JP5749947B2 - Method and kit for measuring feline-derived α1 microglobulin, and antibody and antibody-producing cell line therefor - Google Patents

Description

  The present invention relates to a measurement method and measurement kit for cat-derived α1 microglobulin, and an antibody and an antibody-producing cell line therefor.

  In recent years, with the declining birthrate, the number of households that keep pets is increasing. However, there are not a few cases where the pet is not kept according to the nature of the pet. In particular, as a result of uneven eating, pets cause adult pathological symptoms such as diabetes, and even pets are seen in veterinarians.

Under such circumstances, in recent years, businesses related to pet diagnosis are expanding. If a pet's nephropathy can be detected at an early stage, the veterinarian can give instructions on how to keep the pet, particularly how to eat, by the owner. In general, one marker of nephropathy is α1 microglobulin (α ₁ -m).

  α1 microglobulin, for example, derived from humans is produced in cells of the whole body of humans, and is secreted to the outside of cells with a constant production amount with little influence on changes in the environment inside and outside the cell. In some cases, it is useful as an index for early diagnosis of diabetic nephropathy.

  However, regarding the α1-microglobulin derived from cats, the current situation is that even the amino acid sequence of the protein has not been elucidated, in addition to the presence of an antibody specific to the protein.

  The present inventor has elucidated the amino acid sequence of cat-derived α1 microglobulin and succeeded in producing a monoclonal antibody that specifically binds to the protein, and has filed a patent application (Japanese Patent Application Nos. 2010-033676, In the present specification, this is referred to as “prior application”).

  However, these antibodies were not sensitive enough to measure the concentration of feline-derived α1 microglobulin.

Journal of Veterinary Internal Medicine. 22 (5): 1111-1117, 2008

  The present invention has been made in order to solve the above-mentioned problems, and the object of the present invention is to provide a measurement method and kit using a combination of antibodies that are more sensitive and specific to cat-derived α1 microglobulin, and It is to provide antibodies and antibody-producing cell lines for this purpose.

As a result of diligent research, the present inventors have discovered that the binding strength of cat-derived α1 microglobulin to the antibody differs depending on the location of the mouse obtained in obtaining the antibody-producing cells , thereby completing the present invention. It came to do. That is, the present invention is as follows.

The present invention relates to a first antibody produced by the cell line Mouse-Mouse hybridoma α ₁ -mmAb3 ( Accession No .: FERM P-22051 ), the cell line Mouse-Mouse hybridoma α ₁ -mmAb4 ( Accession No: FERM). P-22052 ) and the second antibody produced by P-22052 ) to provide a method for measuring the concentration of cat-derived α1 microglobulin in a specimen by enzyme immunoassay (sandwich method).

The present invention further includes a first antibody produced by the cell line Mouse-Mouse hybridoma α ₁ -mmAb3 ( accession number: FERM P-22051 ), a cell line Mouse-Mouse hybridoma α ₁ -mmAb4 ( accession number). And a second antibody produced by FERM P-22052 ), which also provides a kit for measuring a feline-derived α1 microglobulin concentration.

The present invention also includes a cell line Mouse-Mouse hybridoma α ₁ -mmAb3 ( accession number: FERM P-22051 ), a cell line Mouse-Mouse hybridoma α ₁ -mmAb4 ( accession number: FERM ), which are novel antibody-producing cell lines. P-22052 ) is also provided.

The present invention further uses a protein having the amino acid sequence represented by SEQ ID NO: 1 as an antigen, cell line Mouse-Mouse hybridoma α ₁ -mmAb3 ( accession number: FERM P-22051 ) or cell line Mouse-Mouse hybridoma α _1- An antibody that specifically binds to cat-derived α1 microglobulin, which is produced by m mAb4 ( Accession Number: FERM P-22052 ) is also provided.

  According to the present invention, the feline-derived α1 microglobulin concentration in a specimen can be measured with higher sensitivity.

2 is a photograph showing the results of SDS-PAGE after expression of a GST fusion protein in Reference Example 1. It is a graph which shows the chromatographic pattern obtained as a result of HPLC in Reference Example 1, the left vertical axis indicates absorbance at a wavelength of 220 nm, the right vertical axis indicates acetonitrile concentration (%), and the horizontal axis indicates time (minutes). Yes. It is a graph which shows the measurement result by the enzyme immunoassay method (sandwich method) of Example 1 and 2, and the vertical axis | shaft on the left side has shown the light absorbency.

According to the present invention, a first antibody produced by the cell line Mouse-Mouse hybridoma α ₁ -mmAb3 ( accession number: FERM P-22051 ) and the cell line Mouse-Mouse hybridoma α ₁ -mmAb4 ( accession number) : FERM P-22052 ) and a method for measuring the feline-derived α1 microglobulin concentration in a specimen by enzyme immunoassay (sandwich method) using the second antibody produced by FERM P-22052 ).

  Here, the specimen includes dialysis fluid used in cat hemodialysis in addition to cat-derived blood, urine, bone marrow fluid, and cat-derived cell suspension.

The cat-derived α1 microglobulin refers to a protein having the amino acid sequence represented by SEQ ID NO: 1. The present inventor first identified a structural gene (referred to herein as an “α ₁ -m gene”) that encodes α1 microglobulin among feline genes, and the cat was identified by the α ₁ -m gene. The amino acid sequence of the derived α1 microglobulin (referred to herein as “α ₁ -m”) has also been analyzed for the first time, and a patent application has been filed (Japanese Patent Application No. 2010-033676). The protein of the present invention having the amino acid sequence shown in SEQ ID NO: 1 is α ₁ -m derived from a cat whose amino acid sequence has been specified for the first time by the present inventors.

In addition, the present inventor has succeeded in producing a cell that produces an antibody using the feline-derived α1 microglobulin as an antigen, and has made a deposit with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Accession number: FERM P-21910, FERM P-21911). The present invention succeeded in measuring α ₁ -m with higher sensitivity than the above-mentioned antibody, and was newly deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of January 12, 2011. ( Accession number: FERM P-22051 , accession number: FERM P-22052 ). The present invention relates to cell lines Mouse-Mouse hybridoma α ₁ -mmAb3 ( accession number: FERM P-22051 ), cell lines Mouse-Mouse hybridoma α ₁ -mmAb4 ( accession number: FERM ), which are the novel antibody-producing cell lines. P-22052 ) is also provided.

In addition, the present invention uses a protein having the amino acid sequence represented by SEQ ID NO: 1 (cat-derived α ₁ -m) as an antigen, and a cell line Mouse-Mouse hybridoma α _1- m mAb3 ( accession number: FERM P-22051 ). The produced antibody that specifically binds to cat-derived α1 microglobulin (first antibody) and the protein having the amino acid sequence represented by SEQ ID NO: 1 (cat-derived α ₁ -m) as an antigen And an antibody (second antibody) that specifically binds to cat-derived α1 microglobulin produced by the cell line Mouse-Mouse hybridoma α ₁ -mmAb4 ( Accession Number: FERM P-22052 ). provide.

  The first antibody and the second antibody of the present invention include enzyme immunoassay (sandwich method), fluorescence immunoassay (sandwich method), radioisotope immunoassay (sandwich method), immunoturbidimetric method and latex immunity. Used as two antibodies for turbidimetry.

  The enzyme immunoassay (sandwich method) is a typical measurement method in protein quantification, and can be easily practiced by those skilled in the art. The enzyme immunoassay (sandwich method) basically uses two types of antibodies: an antibody immobilized on a solid phase (capture antibody) and an antibody that further binds to an antigen bound to the capture antibody (primary antibody). . On top of this, an enzyme label for performing color development or luminescence reaction is applied to the primary antibody. Alternatively, when the primary antibody is not labeled with an enzyme, an antibody (secondary antibody) that can be further bound to the primary antibody and is labeled with an enzyme may be used. As the secondary antibody, there is a possibility that IgG and IgM can be used.

  The enzyme label here includes not only an embodiment in which an enzyme that can be used for color development or luminescence reaction is directly labeled on the primary antibody, but also an embodiment in which the enzyme is indirectly modified during the reaction through a specific binding mode. This is common knowledge for those skilled in the art. As a specific binding mode, for example, avidin-biotin binding is representative. More specifically, a combination of a primary antibody modified with biotin and an avidin-modified peroxidase corresponds to this embodiment, and the peroxidase is indirectly modified to a primary antibody through a binding mode called avidin-biotin bond.

  The enzyme labeled with the primary antibody or the like can be appropriately selected by those skilled in the art, and in addition to peroxidase, alkaline phosphatase, luciferase, β-galactosidase, and the like can be used. The substrate used for the enzyme can also be appropriately selected by those skilled in the art depending on the type of enzyme used. For example, when the enzyme is peroxidase, tetramethylbenzidine, 2,2′-azinobis (3-ethylbezothiazoline) -6-sulfonic acid) ammonium salt, 4-aminoantipyrine, 4-aminoantipyrine hydrochloride, 5-aminosalicylic acid, 2,4-dichlorophenol, N, N-dimethyl-m-toluidine, N, N-dimethylaniline, etc. And when the enzyme is alkaline phosphatase, blue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate disodium salt, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt And when the enzyme is luciferase, D- -)-Luciferin, and when the enzyme is β-galactosidase, 5-bromo-4-chloro-3-indolyl β-D-galactosidase, 5-bromo-6-chloro-3-indolyl β-D-galactosidase, Examples thereof include 5-bromo-3-indolyl β-D-galactosidase and 6-chloro-3-indolyl β-D-galactosidase.

  The first antibody of the present invention may be used as either a capture antibody or a primary antibody, and is not limited. In other words, the second antibody is a primary antibody when the first antibody is a capture antibody, and is a capture antibody when the first antibody is a primary antibody. This point is also demonstrated in the examples described later.

  In addition, the fact that the first antibody and the second antibody of the present invention can be measured by enzyme immunoassay (sandwich method) means that those skilled in the art can perform fluorescence immunoassay (sandwich method), radioisotope immunoassay. It can also be applied to the method (sandwich method), immunoturbidimetry and latex immunoturbidimetry. For example, when a fluorescent immunoassay (sandwich method) is used as a measurement method, a fluorescent label is used instead of the enzyme label in the above-described enzyme immunoassay (sandwich method), and a radioisotope immunoassay (sandwich method). ), A radioisotope label may be applied instead of the enzyme label in the enzyme immunoassay (sandwich method) described above. In addition, for the immunoturbidimetric method and latex immunoturbidimetric method, it is only necessary to use these two antibodies in the reaction system. The latex material in the latex immunoturbidimetric method can be appropriately selected by those skilled in the art, but generally used polystyrene may be used.

  The present invention further provides a measurement diagnostic kit using the first antibody and the second antibody of the present invention described above. The diagnostic kit of the present invention is combined in such a manner that the measurement can be used according to the above-described measurement method. For example, when an enzyme immunoassay (sandwich method) is selected as the measurement method, the antibody of the present invention In addition, it is designed to include wells, chromogenic substrate solutions, reaction stop solutions, washing solutions, standard solutions and the like.

  Hereinafter, the present invention will be described in more detail with reference to reference examples, examples, and experimental examples, but the present invention is not limited thereto.

<Reference example>
Hereinafter, as a reference example, synthesis of cat-derived α ₁ -m, production of an antibody-producing hybridoma using the α ₁ -m, and production of the antibody of the present invention are shown. These reference examples are substantially the same as the method of the prior application.

<Reference Example 1: Synthesis of cat-derived α ₁ -m>
(1) Primer design PCR was performed to amplify the nucleic acid of only the cat-derived α ₁ -m protein region excluding the part considered to be a signal peptide region and the complex protein bikunin. As for the primer, the upstream primer adds EcoRI restriction enzyme site to the 5 'end, and the downstream primer adds XhoI restriction enzyme site and His-Tag to the 3' end, so that each has the following base sequence: Designed.

-Upstream primer: 5'-CACGGATCCAGCCCCGTGCTGACGCCGCCCGATGACATCCAAGTGCAAGAGAACTT-3 '(SEQ ID NO: 2)
-Downstream primer: 5'-CACCTCGAGTTAGTGGTGGTGGTGGTGATGCGTGAGTGGAGAGGGCTCTGGTTCC-3 '(SEQ ID NO: 3)
(2) PCR method Using the upstream primer and downstream primer as described above, 95 ° C 2 minutes 1 cycle, 95 ° C 1 minute, 75 ° C 1 minute, 72 ° C 1 minute 30 cycles, 72 ° C 10 minutes 1 cycle PCR was performed under the conditions described above. The PCR product was subjected to agarose gel electrophoresis, and then DNA was extracted from the agarose gel. The DNA extraction solution was mixed with an equal amount of phenol and centrifuged at 15700 × g for 5 minutes, and then the aqueous layer containing the nucleic acid was separated. The separated aqueous layer was mixed with an equal amount of chloroform, centrifuged at 15700 × g for 5 minutes, and then the supernatant was separated. Next, add 2.5 volumes of 100% ethanol to the solution after separation, let stand at −80 ° C. for 30 minutes, and then centrifuge at 15700 × g for 5 minutes, and then remove the supernatant to obtain a precipitate. It was. 70% ethanol was added to the precipitate, and after centrifugation at 15700 × g for 5 minutes, the supernatant was removed to obtain a concentrated sample of the PCR product. Concentrate the PCR product with 5 μl EcoRI (Takara Bio Inc.), 5 μl XhoI (Takara Bio Inc.), 5 μl H.Buffer (500 mM Tris-HCl, pH 7.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 1000 mM) NaCl) and 35 μl of RNase free H ₂ O were mixed. Also 5 μl (2.5 μg) pGEX6P-1 (GE Healthcare Bioscience) was mixed with 5 μl EcoRI, 5 μl XhoI, 5 μl H.Buffer and 30 μl RNase free H ₂ O. Each solution was treated with a restriction enzyme by incubating at 37 ° C. overnight, then subjected to agarose gel electrophoresis, and each DNA band was extracted using QIAquick Gel Extraction Kit (QIAGEN). Ligation was performed using DNA Ligation Kit (Takara Bio Inc.). That is, 5 μl of Ligation Mix, 1 μl of restriction enzyme-treated α ₁ -m cDNA solution and 4 μl of pGEX6P-1 were mixed and allowed to stand at 16 ° C. overnight to allow ligation of α ₁ -m cDNA. A plasmid vector (pGEX-α ₁ -m) was created. Further, 2.5 μl of this pGEX-α ₁ -m solution was added to 25 μl of E. coli JM109 Competent Cells (Takara Bio Inc.), allowed to stand for 30 minutes on ice, and then heated for 45 seconds in a 42 ° C. thermostat. Give Shock, immediately cool on ice for 2 minutes, then gently add 250 μl of SOC medium (2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO ₄ , 10 mM MgCl ₂ , 20 mM glucose) at 37 ° C. for 1 hour. Keep warm. 100 μl of E. coli solution transfected with pGEX-α ₁ -m was applied to ampicillin-added LB medium, and allowed to stand overnight at 37 ° C., colonies were picked, mixed with 1.2 ml of ampicillin-added LB liquid medium, Cultured overnight at 37 ° C. After culturing, the culture medium is centrifuged at 13400 × g for 1 minute, and the supernatant is completely removed. Extraction of pGEX-α ₁ -m from the resulting sediment using QIAPrep Spin Mini Kit50 (QIAGEN) Went. This pGEX-α ₁ -m was confirmed by agarose gel electrophoresis. Further, the success or failure of subcloning of pGEX-α ₁ -m was performed by base sequence analysis using T7 primer and Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems) and Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems).

(3) Confirmation of His-Tag GST fusion protein expression
After culturing E. coli transfected with pGEX-α ₁ -m in LB medium at 37 ° C overnight, mix 100 µl with 20 µl of Isopropyl-β-D-thiogalactopyranoside (IPTG: 0.1 mM) and add about 2 at 30 ° C. Time-shaking culture (BR40-LF, TAITEC) was performed. The E. coli solution after shaking culture is centrifuged at 15700 × g for 1 minute, then the supernatant is removed, and the solubilized solution (50 μl of 50 mM Tris-Hcl, 100 μl of 1 × RIPA Lysis Buffer (Up State), 140 μl) (Protease Inhibitor, 710 μl of H ₂ O) (30 μl) was solubilized and centrifuged at 15700 × g for 5 minutes to separate the supernatant and sediment. To 30 μl of supernatant, add 30 μl of 2 × SB solution (2% SDS, 40% Glycerol, 0.6% BPB, 25 mM Tris-HCl Buffer (pH 6.8, 20 ° C.)) and 1 μl of 2ME, and at 95 ° C. for 3 minutes Warmed up. 20 μl of SB solution was added to the sediment, and the mixture was crushed with an ultrasonic crusher (UR-20P, TOMY SEIKO CO, LTD) for 5 seconds and then heated at 95 ° C. for 3 minutes. Thereafter, the expression of the His-Tag-attached GST fusion protein and the solubility of the His-Tag-attached GST fusion protein in Escherichia coli were confirmed by SDS-PAGE for the supernatant and the sediment.

(4) SDS-PAGE method
SDS-PAGE was performed according to the Laemmli method using compact PAGE (AE-7300, ATTO), with the following modifications. That is, the composition of the separation gel was 15% Acrylamide, 0.2% N, N-Methylene-bis-Acrylamide, 0.1% SDS, 375 mM Tris-HCl buffer (pH 8.8, 20 ° C.). The gel was prepared using a 2/4 series gel preparation device (AE-7344, ATTO). The composition of the electrode buffer was 0.1% SDS, 129 mM glycine, 25 mM Tris (pH 8.3, 20 ° C.). The composition of the electrophoresis sample (SB) was 1% SDS, 20% Glycerol, 0.3% BPB, 12.5 mM Tris-HCl Buffer (pH 6.8, 20 ° C.). In addition, as a marker, a prestained SDS-PAGE standard (Broad) marker (BIO-RAD) or an SDS-PAGE standard (Broad) marker (BIO-RAD) was used. Electrophoresis was performed in Tris-Gly / PAGE High mode for 30 minutes, then in Tris-Gly / PAGE Low mode, and terminated when the lower ion interface moved to a position 1 to 2 mm above the bottom of the gel. The gel after SDS-PAGE was subjected to silver staining in accordance with the Oakle method. Specifically, the gel was fixed with 30% ethanol and 10% acetic acid solution, washed, and immersed twice in 20% ethanol for 5 minutes. After removing 20% ethanol, the mixture was reacted for 4 minutes with a 5% glutaraldehyde solution, washed with pure water, and then immersed in 20% ethanol twice for 4 minutes. Thereafter, it was washed with pure water, reacted for 5 minutes with an ammoniacal silver nitrate solution, washed with pure water, and then developed with 0.005% citric acid and 0.019% formaldehyde solution. The gel after color development confirmation was fixed with 20% ethanol and 10% acetic acid solution for 5 minutes, immersed in 20% ethanol twice for 5 minutes, and photographed. All silver staining methods were performed under light-shielding conditions.

(5) Expression induction and isolation of GST fusion protein with His-Tag
Escherichia coli in which the expression of the His-Tag-attached GST fusion protein was confirmed was applied to ampicillin-added LB agar medium, colonies were picked, added to 3 ml of ampicillin-added LB liquid medium, and cultured overnight at 37 ° C. with shaking. Subsequently, 3 ml of the culture solution was added to 250 ml of ampicillin-added LB liquid medium, and after shaking culture at 37 ° C. for about 150 minutes, 2.5 ml of 0.1 mM IPTG was added, followed by shaking culture at 25 ° C. for about 2 hours. The culture solution after induction of expression of the His-Tag-attached GST fusion protein was centrifuged at 6000 xg for 15 minutes, and 20 ml of 0.5 mM EDTA, 0.4 M NaCl, 5 mM MgCl ₂ , 5% glycerol, 0.5 mM Phenylmethylsulfonyl fluoride (PMSF) ), 1 mM Dithiothreito (DTT) and 1 mg / ml lysozyme-added 50 mM Tris-HCl (pH 8.0), left at 4 ° C. for 1 hour, and then freeze-thawed twice. Subsequently, 0.5% of Nonidet P-40 was added, crushed for 20 seconds × 5 times with an ultrasonic crusher, centrifuged at 9300 × g for 20 minutes, and the supernatant and sediment were analyzed by SDS-PAGE. As a result of electrophoresis, it was found that the His-Tag-attached GST fusion protein was contained in the supernatant (FIG. 1).

(6) Affinity chromatography method The supernatant obtained above was combined with a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4) of a nickel affinity column (Bio-Scale Mini IMAC Profinity, Bio Rad). ), And then washed well with a binding buffer and eluted with an elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 300 mM imidazole, pH 7.4). Elution was performed using a peristaltic pump (SJ-1211L, ATTO) at a flow rate of 0.5 ml / min. The absorbance of the eluate was monitored at an absorbance wavelength of 220 nm using an ultraviolet absorbance monitor (AC-5100L, ATTO) and recorded with a recorder (R-01A, RIKADENKI). As shown in FIG. 1, the SDS-PAGE image of the eluate was confirmed to have a plurality of bands with the His-Tag-attached GST fusion protein as the main band. Add 2 ml of the His-Tag GST fusion protein eluate so that the concentration is 1 mM, mix, and place in a dialysis membrane (UC30-32-100, Sanko Junyaku Co., Ltd.) with a molecular weight of 13 kDa. The mixture was dialyzed with 2 L of 150 mM NaCl, 1 mM EDTA and 50 mM Tris-HCl (pH 7.5) for about 6 hours. After dialysis, the His-Tag-attached GST fusion protein eluate is quantified with DC Protein Assay (Bio-Rad), and 1 μl of PreScission Protease (GE Healthcare Bioscience) is added to 200 μg of protein. After mixing, the mixture was reacted at 4 ° C for 6 hours or more. In the SDS-PAGE image after the enzyme cleavage, a recombinant α ₁ -m of the cleaved GST and the cat was confirmed. Further, this solution was used as a sample for high performance liquid chromatography (HPLC).

(7) HPLC method
HPLC system includes system controller (SCL-10A VP, Shimadzu), liquid feeding unit (LC-10AD VP, Shimadzu), ultraviolet partial altimeter (SPD-10A VP, Shimadzu), column oven (CTO-10A VP, Shimadzu) And a degassing unit (DGU-14A, Shimadzu), and MightysilRP-18 GP250-4.6 (Kanto Chemical) was used as the column. The HPLC separation conditions were as follows. The column was equilibrated with a 0.1% trifluoroacetic acid (TFA) solution with a mobile phase flow rate of 1 ml / min and a sample addition amount of 400 μl. An 80% liner gradient was used. The eluate was monitored for absorbance at an absorption wavelength of 220 nm, the detected peak was collected, centrifuged for 1 hour in a concentration centrifuge (CC-181, TOMY), and then freeze-dried (FDU-540, EYELA) and stored at -20 ° C. The chromatographic pattern was as shown in FIG. 2, and was separated into roughly 8 peaks, and the protein composition of each eluted fraction was analyzed by SDS-PAGE. As a result of analysis, it was eluted in the f fraction alone. A monoclonal antibody was prepared using this protein as an antigen of recombinant cat α ₁ -m (rFeα ₁ -m).

<Reference Example 2: Production of antibody-producing hybridoma and anti-rFeα ₁ -m antibody>
In preparing a monoclonal antibody using the protein synthesized in Reference Example 1 as an antigen of a recombinant cat α ₁ -m (rFeα ₁ -m), an antibody-producing hybridoma was first prepared.

(1) Production of antibody-producing hybridoma (1-1) Immunization The immunization was carried out by injecting purified rFeα ₁ -m as an antigen subcutaneously into the hind limbs (footpad) of Balb / c mice. Immunization is performed 4 times at 5-day intervals, and from the first to the third immunization, 100 μl (1 mg / ml) of antigen solution and 200 μl of antigen solution (50 μg / foot) emulsified by mixing an equal amount of adjuvant In the final immunization, only 20 μl (10 μg / foot) of the antigen solution was used. Adjuvant Complete Freund (Wako Pure Chemical Industries, Ltd.) was used as the adjuvant for the first immunization, and Adjuvant Incomplete Freund (Wako Pure Chemical Industries, Ltd.) was used for the second to third immunizations.

(1-2) Cell fusion Three days after the final immunization, popliteal lymph nodes were removed, lymphocytes were collected, and then cell fusion was performed using GenomONE-CF (Ishihara Sangyo Co., Ltd.). As myeloma cells, P3X63-Ag8.653 (Dainippon Sumitomo Pharma Co., Ltd.) was used. The fusion method was performed according to the attached protocol. Specifically, first, lymphocytes and myeloma cells were mixed at a cell number ratio of 5: 1, centrifuged at 1000 rpm at 4 ° C. for 5 minutes, and then the supernatant was removed. Thereto was added 1 ml of ice-cold fusion buffer per 10 ⁸ cells of lymphocytes and suspended uniformly, and then 25 μl of ice-cooled HVJ-Envelope suspension was added per ml of cell mixture. The cell suspension was allowed to stand on ice for 5 minutes, then centrifuged at 1000 rpm and 4 ° C. for 5 minutes, and incubated at 37 ° C. for 15 minutes with the cells pelleted without removing the supernatant.

After completion of the incubation, 50 ml of a growth medium heated to 37 ° C. was added per 10 ⁸ lymphocytes, suspended, and then seeded in a 96-well plate (96 Well Cell Culture Plate: Greiner bio-one) at 100 μl / well. As growth medium, RPMI1640 (Invitrogen), penicillin G (PG; Meiji Pharmaceutical Co., Ltd.) 100,000 IU / ml, streptomycin (SM; Meiji Pharmaceutical Co., Ltd.) 100 mg / ml, 7.5% Bri Clone (IL-6, human) No. BR-001, Dainippon Sumitomo Pharma Co., Ltd.), 10% inactivated fetal bovine serum (FBS; Nichirei Co., Ltd.) added, and gently added and suspended. Operated. After culturing for 24 hours, the culture medium was replaced with a HAT medium in which 2% HAT (Invitrogen) was added to the above growth medium.

(2) Screening for antibody-producing hybridomas The resulting hybridomas were subjected to a primary screening using an ELISA method one week after cell fusion. As a result, only those hybridomas in which the reaction was positive were detected using Western blotting. Confirmed by subsequent screening.

(2-1) Primary screening
Primary screening of antibody-producing hybridomas was performed using an ELISA method using rFeα ₁ -m as an antigen. As an ELISA plate, 96 well ELISA Microplate (Greiner bio-one) was used. In addition, an automatic washer (Auto Mini Washer AMW-8, Biotech Co., Ltd.) was used to wash the plate, and PBS (1.37M NaCl, 27 mM KCl, 100 mM Na ₂ HPO ₄ , 18 mM KH ₂ PO ₄ , pH 7.4, 25 ° C.) was used. As a solid phase, rFeα ₁ -m adjusted to 3 μg / ml with PBS was added to the plate at 50 μl / well and reacted overnight at 4 ° C. After the solid-phase reaction was completed, the antigen solution on the plate was discarded, and PBS supplemented with 0.5% Bovine Serum Albumin (BSA; Wako Pure Chemical Industries, Ltd.) was added as a blocking solution at 150 μl / well and reacted at 37 ° C. for 60 minutes. . After completion of the blocking reaction, the plate was washed once, and the culture supernatant of each hybridoma culture was added at 50 μl / well as a primary antibody, and reacted at 37 ° C. for 60 minutes. After completion of the primary antibody reaction, the plate was washed once, and a peroxidase-labeled anti-mouse IgG antibody (SIGMA-ALDRICH) diluted 1000-fold with PBS supplemented with 0.1% BSA as a secondary antibody was added at 50 μl / well at 37 ° C. For 60 minutes. After completion of the secondary antibody reaction, the plate was washed 3 times, and PBS containing 0.04% o-phenylenediamine and 0.04% H ₂ O ₂ was added as a substrate solution at 150 μl / well, and room temperature was kept for 30 to 60 minutes under light shielding. Reacted. After completion of the substrate reaction, 3M H ₂ SO ₄ was added as a reaction stop solution at 50 μl / well, shaken for 1 minute, and the absorbance at a wavelength of 490 nm was measured with a Microplate Reader (Model 550, BIO-RAD). The positive well cells having high absorbance were transferred to a 24-well plate (24 Well Cell Culture Plate; Greiner bio-one) and cultured.

(2-2) Secondary screening
This was confirmed by Western blotting using rFeα ₁ -m as an antigen, and secondary screening of antibody-producing hybridomas was performed. Based on Lowry's method, the protein was quantified by measuring the absorbance at a wavelength of 655 nm with a Microplate Reader using a DC Protein Assay Kit (BIO-RAD). A calibration curve was prepared using BSA. The Western blotting method was performed as follows based on the method of Towbin et al. A polyvinylidene difluoride (PVDF) film (BIO-RAD) was used as the transfer film. The PVDF membrane was infiltrated with 100% methanol for 10 seconds and further with a transfer electrode buffer (25 mM Tris-HCl (pH 8.3, 20 ° C.), 192 mM glycine, 5% methanol) for 30 minutes and subjected to electrophoresis. In assembling the transfer device, a filter paper (BIO-RAD), a PVDF membrane, a gel after completion of SDS-PAGE, and a filter paper were layered in order from the bottom on the anode electrode plate, and the cathode electrode plate was fixed thereon. The filter paper was previously immersed in the electrode buffer for 2 to 3 minutes. The transfer condition was a constant current of 1.9 mA / cm ² for 60 minutes. After the transfer, the PVDF membrane was subjected to blocking operation by adding 0.5% BSA to 10 mM Tris-HCl (pH 7.5, 20 ° C.), 140 mM NaCl, 0.01% Tween 20 (TBST) and shaking at room temperature for 60 minutes. After the blocking, the cells were shaken and washed twice with TBST for 5 minutes, and the cell culture supernatant was used as the primary antibody, and the reaction was shaken at room temperature for 90 minutes. After completion of the primary antibody reaction, the mixture was washed twice with TBST for 5 minutes, and then peroxidase-labeled anti-mouse IgG antibody diluted 1000 times with TBST was shaken at room temperature for 60 minutes. After completion of the secondary antibody reaction, shake and wash twice with TBST for 5 minutes and use 0.06% 3,3-diaminobenzidine tetra-hydrochloride, 0.03% H ₂ O ₂ and 50 mM Tris-HCl (pH 7.6, 20 ° C.) as a substrate. It was used as a reaction solution and allowed to react for 1 to 5 minutes. After completion of the substrate reaction, the reaction was stopped by washing with water, and then dried and stored. Hybridomas that showed positive reaction were cloned by the limiting dilution method described later.

(3) Cloning The limiting dilution method was used for the cloning of the hybridoma. Specifically, the hybridoma after screening was diluted with HAT medium so as to be 2 cells / 100 μl, and seeded in a 96-well plate at 100 μl / well. When hybridomas were semiconfluent, they were expanded in a 24-well plate and cultured until they became semiconfluent again, and then confirmed by Western blotting using rFeα ₁ -m as an antigen as in the secondary screening. This cloning operation was performed twice. In addition, in order to prevent the antibody-producing ability from being reduced by subculturing the hybridoma for a long period of time, it was stored using a cell cryopreservation solution (Cell Banker (BLC-1), Toji Field Co., Ltd.) for each cloning. .

(4) Mass culture of antibody-producing hybridoma and collection and purification of anti-rFeα ₁ -m · mAb A large amount of the cloned hybridoma using a floating cell culture flask (filter top SC flask 250 ml 75 cm ² ; Greiner bio-one) Cultured. The culture was performed at 37 ° C., 5% CO ₂ for 5 days in a CO ₂ incubator (Toji Field Co., Ltd.), and HAT medium was used as the medium. The hybridoma cultured in large volume was suspended in serum-free RPMI and administered into the abdominal cavity of nude mice (Balb / c-nu) at 2 × 10 ⁷ cells / head. Ascites was collected 10-20 days after administration. Ascites collected from nude mice was allowed to stand at room temperature for 1 hour or at 4 ° C. overnight and then centrifuged at 3000 rpm and 4 ° C. for 5 minutes to remove fibrin, hybridoma, erythrocytes, etc. in ascites. The separated supernatant was salted out with 50% ammonium sulfate. Specifically, a saturated ammonium sulfate solution equivalent to the supernatant was gradually added dropwise with stirring on ice, and the mixture was further stirred for 1 hour. This was centrifuged at 10,000 rpm at 4 ° C. for 10 minutes, and the precipitate was dissolved in 20 mM sodium phosphate buffer (pH 7.0). The salted-out globulin solution was desalted using a Sephadex G-25 Fine (GE Healthcare Bioscience) column (inner diameter 1.5 cm, length 30 cm) equilibrated with 20 mM sodium phosphate buffer (pH 7.0). . The chromatography flow rate was adjusted to 0.5 ml / min with a peristaltic pump (SJ-1211L, ATTO). The desalted globulin solution was purified by affinity chromatography using Protein G Sepharose 4 Fast Flow (GE Healthcare Bioscience) packed in an Eco column (inner diameter 2.5 cm, length 10.0 cm: BIO-RAD). Specifically, the desalted globulin solution was added to a column equilibrated with 20 mM sodium phosphate buffer (pH 7.0) at a flow rate of 0.5 ml / min, and then the column was eluted with 100 mM glycine (pH 3.0). I let you. The eluate was immediately neutralized with 1/10 volume of 1M Tris-HCl (pH 9.0). The purified eluate was desalted with a Sephadex G-25 Fine column (inner diameter 2 cm, length 30 cm) equilibrated with 50 mM ammonium acetate (pH 7.0), and then Freeze Dryer (FDU540, EEYLA Tokyo Rika Instrument Co., Ltd.) Company) was lyophilized and stored at -20 ° C.

<Examples 1 and 2>
Table 1 shows combinations of the antibodies of Examples 1 and 2 used in the experimental examples described later.

In Table _1, α 1 -m mAb3 producing antibodies, cell lines _{Mouse-Mouse hybridoma α 1 -m mAb3} ( accession number: FERM P-22051) refers to an antibody produced by, α ₁ -m mAb4 produce antibodies, It refers to the antibody produced by the cell line Mouse-Mouse hybridoma α ₁ -mmAb4 (Accession Number: FERM P-22052 ).

<Experimental example>
Examples 1 and 2 were evaluated by enzyme immunoassay (sandwich method). Specifically, α ₁ -m in the urine of _one healthy cat was measured. Specifically, after capture antibody was solid-phased at 0.5 mg / well and blocked, urine of one healthy cat was reacted for 2 hours. After washing, the biotin-labeled primary antibody was reacted for 2 hours, then avidin peroxidase was reacted for 1 hour, substrate reaction was performed using tetramethylbenzidine, and the absorbance at 450 nm was measured. FIG. 3 is a graph showing the results, and the vertical axis represents the absorbance. As is clear from FIG. 3, the absorbance by the combination of the antibodies of Examples 1 and 2 was sufficiently high. From this result, the combination of antibodies of the present invention are shown to exhibit high avidity than against feline alpha ₁ -m, it showed that it is possible to measure the sensitivity of the cat alpha ₁ -m .

  The embodiments and experimental examples disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

Claims (6)

Cell lines _{Mouse-Mouse hybridoma α 1 -m mAb3} ( accession number: FERM P-22051) and the first antibody produced by the cell line _{Mouse-Mouse hybridoma α 1 -m mAb4} ( accession number: FERM P-22052) using a second antibody produced by the enzyme immunoassay (sandwich method), fluorescence immunoassay (sandwich method), radioisotope immunoassay (sandwich method), turbidimetric immunoassay and latex immunoturbidimetric A method of measuring a feline-derived α1 microglobulin concentration in a specimen by any method selected from the group consisting of turbidity methods. Cell lines _{Mouse-Mouse hybridoma α 1 -m mAb3} ( accession number: FERM P-22051) and the first antibody produced by the cell line _{Mouse-Mouse hybridoma α 1 -m mAb4} ( accession number: FERM P-22052) A kit for determining the concentration of cat-derived α1 microglobulin, comprising the second antibody produced by the method. Cell line Mouse-Mouse hybridoma α ₁ -m mAb3 ( Accession number: FERM P-22051 ). Cell line Mouse-Mouse hybridoma α ₁ -m mAb4 ( Accession number: FERM P-22052 ). Specific to the cat-derived α1 microglobulin produced by the cell line Mouse-Mouse hybridoma α ₁ -mmAb3 ( Accession Number: FERM P-22051 ) using the protein having the amino acid sequence represented by SEQ ID NO: 1 as an antigen Binding antibody. Specific to the cat-derived α1 microglobulin produced by the cell line Mouse-Mouse hybridoma α ₁ -mmAb4 ( Accession Number: FERM P-22052 ) using the protein having the amino acid sequence represented by SEQ ID NO: 1 as an antigen Binding antibody.