JP4597172B2 - Antibody to bovine myoglobin partial peptide, test method using the antibody, and test kit - Google Patents

Description

  The present invention relates to a monoclonal antibody and a polyclonal antibody against a bovine myoglobin partial peptide, a hybridoma producing the monoclonal antibody, a method for detecting bovine myoglobin using the monoclonal antibody and / or a polyclonal antibody, and a method for detecting a bovine-derived component in food , As well as kits.

  In recent years, there has been an increasing interest in food safety. In particular, food allergies can cause fatal disabilities in severe cases, so not only consumers with food allergies but also food manufacturers and supervisory authorities are in a position to preserve food safety. This is an extremely important issue. To date, the Ministry of Health, Labor and Welfare has stated that for processed foods containing five items (hereinafter referred to as “specified raw materials”) of eggs, milk, wheat, buckwheat, and peanuts, these specified raw materials are included in accordance with the Food Sanitation Law Enforcement Regulations. The manufacturer is obliged to display the label. In addition, abalone, squid, how much, shrimp, orange, crab, kiwi fruit, beef, walnut, salmon, mackerel, soy, chicken, banana, pork, matsutake, peach, yam, apple, gelatin In regard to processed foods that contain these as raw materials, the Food Hygiene Law Enforcement Regulations recommends that as much as possible the labeling that these are included as raw materials should be made as much as possible. Patent Document 1).

To date, an inspection method has been established for five specified raw materials, and it is now possible to scientifically verify whether or not the specified raw materials are contained in food (Patent Documents 1, 2, 3, 4). ). However, 20 items that conform to the specified raw materials have not been fully studied, and scientific verification methods have not been established. Of the 20 items, particularly beef, the BSE problem has become a social concern in recent years, and a simple detection method has been awaited.
JP 2006-22078 A JP 2003-294748 A JP 2003-294738 A JP 2003-294737 A About notice about food containing allergic substances, notice of food safety department planning department, food health department planning section manager of Ministry of Health, Labor and Welfare, food health department No. 2 food supervision No. 46

  This invention makes it a subject to detect easily the cow origin component resulting from beef especially in foodstuffs.

  In order to detect a specific food from processed food, an immunological technique using an antibody that specifically reacts with a protein serving as an index in the food is used. Also in the present invention, it is necessary to first select an index protein from beef protein, but the amino acid sequences of beef myofibrillar protein and myoplasmic protein have high homology with other animal species, and as an immunogen. When used, there is a high possibility of crossing with many livestock species. Cattle, pigs, chickens, etc. are listed as the main animal species used as food, but since pigs are mammals closely related to cattle, there is a relatively high possibility that they will be crossed. Unless it is resolved, specific detection cannot be expected. Furthermore, when a protein having high homology with other animal species is used as an immunogen, the immunized animal may not recognize it as an antigen, and it may not be possible to produce antibodies.

  Therefore, the present inventors selected myoglobin having a low amino acid sequence homology among animal proteins among the meat proteins as the index protein. Although the amino acid sequence homology of myoglobin in cattle and mice is somewhat low, the titer is difficult to increase, and various improvements such as changing the adjuvant and the number of immunizations were made. As a result, a monoclonal antibody against bovine myoglobin could be produced. A polyclonal antibody against bovine myoglobin was prepared from antiserum obtained by immunizing rabbits. When the reactivity was examined, it was found that both antibodies recognize bovine myoglobin. In addition, in order to eliminate cross-reactivity between pork and chicken, which are frequently used in other livestock breeds, especially meat processing, we searched for sites that differ in amino acid sequence from porcine and avian myoglobin. , Aevkhlaeshan (SEQ ID NO: 1) and aqykvlgfhg (SEQ ID NO: 2) were found. Furthermore, antibodies against peptides having the amino acid sequence were prepared, and these antibodies were found to have high specificity and reactivity. In addition, the inventors have found that beef protein can be specifically detected by immunological measurement using the above polyclonal antibody and monoclonal antibody, and have completed the present invention.

Therefore, the present invention uses the following items 1 to 9, for example, bovine myoglobin in foods under various processing conditions, monoclonal antibodies and polyclonal antibodies having high reactivity, hybridomas producing the monoclonal antibodies, and the antibodies. A method for detecting bovine myoglobin with high sensitivity and specificity from a protein extraction solution and a kit comprising the antibody are provided.
Item 1. A monoclonal antibody against bovine myoglobin obtained using a peptide having an amino acid sequence of aevkhlaeshan (SEQ ID NO: 1) or aqykvlgfhg (SEQ ID NO: 2).
Item 2. Produced from hybridomas consignment number FERM P -21,336, monoclonal antibodies against bovine myoglobin.
Item 3. A hybridoma producing the monoclonal antibody according to claim 1.
Item 4. The hybridoma of the consignment number FERM P -21336.
Item 5. A polyclonal antibody against bovine myoglobin obtained by using a peptide having an amino acid sequence of aevkhlaeshan (SEQ ID NO: 1) or aqykvlgfhg (SEQ ID NO: 2).
Item 6. A method for detecting bovine myoglobin by immunological measurement using the monoclonal antibody according to claim 1 or 2 and / or the polyclonal antibody according to claim 5.
Item 7. A method for detecting bovine-derived components in foods by immunological measurement using the monoclonal antibody according to claim 1 or 2 and / or the polyclonal antibody according to claim 5.
Item 8. The method according to claim 6 or 7, wherein the immunological measurement is performed by ELISA or immunochromatography.
Item 9. A kit for detecting bovine myoglobin, comprising the monoclonal antibody according to claim 1 and / or the polyclonal antibody according to claim 5.
Item 10. A kit for detecting bovine-derived components in food, comprising the monoclonal antibody according to claim 1 and / or the polyclonal antibody according to claim 5.

Antibodies of the present invention is a monoclonal antibody obtained by using a peptide having the amino acid sequence of Aevkhlaeshan (SEQ ID NO: 1) or Aqykvlgfhg (SEQ ID NO: 2), preferably, the hybridoma of consignment number FERM P -21 336 (the hybridoma is, consignment institutions: the National Institute of Advanced industrial Science and technology, Patent microorganisms Depositary Center, was commissioned on August 10, 2007) is a monoclonal antibody produced.

  The antibody of the present invention is also a polyclonal antibody obtained using a peptide having an amino acid sequence of aevkhlaeshan (SEQ ID NO: 1) or aqykvlgfhg (SEQ ID NO: 2).

  These antibodies of the present invention have high reactivity and specificity for bovine myoglobin. The antibody of the present invention also has high reactivity and specificity for bovine myoglobin contained in an extract subjected to heat treatment and denaturation treatment with chemicals or the like.

The hybridoma of the present invention is a hybridoma producing a monoclonal antibody obtained by using a peptide having the amino acid sequence of Aevkhlaeshan (SEQ ID NO: 1) or Aqykvlgfhg (SEQ ID NO: 2), preferably hybridoma trustees number FERM P -21,336 It is.

  The antibody and hybridoma of the present invention are produced as follows.

The monoclonal antibodies of the invention, for example, a peptide having an amino acid sequence of Aevkhlaeshan (SEQ ID NO: 1) or Aqykvlgfhg (SEQ ID NO: 2), produced from the resulting hybridomas by immunizing an animal, preferably a consignment number FERM Produced from hybridoma of P- 21336.

  The polyclonal antibody of the present invention is prepared from an antiserum obtained by immunizing an animal with a peptide having an amino acid sequence of aevkhlaeshan (SEQ ID NO: 1) or aqykvlgfhg (SEQ ID NO: 2), for example.

The hybridoma of the present invention is a hybridoma that produces the monoclonal antibody of the present invention, obtained by immunizing an animal with a peptide having the amino acid sequence of aevkhlaeshan (SEQ ID NO: 1) or aqykvlgfhg (SEQ ID NO: 2), for example. There, particularly preferably hybridoma consignment number FERM P -21336.

  Peptides used for immunization can be obtained by chemical synthesis methods, genetic recombination methods using organisms such as microorganisms as factories, or synthesis methods using cell-free synthesis systems, with chemical synthesis methods being particularly preferred.

  Since the peptide has such a low molecular weight that it cannot cause an immune response as it is, it is conjugated to an appropriate protein and used as an immunogen. Conjugates used include bovine serum albumin, ovalbumin, and coconut hemocyanin (KLH), with coconut hemocyanin (KLH) being particularly preferred.

  Examples of animals used for immunization include mice, rats, rabbits, goats, monkeys and the like as mammals, with mice and rabbits being particularly preferred.

  The monoclonal antibody of the present invention reacts with bovine myoglobin, more preferably denatured bovine myoglobin, after preparing a hybridoma using, for example, a peptide having the amino acid sequence of SEQ ID NO: 1 or 2 conjugated with KLH as an immunogen. It is obtained by selecting a hybridoma that produces an antibody and purifying the monoclonal antibody that it produces. Induction of immunity can usually be performed by an operation in which an immunogen in an amount of 1 ng to 10 mg is divided into 1 to 5 times with 10 to 14 days. After sufficient immunization, organs capable of producing antibodies (spleen and lymph nodes) are aseptically removed from the animals and used as parent strains for cell fusion. The spleen is most preferable as an organ to be removed. Myeloma cells are used as cell fusion partners. Myeloma cells include mouse origin, rat origin, human origin, etc., but mouse origin is preferred. Examples of cell fusion include methods using polyethylene glycol, cell electrofusion, and the like, but methods using polyethylene glycol are simple and preferred. Selection of spleen cells or myeloma cells that have not undergone cell fusion and hybridomas can be performed, for example, by culturing in a serum medium supplemented with HAT supplements (hypoxanthine-aminopterin-thymidine).

  Selection of a hybridoma producing an antibody against bovine myoglobin is preferably performed by direct ELISA on an EIA plate in which bovine myoglobin, more preferably denatured bovine myoglobin, is collected by collecting the culture supernatant described above. A well showing strong color development as a result of direct ELISA is selected, and the cells in the well are subjected to cloning. The hybridoma corresponding to the strongly colored culture supernatant is selected as a hybridoma producing an antibody that reacts with bovine myoglobin, more preferably with denatured bovine myoglobin. Furthermore, an operation (cloning) for selecting and unifying antibody-producing hybridomas is necessary, and there are a limiting dilution method, a fibrin gel method, a method using a cell sorter, etc., but the limiting dilution method is simple and preferable. Thereby, the hybridoma which produces the target monoclonal antibody is acquirable.

  By culturing the hybridoma obtained by the above method, a monoclonal antibody can be obtained in the culture supernatant. Furthermore, in order to obtain a large amount of monoclonal antibody, there are in vivo and in vitro methods, but an in vivo method, particularly a method using mouse ascites is preferred. Monoclonal antibodies are purified from the culture supernatant or mouse ascites by ammonium sulfate salt folding, affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, etc., but affinity chromatography is used in consideration of purification purity and simplicity. Is most preferred. When it is necessary to obtain a highly pure monoclonal antibody, gel filtration chromatography, ion exchange chromatography or the like is preferably performed as a final purification after affinity chromatography.

  In addition, the monoclonal antibody of the present invention is, for example, Nishinaka, S. et al. Int Arch Allergy Appl Immunol. 1989; 89 (4): 416-9, Nishinaka, S. et al. J Vet Med Sci. 1996 Nov; 58 (11): According to the method described in 1053-6 and the like, it can also be prepared using birds such as chickens that produce IgY.

  For example, the polyclonal antibody of the present invention is obtained by immunizing a rabbit with a peptide having the amino acid sequence of SEQ ID NO: 1 or 2 conjugated to KLH, and immunizing the rabbit with each polyclonal antibody. Can be obtained by purifying Purification from the antiserum can be performed in the same manner as the purification of the monoclonal antibody.

  The polyclonal antibody of the present invention can also be produced using birds such as ducks that produce IgY, for example, according to the method described in the patent literature (Japanese Patent Application Laid-Open No. 2002-30100).

  The method of the present invention relates to a method for detecting bovine myoglobin by immunological measurement using the monoclonal antibody and / or polyclonal antibody of the present invention. Immunological measurement methods include Western blotting (WB), enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), etc. Antibodies or antigens that are labeled with enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), radioactive isotopes (RI) such as iodine 125, luminescent substances such as acridinium compounds, or fluorescent substances such as fluorescein isothiocyanate However, it is not limited to these. In the method of the present invention, an enzyme immunoassay (ELISA) which is a kind of enzyme immunoassay (EIA) is particularly preferably used, and ELISA is more preferable. Further, an immunochromatography method that is excellent in convenience is also suitable.

  The method for detecting bovine myoglobin of the present invention uses a composition that may contain the antibody of the present invention and a bovine-derived component. Examples of the composition include foods (particularly processed foods), feeds, laboratory reagents, cosmetics, pharmaceuticals, quasi drugs, medical devices, and the like. Since bovine myoglobin is abundantly contained in beef, it is contained in, for example, food (especially processed food), feed, laboratory reagents, cosmetics, pharmaceuticals, quasi-drugs, medical devices, etc. by the detection method of the present invention. It is also possible to detect bovine-derived components resulting from beef.

  The method of the present invention can detect bovine myoglobin at a concentration of 1 ng / ml or more. In particular, it is possible to detect bovine myoglobin at a concentration of 1 to 200 ng / ml, preferably 1 to 100 ng / ml.

  In the present specification, “detect bovine myoglobin” means “detect the presence of bovine myoglobin”, and detect the presence of bovine myoglobin by detecting a partial peptide of bovine myoglobin. including.

  In general, water, phosphate buffered saline, alcohol or the like is used for protein extraction from food. However, since proteins in foods are denatured through various processing steps, protein extraction may be difficult with only the above solvents. Improvement of protein extraction efficiency from foods can be achieved by adding surfactants such as urea and SDS, protein denaturants such as guanidine hydrochloride, and reducing agents such as mercaptoethanol and dithiothreitol to the above solvents. it can. Therefore, protein extraction from food used in the method for detecting bovine myoglobin of the present invention is performed by adding the above-mentioned denaturing agent and reducing agent (preferably SDS and mercaptoethanol) and performing shaking extraction for 12 hours or more. Can be broken. By this extraction treatment, bovine myoglobin in foods with different processing conditions becomes denatured in the extraction solution, and bovine myoglobin in foods is converted by immunological techniques such as ELISA and Western blotting using the antibody of the present invention. Can be detected.

  A kit for detecting bovine myoglobin comprising the antibody of the present invention is also included in the scope of the present invention. The kit comprises the monoclonal antibody and / or polyclonal antibody of the present invention, and preferably comprises the necessary amount of reagents necessary for the immunoassay used. An example of such a kit is a kit for detecting bovine myoglobin using an ELISA method, wherein the monoclonal antibody and / or polyclonal antibody of the present invention is an antibody for solid phase adsorption and / or a label for detection. The detection labeled antibody used as an antibody is labeled with HRP, and a kit provided with reagents necessary for other ELISA methods (for example, a microplate, an extraction solution, a buffer solution, etc.) can be mentioned. Alternatively, when the antibody moves on the membrane due to capillary action, an antigen-antibody reaction complex is formed by the antigen, labeled antibody, and supplementary antibody in the specimen, and the labeling of the labeled antibody is confirmed. A kit for detecting bovine myoglobin using an immunochromatography method, wherein the monoclonal antibody and / or the polyclonal antibody of the present invention is used as a supplementary antibody and / or a labeled antibody for detection. Gold colloid is used. Examples include kits equipped with reagents necessary for other immunochromatography methods (for example, immunochromatographic test plates, extraction solutions, buffer solutions, etc.).

  Moreover, since bovine myoglobin is abundantly contained in beef, the kit contains, for example, food (especially processed food), feed, laboratory reagents, cosmetics, pharmaceuticals, quasi drugs, medical devices, etc. It is also possible to detect a bovine-derived component caused by beef.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, a present Example does not limit this invention at all. Hereinafter, a peptide having the amino acid sequence of aevkhlaeshan (SEQ ID NO: 1) is referred to as “peptide A”, and a peptide having the amino acid sequence of aqykvlgfhg (SEQ ID NO: 2) is referred to as “peptide B”.

Example 1: Preparation of anti-peptide antibody Two sites (peptide A and peptide B) having different amino acid residues from other livestock species were selected from bovine myoglobin (Fig. 1), and the N-terminus of peptide A was obtained by chemical synthesis. And a peptide in which methionine-cysteine is further added to the N-terminus of peptide B (having the amino acid sequence of mcakykvlgfhg in the amino acid one-letter code) Two types of synthetic peptides were prepared. The reason for adding the amino acid to the N-terminus of peptide A and peptide B is that it is necessary to introduce a cysteine residue into the peptide in order to conjugate these peptides to coconut shell hemocyanin (KLH). In the production of monoclonal antibodies, these peptides conjugated to coconut hemocyanin (KLH) were used as immunogens, and each immunogen was immunized to Balb / c mice (female, 8 weeks old). An emulsion for immunization was prepared by mixing with an equal volume of Freund's complete adjuvant. In the first immunization, this emulsion was intraperitoneally administered so that KLH was 50 μg per mouse. In the secondary immunization, an emulsion mixed with Freund's incomplete adjuvant was administered intraperitoneally two weeks after the first immunization so that KLH was 50 μg per mouse. On the 10th day after the second immunization, the antibody titer was measured, and for mice that had a sufficient antibody titer, 2 weeks after the second immunization, administered to the tail vein so that KLH was 50 μg per mouse, Final immunization was performed.

  Two days after the final immunization, the spleen was aseptically removed to obtain spleen cells, and cell fusion was performed with cultured myeloma cells under 50% polyethylene glycol conditions to prepare hybridomas. After cell fusion, incubate for about 2 weeks in serum medium supplemented with HAT supplements to select hybridomas, and then use the EIA plate to solidify bovine myoglobin denatured by SDS and heat treatment. The titer was measured, and a well containing a hybridoma producing an antibody that reacts with denatured bovine myoglobin was selected.

  After counting the number of cells in the wells where hybridomas producing antibodies that react with denatured bovine myoglobin are present, the hybridomas are cloned by limiting dilution in a serum medium supplemented with 10% cloning reagent (BM condimed H1). By repeating the operation twice, several hybridomas producing monoclonal antibodies that react with denatured bovine myoglobin were obtained. Finally, anti-peptide A monoclonal antibody and anti-peptide B monoclonal antibody were purified from ascites using an affinity column. On the other hand, polyclonal antibodies can be prepared by immunizing rabbits with each immunogen using peptide A or B conjugated to KLH as an immunogen and purifying each polyclonal antibody from the resulting antiserum using an affinity column. I went there.

Example 2: Purification of bovine myoglobin Myoglobin was purified using bovine thigh meat. Fat was removed from beef leg meat and minced and mixed well. 100 g was weighed, doubled amount of distilled water was added and mixed well, and stirred in ice for 30 minutes. After centrifugation at 4,500 × g for 15 minutes at 4 ° C., the supernatant was filtered, and the filtrate was used as a meat extract. Ammonium sulfate (ammonium sulfate) was added to the meat extract to a concentration of 90%, and the mixture was stirred for 1 hour in ice. After centrifugation at 20,000 xg for 15 minutes at 4 ° C, the supernatant is filtered, the pH of the filtrate is adjusted to 6.85, ammonium sulfate is added so that the ammonium sulfate concentration becomes 95%, and the mixture is stirred in ice for 1 hour. did. After centrifuging at 20,000 × g for 15 minutes at 4 ° C., the supernatant was removed, and distilled water was added to the precipitate to dissolve it. This was used as a crude myoglobin fraction. After replacing the crude myoglobin fraction with 20 mM Tris-HCl (pH 8.3) (solution A), it was injected into a QAE column (Toyopearl QAE 550M) equilibrated with the same buffer, and from solution A to solution B (20 mM Tris- A gradient of HCl and 0.5 M NaCl (pH 8.3) was applied, and separation was performed according to ionic strength, and a fraction with a peak was collected. The fraction collected was confirmed to be bovine myoglobin from the N-terminal amino acid sequence after confirming the molecular weight and purity of the band by electrophoresis.

Reference Example 1: Preparation of denatured bovine myoglobin antibody Sandwich ELISA requires antibodies with different epitope portions for the immobilized antibody and the labeled antibody, respectively. Although the degree varies depending on the food, an antibody that reacts with the denatured protein is desirable because the protein in the food after the processing step is denatured. SDS was added to bovine myoglobin 1 mg / ml purified in Example 2 to 1%, and then heat-treated at 95 ° C. for 10 minutes to obtain denatured bovine myoglobin. Using this as an immunogen, Balb / c mice were immunized to obtain monoclonal antibodies. An emulsion for immunization was prepared by mixing with an equal volume of Freund's complete adjuvant. In the first immunization, this emulsion was intraperitoneally administered so that the modified bovine myoglobin was 50 μg per mouse. For secondary immunization, an emulsion mixed with Freund's incomplete adjuvant was administered intraperitoneally two weeks after the first immunization so that the modified bovine myoglobin was 50 μg per mouse. The antibody titer was measured on the 10th day after the second immunization, and mice with sufficient antibody titer were administered to the tail vein so that the modified bovine myoglobin was 50 μg per mouse 2 weeks after the second immunization. The final immunization was performed.

  Two days after the final immunization, the spleen was aseptically removed to obtain spleen cells, and cell fusion was performed with cultured myeloma cells under 50% polyethylene glycol conditions to prepare hybridomas. After cell fusion, the hybridoma is selected by culturing in serum medium supplemented with HAT supplement for about 2 weeks, and then the antibody titer of the culture supernatant is measured using an EIA plate on which denatured bovine myoglobin is immobilized. Wells containing hybridomas producing antibodies reactive with myoglobin were selected. After counting the number of cells in the wells where hybridomas producing antibodies that react with denatured bovine myoglobin are present, the hybridomas are cloned by limiting dilution in a serum medium supplemented with 10% cloning reagent (BM condimed H1). By repeating the operation twice, several hybridomas producing monoclonal antibodies that react with denatured bovine myoglobin were obtained.

  Finally, anti-denatured bovine myoglobin monoclonal antibody was purified from ascites using an affinity column. On the other hand, for production of a polyclonal antibody, rabbits were immunized with denatured bovine myoglobin, and the polyclonal antibody was purified from the obtained antiserum using an affinity column.

Example 3: Detection of bovine myoglobin by immunoassay
Western Blot Western blot was performed to evaluate the specificity of the obtained anti-peptide antibody. Meat extract proteins of beef leg, pork leg and chicken leg were prepared as follows. That is, cow thigh meat, pork thigh meat and chicken thigh meat were mixed with each other by removing fat and mincing. 100 g was weighed, added twice the amount of distilled water and homogenized, and then freeze-dried to obtain each dried meat powder. Add 20 ml of protein extraction solution (PBS (pH 7.2), 0.5% SDS, 2% mercaptoethanol) to 400 mg of each dried meat powder, extract by shaking for 12 hours or more, and centrifuge the supernatant after centrifugation at 20,000 xg for 15 minutes. It was set as each meat extraction protein. Moreover, porcine myoglobin and trimyoglobin were purified in the same manner as the above-described method for purifying bovine myoglobin. After measuring the protein concentration, prepare meat extract protein at 1 mg / ml and various myoglobin at 100 μg / ml. Sample buffer (0.125M Tris-HCl (pH 6.8), 10% mercaptoethanol, 4% SDS, 10% % Sucrose, 0.004% Bromophenol Blue) and heat-treated at 95 ° C. for 10 minutes as a sample for electrophoresis.

SDS-PAGE was performed according to the method of Laemmli (Laemmli UK, Nature. 1970; 227: 332-337) using a separation gel with an acrylamide concentration of 15% and a concentrated gel with 4.5%. Each meat extract protein was applied to the gel so as to be 10 μg / lane and each myoglobin was 1 μg / lane. Proteins were transferred from the separation gel subjected to SDS-PAGE electrophoresis to a PVDF membrane using a semi-dry blotting apparatus. The protein-transferred membrane is immersed in Tween-PBS (0.137M NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO ₄ · 12H ₂ O, 1.5 mM KH ₂ PO ₄ , 0.1% Tween) and allowed to stand at 4 ° C. overnight. And blocking. After blocking, appropriately diluted anti-denatured bovine myoglobin polyclonal antibody, anti-peptide A polyclonal antibody, and anti-peptide B monoclonal antibody were reacted for 1 hour with shaking. After washing with Tween-PBS, ALP-labeled anti-mouse IgG or ALP-labeled anti-rabbit IgG appropriately diluted as a secondary antibody was used for reaction for 1 hour with shaking. After washing with Tween-PBS and equilibrating with 0.1 M Tris-HCl (pH 9.5) for 15 minutes, color was developed using Alkaline Phosphatase Substrate Kit IV (Vecter, BCIP / NBT substrate).

  These electrophoretic images (CBB staining) are shown in FIG. 2-1, and the results of Western blotting using anti-denatured bovine myoglobin polyclonal antibody, anti-peptide A polyclonal antibody, and anti-peptide B monoclonal antibody as primary antibodies are shown in FIG. It is shown in 2-3, 2-4. Anti-peptide A polyclonal antibody and anti-peptide B monoclonal antibody reacted only with bovine myoglobin and did not show cross-reactivity with porcine and avian myoglobin. On the other hand, the anti-denatured bovine myoglobin polyclonal antibody did not react with trimyoglobin, but a cross reaction with porcine myoglobin was observed. That is, it was proved that the antibody of the present invention has higher specificity than the polyclonal antibody against bovine myoglobin.

Sandwich ELISA
Each of the obtained antibodies was immobilized on a plate and labeled with HRP enzyme, and a combination of sandwich ELISA using denatured bovine myoglobin as a standard protein was investigated. In addition, the cross-reactivity of swine with avian myoglobin was investigated. The immobilized antibody was adjusted to 5 μg / ml with 0.1 M sodium phosphate (pH 7.2), dispensed 100 μl onto an EIA plate, allowed to stand overnight at 4 ° C., and adsorbed onto the plate. Thereafter, 260 μl of blocking solution was added and allowed to stand at 37 ° C. for 90 minutes. After washing, add 100 μl of a standard solution of denatured myoglobin prepared by diluting bovine, porcine, and avian myoglobin denatured under the above conditions from 200 ng / ml to each well, and let stand at room temperature for 1 hour. did. After washing, 100 μl of a labeled antibody appropriately diluted as a secondary antibody was added and allowed to stand at room temperature for 1 hour to react with denatured myoglobin reacted with a solid-phased antibody. After washing, 100 μl of TMB enzyme substrate was added and reacted at room temperature for 10 minutes, and then 50 μl of 1M sulfuric acid was added to stop the enzyme reaction, and the absorbance at 450 nm was measured.

With a combination using an anti-denatured bovine myoglobin polyclonal antibody as the immobilized antibody and an anti-denatured bovine myoglobin polyclonal antibody as the labeled antibody, a calibration curve up to about 10 ng / ml could be drawn. However, there was some cross-reactivity with porcine myoglobin. In general, the cross-reaction can be reduced by using an antibody with a high specificity for the immobilized antibody. Therefore, an anti-peptide antibody was used as the immobilized antibody, and a combination using an anti-peptide A polyclonal antibody as the immobilized antibody and an anti-denatured bovine myoglobin polyclonal antibody as the labeled antibody was examined. About 5 ng / ml A standard curve was obtained, and there was no cross-reaction between swine and avian myoglobin (Figure 3). Further, the sensitivity can be further increased by using the monoclonal antibody of the present invention for both the immobilized antibody and the labeled antibody, and bovine myoglobin can be detected with a sensitivity of about 1 ng / ml or more. It is believed that there is.
As described above, it was proved that bovine myoglobin can be detected specifically and highly sensitively by immunological measurement using the antibody of the present invention.

FIG. 1 shows a multiple alignment of the myoglobin amino acid sequence of each animal. FIG. 2 shows each animal meat extract fraction and SDS-PAGE image of myoglobin (CBB staining), and Western using anti-denatured bovine myoglobin polyclonal antibody, anti-peptide A polyclonal antibody and anti-peptide B monoclonal antibody as primary antibodies. An image of the blot results is shown. FIG. 3 shows the results of sandwich ELISA using an anti-peptide A polyclonal antibody as the immobilized antibody and an anti-denatured bovine myoglobin polyclonal antibody as the labeled antibody.

Claims (9)

A monoclonal antibody against bovine myoglobin obtained by conjugating mice with coconut hemocyanin (KLH) and using it as an immunogen by conjugating a peptide consisting of the amino acid sequence of mcakykvlgfhg in amino acid single letter code . A monoclonal antibody against bovine myoglobin produced from a hybridoma having the deposit number of FERM P-21336. A hybridoma producing the monoclonal antibody according to claim 1. Hybridoma with accession number FERM P-21336. The immunoassay using a monoclonal antibody according to claim 1 or 2, the method of detecting bovine myoglobin. The immunoassay using a monoclonal antibody according to claim 1 or 2, the method of detecting bovine-derived components in the food product. The method according to claim 5 or 6 , wherein the immunological measurement is performed by ELISA or immunochromatography. It characterized in that it comprises a monoclonal antibody according to claim 1 or 2, a kit for detecting bovine myoglobin. It characterized in that it comprises a monoclonal antibody according to claim 1 or 2, a kit for detecting bovine-derived components in the food product.

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