JP4139818B2 - Monoclonal antibody specific for mussel larvae - Google Patents

Description

  The present invention relates to a hybridoma that produces a monoclonal antibody specific for mussel-attached larvae, or a method for producing the same, a monoclonal antibody or fragment thereof specific to mussel-attached larvae, and a reagent for detecting mussel-attached larvae, or a detection method , A detection kit, and a detector.

  Discrimination and identification of the species of bivalve larvae were performed by experts based on morphological techniques. However, the larvae of various bivalves are very similar in form, and it is difficult for specialists to identify and identify species. Therefore, there is a need for the development of a technique that can easily discriminate and identify the bivalve adhesion stage larvae.

Conventionally, as a method for identifying clam floating larvae, monoclonal antibodies specific to clam floating larvae have been developed (see Patent Document 1). However, a monoclonal antibody specific to the mussel adhesion stage larva has not been developed, and a method for quickly and simply identifying the mussel adhesion stage larva has not been developed.
JP-A-11-196866

  The present invention has been made in view of the above problems, a monoclonal antibody specific to mussel adhesion stage larvae and fragments thereof, a hybridoma producing a monoclonal antibody specific to mussel adhesion stage larvae, a method for producing the same, and It is an object of the present invention to provide a reagent, a detection method, a detection kit, and a detector for detecting mussel adhesion stage larvae.

  In order to solve the above-mentioned problems, the present inventors fused spleen cells of mice immunized by intraperitoneal injection of mussel adhesion larvae (Pediberger larvae) and mouse myeloma cells. When monoclonal antibodies were prepared from hybridomas, it was found that there were many antibodies that cross-reacted not only with mussel-adhered larvae but also with oyster-adhered larvae. Therefore, by identifying a hybridoma that produces a monoclonal antibody that does not react to oyster-adherent larvae but reacts to mussel-adherent larvae, it is possible to select a hybridoma that produces a monoclonal antibody that specifically reacts to mussel-adherent larvae. The present inventors have found that this can be done and have completed the present invention.

  That is, the method for producing a hybridoma according to the present invention is a method for producing a hybridoma that produces a monoclonal antibody that specifically reacts with mussel-adhered larvae, which is derived from mammals other than humans immunized with mussel-adherent larvae. The method includes selecting a hybridoma that produces a monoclonal antibody that reacts with the mussel adhesion stage larvae but does not react with the oyster adhesion stage larvae, from a hybridoma obtained by fusing spleen cells and myeloma cells. The monoclonal antibody produced by the hybridoma is preferably one that does not react with crustacean plankton or barnacle adhering larvae.

  The hybridoma according to the present invention is characterized by producing a monoclonal antibody that reacts specifically with mussel-adhered larvae but does not react with oyster-adhered larvae, clams-adhered larvae, and mussel-adherent larvae.

  The monoclonal antibody or fragment thereof according to the present invention is characterized in that it specifically reacts with mussel-adhered larvae and does not react with oyster-adhered larvae, clams-adhered larvae, and snail-adherent larvae.

  The reagent for detecting mussel-adhered larvae according to the present invention specifically reacts with mussel-adhered larvae, and does not react with oyster-adhered larvae, clams-adhered larvae, or mussel-adherent larvae, or the like The fragment is contained as an active ingredient.

  Furthermore, the method for detecting mussel adhesion stage larvae according to the present invention is a monoclonal antibody that reacts specifically with the mussel adhesion stage larvae and does not react with oyster adhesion stage larvae, clam attachment stage larvae, and mussel adhesion stage larvae. Or adding a fragment thereof.

  The kit for detecting mussel-attached larvae according to the present invention comprises a monoclonal antibody or fragment thereof that specifically reacts with mussel-attached larvae but does not react with oyster-attached larvae, clams-attached larvae, and mussel-attached larvae .

  Further, the detector of the mussel adhesion stage larvae according to the present invention specifically reacts with the mussel adhesion stage larvae, and does not react with the oyster adhesion stage larvae, clams attachment stage larvae, and the snail adhesion stage larvae, or a fragment thereof Is fixed.

  Here, as the monoclonal antibody that reacts specifically with the mussel adhesion stage larvae and does not react with the oyster adhesion stage larvae, clam attachment stage larvae, and the snail adhesion stage larvae, for example, the National Institute of Advanced Industrial Science and Technology Patent Mention may be made, for example, of monoclonal antibodies produced from the hybridoma cell line deposited at the biological deposit center under the deposit number FERM AP-20220. The monoclonal antibody preferably further does not react with crustacean plankton or barnacle adhering larvae. Examples of such monoclonal antibodies include hybridoma cell lines deposited under the accession numbers FERM P-20216, FERM P-20218, FERM P-20219, etc. at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. be able to.

  According to the present invention, a monoclonal antibody specific to mussel adhesion stage larvae and fragments thereof, a hybridoma producing a monoclonal antibody specific to mussel adhesion stage larvae, a method for producing the same, and a reagent for detecting mussel adhesion stage larvae, Detection methods, detection kits, and detectors 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 attached stage larvae of bivalves (especially oysters, clams, and snails) other than mussels, or a lysate obtained by treating these attached stage larvae with an ultrasonic device, a homogenizer, etc. It is obtained from a hybridoma that produces a monoclonal antibody that does not react with the crude mussel adhesion stage or reacts with the crude extract (hereinafter referred to as “hybridoma according to the present invention”). it can. Production of the monoclonal antibody using the hybridoma according to the present invention may be carried out by culturing the hybridoma in an appropriate culture medium and collecting the culture supernatant. Rats, hamsters, rabbits, pigs, cows, horses, dogs, monkeys, etc.), and ascites may be collected. 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.

  Examples of the hybridoma include a cell line deposited under the accession number FERM P-20216, FERM P-20218, FERM P-20219, or FERM P-20220 at the National Institute of Advanced Industrial Science and Technology (AIST). Can be used.

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, for example, by partially digesting the above-described monoclonal antibody with 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, mussel adhesion stage larvae or a crude extract thereof is used as an antigen, and an appropriate amount of antigen (adjuvant may be used) is used as a mammal (eg, mouse, rat, hamster, rabbit, pig, cow, horse). Intravenous, subcutaneous, intraperitoneal (1 to several times) administration (1 to several times) in a dog, monkey, etc. Thereafter, antibody-producing cells are collected from the immunized animal and fused with myeloma cells (myeloma cells) to produce hybridomas. From the obtained hybridomas, a hybridoma that does not show reactivity to oyster-adherent larvae or their crude extract but produces a monoclonal antibody that shows reactivity to mussel-adherent larvae or its crude extract should be selected. Thus, a hybridoma that produces a monoclonal antibody specific to the mussel adhesion stage 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 the selection of the hybridoma according to the present invention, it is possible to select a hybridoma that produces a monoclonal antibody highly specific for the mussel adhesion stage larvae or a crude extract thereof by repeating the above screening and cloning.

  In the above-mentioned hybridoma screening, it is sufficient to examine at least the adhesion stage larvae of mussels and oysters or their reactivity with the crude extract, and further, various adhesion stage larvae of clams such as clams and snails or those It is preferable to examine the reactivity with the crude extract of other species. In addition to the bivalve adhering larvae, other larvae such as barnacle adhering larvae (Cypris larvae), crustacean plankton, or their crude extract and It is more preferable to examine the reactivity. As a result, hybridomas producing monoclonal antibodies with higher specificity for the mussel-adhered larvae or crude extracts thereof can be obtained, and various larvae or plankton can be obtained by using the monoclonal antibodies produced from the hybridomas. It is possible to specifically detect blue mussel adherent stage larvae in samples containing seawater (for example, seawater), and to identify blue mussel adherent stage larvae in samples containing various larvae or plankton (eg, seawater). Detection, that is, confirmation of the presence of mussel-adhered larvae, identification of mussel-adherent larvae, measurement of the amount of mussel-adherent larvae, and the like.

== Use of monoclonal antibodies or fragments thereof ==
The monoclonal antibody according to the present invention does not show reactivity to the attachment larvae of bivalves (especially oysters, clams, and snails) other than mussels or their crude extracts, but the mussel attachment larvae or their crude extractions The monoclonal antibody or fragment thereof according to the present invention is considered to be useful for specific detection of mussel adhesion stage larvae because of its specific reactivity with liquid.

  In addition, the monoclonal antibody or fragment according to the present invention is considered to be useful for recovering mussel adhesion stage larvae. The mussel adhesion stage larvae can be collected by utilizing the affinity with the monoclonal antibody or a fragment thereof. For example, the monoclonal antibody or fragment thereof according to the present invention to which a magnetic substance is bound can be allowed to act on mussel adhesion stage larvae, and then the monoclonal antibody or fragment thereof according to the present invention can be recovered 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), panning, etc. may be used for collecting mussel-adherent larvae.

  Detection of mussel adhesion stage larvae according to the present invention is performed by adding the monoclonal antibody or fragment thereof according to the present invention to a sample (for example, seawater or river water or a lysate obtained by treating it with an ultrasonic device, a homogenizer, etc.). It can be carried out by reacting with the mussel adhesion stage larvae in the sample or a lysate thereof, and analyzing the mussel adhesion stage larvae and the lysate to which the monoclonal antibody or fragment thereof is bound by an immunological technique.

  Therefore, a reagent, kit, or instrument containing a monoclonal antibody or a fragment thereof that specifically reacts with mussel adhesion stage larvae but does not react with oyster adhesion stage larvae, clam attachment stage larvae, and mussel adhesion stage larvae, It can be said that it is useful as a larval detection reagent, detection kit, and detector.

  The reagent for detecting mussel adhesion stage larvae according to the present invention may be any reagent as long as it contains the monoclonal antibody or fragment thereof according to the present invention. For example, a buffer solution (for example, phosphate, carbonate, 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.

  The mussel adhesion stage larvae detector according to the present invention is 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, etc.), substances for suppressing non-specific reactions (for example, block ace, gelatin, skim milk, etc.), substances necessary for detecting mussel adhesion stage larvae by immunological techniques ( For example, labeling substances, chromogenic substrates, secondary antibodies, chromogenic enhancers, etc.), stabilizers (eg BSA, goat serum, etc.) Etc. of common 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 mussel adhesion stage larva detector according to the present invention, a sample dripping portion or a first portion soaking the sample, a second portion on which 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 its fragment labeled with a labeling substance such as colored latex particles. And a chromatographic medium containing a reagent. 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 the mussel adherent stage larvae are present in the sample. The principle is that when a sample containing the mussel adhesion stage larvae or a lysate thereof is dropped or immersed in the first part of the chromatographic medium, the mussel adhesion stage larvae or a lysate thereof in the sample, 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 captured by the above-mentioned monoclonal antibody or fragment thereof immobilized in the second part, and the mussel adhesion stage larvae can be detected by the labeling substance at that position. In contrast, in the sample that does not contain the mussel adhesion 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 It moves to the third part and is captured by the above-mentioned secondary antibody immobilized in the third part, and it becomes clear that the mussel adherent stage larvae did not exist by the labeling of the labeling substance. In this way, it is possible to detect whether the mussel adherent stage larvae are present in the sample based on whether the second part is labeled or only the third part is labeled.

  On the other hand, the blue mussel adhesion stage larvae detection kit according to the present invention may be any kit containing the monoclonal antibody according to the present invention or a fragment thereof. , 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 mussel adherent larvae by immunological techniques A general substance to be included in an antigen detection kit such as serum), or the monoclonal antibody according to the present invention or a Detector instrument 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.), colloidal particles such as gold, 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 to these, 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.

  In addition, 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 method for detecting mussel adhesion stage larvae according to the present invention, a detection reagent, a detection kit, or a detector, confirmation of the presence of mussel adhesion stage larvae, identification of mussel adhesion stage larvae, It is possible to separate and measure the amount of larvae adhering to the mussel.

  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, mussel-adherent larvae (pediberger larvae) obtained by indoor breeding were used as antigens.

  Inject the antigen into the abdominal cavity of 7-week-old BALB / c mice 6 times (1-5th injection: 300 larvae / 100-150 μl PBS, 6th injection (final immunization): 1000 larvae / 200 μl PBS The second dose on day 22 from the first dose, the third dose on day 24 from the second dose, the fourth dose approximately half a year after the third dose, from the fourth dose Half a month later, the fifth dose was given and the sixth dose was given approximately 80 days after the fifth dose.) The mice were immunized.

Three days after the final immunization, the mice were dissected and the spleen removed, and sterilized in RPMI1640 (GIBCO) medium containing 10% FBS (Fetal Bovine Serum; GIBCO) and 1% antibiotics (Antibiotic-Antimycotic; GIBCO). The cells were lined with a stainless mesh and then suspended and dispersed to obtain floating cells. The suspension 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, the myeloma cell line P3U1 (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 stirred while slowly adding it, and then gently stirred for 1 minute. Thereafter, 2 ml of FBS ^- medium was slowly added and stirred for 1 minute to perform cell fusion.

After the cell fusion treatment, 8 ml of FBS ^- medium (37 ° C.) was further slowly added over 1 minute, followed by centrifugation (1000 rpm × 5 minutes), and the supernatant was removed by suction. 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 of each of the 5 laboratory petri dishes containing RPMI1640 medium (GIBCO) containing 0.5 was inoculated and cultured for 5 days under conditions of 37 ° C. and 5% CO ₂ .

  Pick up colonies of hybridoma cells that have been clearly proliferated in each cell, and contain HT medium (10% conditioned medium from J774A.1 cells, HYBRIMAX; SIGMA), 100 μM hypoxanthine and 16 μM thymidine. The cells were transferred to a 96-well plate containing RPMI1640 medium), and hybridomas were cultured. Thereafter, the culture supernatant was collected from wells in which the growth of clear hybridoma was confirmed, 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) By adding 600 μl of 50 mM Tris-HCl (pH 7.5) to 30,000 mussel pediberger larvae, homogenizing and sonicating (several seconds x 5 times) in ice, and centrifuging at 5000 rpm for 20 minutes, A blue mussel larvae crude extract (protein concentration: 39.5 mg / ml) was prepared.
(2) 50 μl of the blue mussel larvae crude extract (100 μg / ml) obtained in (1) was added to each well of a 96-well Iwaki ELISA plate, and the antigen was adsorbed to the bottom of the well at 4 ° C.
(3) Each well subjected to the adsorption treatment in (2) was washed with 200 μl of TBS (0.5 M NaCl and 20 mM Tris-HCl (pH 7.5)).
(4) Each well was blocked with 100 μl of TBS containing 1% BSA for 1 hour.
(5) Each well was washed 3 times with 200 μl of TTBS (TBS containing 0.05% Tween20).
(6) 50 μl of the culture supernatant (primary antibody) obtained by culturing the hybridoma was added to each well and allowed to react for 1 hour.
(7) Each well was washed 4 times with 200 μl of TTBS.
(8) To each well was added 50 μl of a secondary antibody (alkaline phosphatase-labeled anti-mouse IgG (H + L) goat antibody; ZYMED laboratory) solution (1000-fold diluted with TBS) and allowed to react for 1 hour.
(9) Each well was washed 5 times with 200 μl of TTBS.
(10) 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.
(11) 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) Hybridomas (A12-1-2b, B2-3-4-4a, C4-3-4c, C4-3-4b, and F10-) that show higher antibody-producing ability to the crude mussel larvae crude extract 4-1c) was obtained.

[Example 2]
Next, in order to examine whether or not the monoclonal antibodies contained in the culture supernatants of the five hybridomas obtained in Example 1 specifically react with the mussel pediberger larvae, other than the mussel pediberger larvae and mussels Larvae of pediberger larvae, clam plankton (Copepoda, Artemia salina nauplius larvae), Chthamalus challengeri Hoek cypris larvae, and Akafuji acupuncture larvae The reactivity with the protein concentration (100 μg / ml) was confirmed in the same manner as in the ELISA method described in Example 1. Each crude extract was prepared by adding each larvae or plankton to 50 mM Tris-HCl (PH7.5), homogenizing and sonicating (several seconds × 5 times), and centrifuging at 5000 rpm × 20 minutes. . The results of ELISA (OD ₄₀₅ value) are shown in Table 1.

[Table 1]

  As shown in Table 1, the supernatants of the hybridoma cell lines A12-1-2b, C4-3-4c, and C4-3-4b have a very strong reactivity to the crude extract of the mussel pediberger larvae. Reactive to crude extracts of pediberger larvae of bivalves other than mussels such as oysters, clams, and mussels, and larvae other than bivalves and crude extracts of plankton. Not shown. In addition, the supernatant of the hybridoma cell line F10-4-1c shows a very strong reactivity to the crude extract of the mussel pediberger larvae, and the morphologically different crude copepoda larvae from the mussel pediberger larvae. It became clear that it showed very weak reactivity to the extract and no reactivity to other larvae crude extracts.

  On the other hand, the supernatant of the hybridoma cell line B2-3-4-4a showed very strong reactivity to the crude extract of oyster pediberger larvae in addition to the crude extract of mussel pediberger larvae. It was found that there was no reactivity to all larvae other than larvae and the crude plankton extract.

  Based on the above, the hybridoma cell lines A12-1-2b, C4-3-4c, C4-3-4b, and F10-4-1c are capable of producing monoclonal antibodies that react specifically with mussel pediberger larvae. It was revealed that the larvae of the mussel pediberger can be specifically detected and recovered by using the 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 (A12-1-2b: accession number FERM P-20216, C4-3-4c: accession number FERM P-20218, C4-3-4b: FERM P-20219, F10-4-1c: FERM P-20220).

Claims (20)

A method for producing a hybridoma that produces a monoclonal antibody that specifically reacts with mussel adhesion stage larvae and copepods ,
The spleen cells and myeloma cells from mammalian non-human animal immunized from fused hybridoma mussel adhesion stage larvae, which reacts to the mussel adhesion stage larvae and copepod acids, oyster deposition stage larvae, clams deposition stage larvae and mactra chinensis A method for producing a hybridoma, comprising a step of selecting a hybridoma that produces a monoclonal antibody that does not react with adherent larvae . The method for producing a hybridoma according to claim 1, wherein the monoclonal antibody further does not react with Artemia naprius larvae or barnacle adhesion stage larvae. A hybridoma that produces a monoclonal antibody that reacts specifically with mussel-adherent larvae and copepods but does not react with oyster-adherent larvae, clams-adherent larvae, and mussel-adherent larvae.   The hybridoma according to claim 3, which is deposited under the deposit number FERM P-20220. The hybridoma according to claim 3, wherein the monoclonal antibody further does not react with Artemia nauplius larvae or barnacle attachment larvae. A monoclonal antibody or a fragment thereof that specifically reacts with mussel-adhered larvae and copepods but does not react with oyster-adherent larvae, clams-adhered larvae, and mussel-adherent larvae. The monoclonal antibody or fragment thereof according to claim 6 , which is produced from a hybridoma deposited under accession number FERM P-20220. Furthermore, the monoclonal antibody or the fragment thereof according to claim 6 , which does not react with Artemia naprius larvae or barnacle adhesion stage larvae. A reagent for detecting larvae and copepods at the time of mussel adhesion,
A detection reagent comprising a monoclonal antibody or a fragment thereof that reacts specifically with mussel-adhered larvae and copepods but does not react with oyster-adherent larvae, clams-adhered larvae, and mussel-adherent larvae. The detection reagent according to claim 9 , wherein the monoclonal antibody is produced from a hybridoma deposited under accession number FERM P-20220. The detection reagent according to claim 9 , wherein the monoclonal antibody further does not react with Artemia naprius larvae or barnacle attachment larvae. A method for detecting larvae and copepods at the mussel adhesion stage,
A detection method characterized by adding to a sample a monoclonal antibody or a fragment thereof that reacts specifically with mussel-adhered larvae and copepods and does not react with oyster-adhered larvae, clams-adhered larvae, and mussel-adherent larvae . The detection method according to claim 12 , wherein the monoclonal antibody is produced from a hybridoma deposited under accession number FERM P-20220. The detection method according to claim 12 , wherein the monoclonal antibody further does not react with Artemia naprius larvae or barnacle attachment larvae. A detection kit for larvae and copepods at the mussel adhesion stage,
A detection kit comprising a monoclonal antibody or a fragment thereof that reacts specifically with mussel-adhered larvae and copepods but does not react with oyster-adhered larvae, clams-adhered larvae, and mussel-adherent larvae. 16. The detection kit according to claim 15 , wherein the monoclonal antibody is produced from a hybridoma deposited under accession number FERM P-20220. The detection kit according to claim 15 , wherein the monoclonal antibody further does not react with Artemia nauplius larvae or barnacle adhesion stage larvae. A detector for mussels larvae and copepods ,
Detection characterized by immobilization of a monoclonal antibody or fragment thereof that reacts specifically with mussel-adherent larvae and copepods but does not react with oyster-adherent larvae, clam-adherent larvae, and mussel-adherent larvae vessel. The detector according to claim 18 , wherein the monoclonal antibody is produced from a hybridoma deposited under accession number FERM P-20220. 19. The detector according to claim 18 , wherein the monoclonal antibody does not further react with Artemia naprius larvae or barnacle attachment stage larvae.