacts with red larvae-adherent larvae but does not react with vertical larvae-adherent larvae or staghorn acupuncture-adherent larvae. The monoclonal antibody is produced from a hybridoma deposited under any accession number selected from the group consisting of FERM P-20278, FERM P-20279, and FERM P-20281. The detection kit as described. A method for detecting red larvae attached larvae,
A detection method comprising adding, to a sample, a monoclonal antibody or a fragment thereof that reacts specifically with a red larvae-adherent larvae but does not react with a vertical larvae-adherent larvae or staghorn acupuncture-adherent larvae.   The monoclonal antibody is produced from a hybridoma deposited under any accession number selected from the group consisting of FERM P-20278, FERM P-20279, and FERM P-20281. The detection method described.