JP2008094721A - Antibody and ligand capturing reagent using the same antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody useful for capturing a biotin-labeled substance used for immunity measurement etc. <P>SOLUTION: A spacer comprising a spacer moiety which is between a biotin moiety and an active group and contains a longer peptide bond than a conventionally used compound is used and bound to N-hydroxysuccinimide through an ester linkage. The resultant ester is bound to a carrier protein and used as an immunogen to carry out immunization. Thereby, the resultant hybridoma produces a monoclonal antibody having high reactivity to the antibody in which the biotin is bound and low reactivity to free biotin. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention is based on a biological specific reaction such as an antigen-antibody reaction, DNA-DNA, DNA-RNA, RNA-RNA hybridization, etc., and a method for measuring a substance involved in the specific reaction and a ligand capture agent used in the measurement method And the scavenger.

In the solid phase immunoassay method, depending on the nucleic acid, antigen or antibody to be measured, an immobilized probe corresponding to the corresponding nucleic acid, an immobilized antibody solid phase corresponding to the corresponding antigen or immobilization to the corresponding antibody Conventionally, a method of preparing an antigen solid phase and selecting the solid phase corresponding to the target substance for each measurement has been common. In such a method, the number of types of solid phase required for each substance to be measured is innumerable, so the preparation of the solid phase is extremely complicated, and the measurement target is irrespective of the mechanical operation or the method used. The complication of having to select for each appropriate solid phase depending on the substance was overlaid.

Attempts have already been made to solve the above problems. For example, using one type of ligand capture agent-immobilized solid phase, selecting a ligand-labeled physiologically active substance according to the substance to be measured, and ligand-labeled physiologically active substance required for measurement on the ligand capture agent-immobilized solid phase That is, a method based on capturing a ligand-labeled probe, antigen or antibody on a solid phase is known. Also, prepare the necessary solid phase, or add the target nucleic acid, antigen, or target antibody and ligand-labeled antibody or ligand-labeled antigen in the sample onto the ligand-capturing agent-immobilized solid phase and capture them. Thus, a method that enables measurement of an arbitrary measurement target substance that is measured immunologically is also known. (For example, refer to Patent Document 1 and Patent Document 2) There is also known a method for obtaining measurement sensitivity at the level of nanogram per ml in an extremely short time. (For example, refer to Patent Document 3) Further, the ligand capture agent and the physiologically active substance are immobilized on the same first binding particle carrier to cause an immunological reaction with the detection reagent, and then immobilized on the second carrier. A method is also known in which the particle carrier for binding itself is captured by the ligand thus made to be more sensitive than conventional immunochromatography. (For example, see Patent Document 4)

In addition, polyclonal antibodies and monoclonal antibodies are known for antibodies against biotin. (For example, in the case of a monoclonal antibody, see Non-Patent Document 1 and Non-Patent Document 2. In these documents, an immunogen was prepared by binding biotin-N-hydroxysuccinimide ester to bovine serum albumin or keyhole limpet hemocyanin. Avidin, streptavidin, etc. are known as those having a binding property to biotin, and in particular, the complex of avidin and biotin is extremely stable and its dissociation constant is as high as 10 ⁻¹⁵ M. It has been known.
Japanese Examined Patent Publication No. 4-49657 Japanese Patent Laid-Open No. 2-145967 JP 2001-235471 A JP 2004-219241 A Dakshinamurti et al. Biochemical Journal 1986, 237, p477 Methods in Enzymology 1997, 279, p451

When biotin is used for ligand labeling in immunoassays that require high sensitivity, sufficient sensitivity for measurement cannot be obtained even if avidin or streptavidin that has a high binding property to biotin is used as the ligand capture agent. There was a case. Even with a commercially available monoclonal antibody against biotin, sufficient sensitivity cannot be obtained, and acquisition of an antibody having high reactivity as a ligand capture agent has been desired.

As a result of diligent research to solve the above-mentioned problems, the present inventors have found that the compound described in Formula I is longer than the compound in which the spacer portion between the biotin moiety and the active group is conventionally used as a compound to be bound to the carrier protein. Hybridomas were prepared by immunization using the bound carrier protein as an immunogen, resulting in the production of hybridomas that produce monoclonal antibodies with high reactivity to antibodies bound to biotin and low reactivity to free biotin. The invention has been reached.

That is, the present invention relates to the following (1) to (6).
(1) As an immunogen, chemical formula I

Antibody (2), wherein the antibody described in (1) is obtained by using a carrier protein to which the compound described above is bound Monoclonal antibody having the following properties (A) Isoelectric point 6.4-6. 8
(B) Isotype IgG1 κ
(C) Reactive to a carrier protein labeled with biotin (3) A hybridoma deposited as deposit number FERM P-20979 at the National Institute of Advanced Industrial Science and Technology (AIST) Ligand capture reagent using the antibody described in (4) (1) (5) The ligand capture reagent characterized in that the antibody described in (4) is a monoclonal antibody having the following properties: (A) Isoelectric point 6.4-6.8
(B) Isotype IgG1 κ
(C) Reactive to biotin-labeled carrier protein (6) Obtained using a hybridoma deposited under the accession number FERM P-20979 at the National Institute of Advanced Industrial Science and Technology (AIST) Ligand capture reagent characterized by using antibody and antibody fragment thereof

The monoclonal antibody produced by the hybridoma obtained by the method for obtaining a hybridoma according to the present invention has a high capture ability for a substance labeled with biotin. This property is based on biologically specific reactions, and is useful for measuring substances involved in specific reactions and ligand capture agents used in the measurement methods. I think it is possible to be sensitive.

The antibody of the present invention has chemical formula I as an immunogen.

The immunized animal can be immunized by a known method using an antigen in which a compound (biotinylation reagent) having a spacer moiety between the biotin moiety and the active group shown in FIG. In the case of a monoclonal antibody, immune cells are obtained by a known method after immunization, and then cell fusion with myeloma is performed by a known method, and fused cells and non-fused cells are selected by a known method. It is obtained from a hybridoma in which screening / cloning of cells producing the antibody is performed. The antibody may be either IgM or IgG, but it should be used to reduce the molecular weight using known proteolytic enzymes or gene recombination techniques, for example, Fab fragment, F (ab ') 2 fragment, single chain Fv fragment, etc. You can also. If it is a polyclonal antibody, it can be obtained by obtaining antiserum by a known method after immunization and purifying it if necessary.
The carrier protein used for immunogen preparation is not particularly limited as long as it has a functional group capable of reacting with a biotinylation reagent and has immunogenicity, but is preferably a high-purity product or one that can be highly purified by purification, such as bovine serum albumin (Bovine Serum Albamin, hereinafter abbreviated as BSA), keyhole limpet hemocyanin (hereinafter, abbreviated as KLH), goat immunoglobulin G, and the like can be used.
The biotinylation reagent has chemical formula I

Commercially available biotinylation reagents shown in (1) can be used. Chemical formula II having a succinimidyl group having no sulfonic acid group instead of a sulfosuccinimidyl group that reacts with an amino group of a carrier protein

It is considered that a similar effect can be obtained even in the case of the compound represented by the above, since the structure bonded to the carrier protein does not change. The antigen biotinylation procedure can be biotinylated by a known method. For example, a carrier protein dialyzed in advance with 50 mM carbonate buffer pH 8 is prepared, and after dissolving the biotinylation reagent in a suitable solvent, the carrier protein molar ratio is determined to be excessive. Add, for example, 20 times the amount to the carrier protein solution, stir and react at 25 ° C. for 2 hours, and then remove the unreacted biotinylated reagent by dialysis against 10 mM phosphate physiological buffer to obtain a biotinylated antigen. be able to. A commercially available biotin labeling kit can also be used for antigen biotinylation. The spacer distance between biotin and amino group, which is different from the conventional technology, is considered to be important for antigen presentation to immune cells. Even if it is a compound having a similar structure with maleimide group or hydrazide group, it is a carrier. If it is combined with a protein and used for immunization, the same effect as in the present invention can be obtained.

As the immunized animal used for animal immunization, mammals used in known antibody production methods can be used. Specific examples include mice, rats, goats, sheep, cows and horses. However, from the viewpoint of easy availability of myeloma cells to be fused with the extracted antibody-producing cells, it is preferable to use mice and rats as immunized animals. In addition, although mouse and rat strains to be actually used are not particularly limited, for example, BALB / c female 4 to 12 weeks old can be used.

Animal immunization can be carried out by administering the above-described immunogen intradermally, intraperitoneally, intramuscularly or intramuscularly. Although the administration schedule varies depending on the type of immunized animal and individual differences, in general, the number of antigen administrations is 2 to 6 times, and the administration interval is preferably 1 to 2 weeks. The dose of antigen varies depending on the kind of animal and individual differences. Generally, it is said to be about 10-100 μg / animal / dose, but immunization can be performed by changing the dose, and the immunized animal having the highest antibody titer in serum or plasma can be selected. When administered, it may be administered together with an immunostimulatory substance called an adjuvant. Examples of adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, CpG DNA, muramyl dipeptide, lipopolysaccharide and the like.

The monoclonal antibodies and hybridomas of the present invention are immunized as described above, and then known monoclonal antibody production methods, for example, monoclonal antibody experiment manuals Tomita Toni, Ando Tamie / Edition Kodansha Scientific 1987, Immunology Research Handbook, Fujiwara Univ. Beauty, Shinji Sakurai / hen Chugai Medical Co., Ltd. 1996, tissue culture technology [3rd edition] application edition The Japanese Society for Tissue Culture / hen Asakura Shoten 1999 can be prepared according to the description.

In the case of in vivo immunization, in the case of in vivo immunization, spleen cells or lymphocytes containing antibody-producing cells are aseptically removed from the immunized animal 1 to 5 days after the last immunization day of the antigen administration schedule. Separation of antibody-producing cells from these spleen cells or lymphocytes can be performed according to a known method.

The above antibody-producing cells and myeloma cells are used for cell fusion. The myeloma cells are not particularly limited and can be appropriately selected from known cell lines. However, in consideration of convenience when selecting hybridomas from the fused cells, it is preferable to use a strain lacking HGPRT (Hypoxanthine-guanine phosphoryltransferase) in which the selection procedure has been established. That is, X63-Ag8 (X63), NS1-Ag4 / 1 (NS-1), P3X63-Ag8. U1 (P3U1), X63-Ag8.653 (X63.653), SP2 / 0-Ag14 (SP2 / 0), F0, and the like.

Fusion of antibody-producing cells and myeloma cells can be appropriately performed according to a known method under conditions that do not extremely reduce cell viability. As such a method, for example, a chemical method of mixing antibody-producing cells and myeloma cells in a high-concentration polymer solution such as polyethylene glycol, a physical method using electrical stimulation, or the like can be used.

The selection of fused cells and non-fused cells is preferably performed by, for example, a known HAT (hypoxanthine / aminopterin / thymidine) selection method. As a medium used for cell culture such as HAT medium or cloning described later, a known medium may be used, and for example, RPMI1640, DMEM, eRDF, IMDM and the like can be used. At the same time, protein such as animal serum, growth factor, conditioned medium, antibiotics, etc. may be added, but when cloning is performed by limiting dilution, it is preferable to add these in combination. In the case of animal serum, for example, fetal bovine serum may be added in an amount of 0.1 to 20%. Sera with a pre-reduced IgG content may be used. For example, IL-6, insulin, ethanolamine, selenium, 2mercaptoethanol, pyruvic acid, and non-essential amino acids may be added for growth factors. For condition medium, for example, on culture after thymocyte culture. For example, gentamicin, kanamycin, streptomycin, penicillin may be added for antibiotics, and bovine serum albumin, casein, transferrin may be added for proteins. May be. The temperature for culturing the cells may be any temperature at which the cells grow, but can be performed at 37 ° C., for example. When sodium hydrogen carbonate is added to the medium, it is preferably cultured in the presence of carbon dioxide gas, for example, at 5% to 10% carbon dioxide gas at which the pH in the medium becomes neutral.

Screening of the hybridoma cells producing the target monoclonal antibody can be performed by a known antibody screening method, for example, an enzyme immunoassay (EIA), an agglutination method, a cell sorter or the like. In the case of EIA, biotin is immobilized by detecting the presence or absence of an antibody having biotin-binding activity contained in a cell culture supernatant obtained using a carrier on which biotin is immobilized. The presence or absence of an antibody having biotin-binding activity is determined by the presence or absence of an aggregation-inhibited image observed by reacting biotinylated sheep blood cells obtained by reacting particles such as sheep erythrocytes with a biotinylation reagent and a cell culture solution on a round bottom plate. By detecting, if a cell sorter is used, cells that produce a desired antibody can be screened by mixing a fluorescently labeled biotin reagent with a hybridoma and discriminating and separating the cells based on the presence or absence of fluorescence on the cell surface.

Hybridoma cells selected by screening are cloned by a known method such as methylcellulose method, soft agarose method, limiting dilution method, and used for antibody production.

The hybridoma cells obtained by the method as described above can be stored in a frozen state in liquid nitrogen or in a freezer at -80 ° C or lower. The cell concentration at the time of cell freezing is preferably in the range of 1 × 10 ⁶ / ml to 1 × 10 ⁷ / ml, and 5 to 10% (v / v) dimethyl sulfoxide may be added to the medium as a stabilizer at the time of freezing. You may cryopreserve using a cell cryopreservation agent.

The monoclonal antibody of the present invention can be obtained by culturing and purifying the hybridoma cells produced by the above-described method using known methods, for example, mass-culture of animal cells and production of useful substances, using the method described by Michio Oishi CM 1986. The monoclonal antibody can be obtained. For example, in the case of a hybridoma in which BALB / cA mice are immunized and fused with BALB / cA-derived myeloma, monoclonal antibodies can be obtained in 1 to 3 weeks after transplanting the cells into the abdominal cavity of the same strain mice previously administered with mineral oil such as pristane. Ascites containing is obtained. For example, when a hybridoma is cultured in a medium, a monoclonal antibody is secreted from the hybridoma, and a culture supernatant containing the antibody is obtained. As necessary, ascites and culture supernatant containing the antibody can be obtained with a higher purity monoclonal antibody by a known antibody purification technique. When a commercially available serum-free medium or serum-free protein-free medium that does not contain serum is used as the culture medium, a monoclonal antibody with higher purity can be obtained. If necessary, known antibiotic / antifungal agents, carbohydrates, lipids and amino acids can be added to the medium. If it is an antibiotic / antifungal agent, for example, gentamicin sulfate, penicillin, streptomycin and amphotericin B can be added to the extent that they do not affect cell growth. For example, glucose, maltose, and xylose can be added as long as they are carbohydrates. If it is an amino acid, for example, L-glutamine can be added. L-glutamine is unstable in the culture medium and can be added up to about 8 mM. However, L-glycyl-L-glutamine and L-alanyl-L-glutamine which are stable glutamine derivatives in the solution can be added. May be. As a result, the hybridoma culture environment can be improved, and the amount of antibody secretion in the culture medium can be increased. When cell culture antibody production is performed by applying a medium different from that used when preparing the hybridoma, it is preferable to use a hybridoma previously conditioned to the same medium. For example, the hybridoma is directly suspended in the same medium, the cells and the medium are separated by centrifugation, and then resuspended to a relatively high cell concentration of 0.5 to 1 × 10 ⁶ / ml in the same medium, doubling rate and survival rate. Repeat the culture while observing cell conditions. If the medium cannot be acclimatized directly, it can be carried out while gradually increasing the mixing ratio from the conventional medium.

The antibody of the present invention is obtained by obtaining a gene or gene fragment encoding the antibody expressed in the above hybridoma by a known technique and culturing a transformant in which an expression vector is incorporated by a known gene recombination technique. Desired antibodies can be obtained. When the antibody is accumulated in the transformant by antibody production, a disrupted solution containing the antibody can be obtained by recovering and disrupting the transformant by a known method. When the antibody is secreted into the medium in which the transformant is cultured by antibody production, a culture supernatant containing the antibody is obtained by transformant culture. If necessary, a highly purified antibody can be obtained from the disrupted solution or culture supernatant containing the antibody by a known antibody purification method.
In addition, the antibody of the present invention is obtained from a gene or gene fragment encoding the antibody expressed in the above hybridoma by a known technique, and a messenger RNA encoding a protein portion having binding activity from the antibody gene or gene fragment is known. The antibody can also be produced by a known cell-free protein synthesis method. A protein synthesis reaction solution containing the antibody is obtained by antibody production. If necessary, a protein synthesis reaction solution containing the antibody can obtain a higher purity antibody by a known antibody purification method.

Examples of antibody purification methods include ammonium sulfate salting out, UF membrane concentration, and column chromatography. Examples of resin carriers that can be used in column chromatography include anion exchange resins, protein A, protein G, Protein L, affinity chromatographic resins to which biotin compounds are bound, hydroxyapatite resins, hydrophobic chromatographic resins, and the like, and crosslinks. Examples thereof include gel filtration carriers that utilize differences in intra-resin mobility depending on molecular weight, such as dextran resin and agarose resin. In particular, when using affinity chromatography resin, column washing operation in the process from antibody adsorption to antibody elution is important, and it is desirable to pass a buffer solution at least 5 times the column size. More preferably, it is desirable to pass 10 to 20 times. Before performing these purification operations, it is desirable to examine the subtype of the monoclonal antibody and select an appropriate purification means. The purified antibody is preferably buffer-exchanged with a pH-neutral buffer by a method such as dialysis. As the pH-neutral buffer, for example, a physiological saline phosphate buffer (PBS (-)) or physiological Tris buffer (TBS) at a normal salt concentration can be used. The pH of this buffer is preferably between 5.5 and 8.5.

The monoclonal antibody thus obtained can be stored in a solution state or a frozen state. When stored in liquid form, it is preferable to add a preservative or store it in a sterile container after aseptic filtration with a 0.22 μm mesh sterile membrane filter. For example, 0.05% sodium azide can be added as a preservative. The container may be sterilized by a known method, and examples thereof include γ-ray irradiation sterilization, UV irradiation sterilization, autoclave sterilization, and ethylene oxide gas sterilization. As the storage container, an inert material that does not react with the antibody during storage can be used, and examples thereof include glass, polyethylene, and polypropylene. If the method does not impair the binding activity of the antibody, a container that has been surface-treated so as not to be adsorbed on the surface of the storage container may be used. The liquid storage temperature may be any temperature that does not freeze, but it is preferably as low as possible to suppress protein denaturation, and more preferably 2 to 10 ° C. In addition, when an antifreezing agent is added at a concentration capable of preventing freezing, it can be stored at a lower temperature. For example, when glycerol having a final concentration of 50% is added, it can be stored in a liquid state without being frozen even at −20 ° C. . When storing in a frozen state, it is preferable to store at -20 ° C or lower, more preferably at -30 ° C or lower. The antibody concentration during storage can be set to a concentration that does not cause precipitation, but is preferably 0.1 to 5 mg / ml. Proteins, water-soluble polymer polymers, surfactants, saccharides and sugar alcohols can also be added as stabilizers. Examples of stabilizing proteins include bovine serum albumin and gelatin, which can be added at a concentration that does not cause precipitation during storage. The concentration range is more preferably 0.1 to 5 mg / ml.

The isoelectric point of the monoclonal antibody can be analyzed by a known analysis means, for example, polyacrylamide gel isoelectric focusing. The isotype of the monoclonal antibody can be analyzed by a known analysis means. For example, in the case of a mouse, the binding property of an antibody that specifically binds to each of the IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, kappa chain, and lambda chain isotypes. It can be confirmed by the presence or absence, and a commercially available antibody isotyping kit may be used.

The ligand capture reagent of the present invention takes a form in which the antibody of the present invention is bound to a carrier. However, the ligand capture reagent may be either bound in advance to the carrier or bound to the carrier after capturing the ligand. A known material can be used as the carrier, and examples thereof include polystyrene, glass, silica, cellulose, polyvinyl, nylon, nitrocellulose, PVDF, magnetite, gold, platinum, crystal, lipid, protein, and latex. The antibody immobilization method can be applied as long as the desorption of the once immobilized antibody is at a level that does not cause a problem in use, and a known method can be applied. The buffer solution used for the antibody solution used for immobilization is not particularly limited, but can be used as long as the antibody is not inactivated in the preparation process and does not inhibit the immobilization reaction. If the concentration is in the range of 3 to 10, a concentration in the range of 1 mM to 200 mM can be used. For example, in the case of a polystyrene Eliser plate, an antibody solution diluted to an appropriate concentration with Dulbecco's phosphate buffer (PBS (-)), phosphate buffer pH 7, and carbonate buffer pH 9.6 at a temperature range of 2 to 40 ° C. It can be immobilized by contact with a solid phase for 30 minutes to overnight. For example, when immobilized on glass fiber, an antibody solution diluted to an appropriate concentration with a citrate buffer pH 3 is used for 0.5 minutes to overnight, more preferably 5 minutes to 30 in the range of 2 to 40 ° C. It can be immobilized by contacting with a solid phase. Alternatively, a cross-linking reagent such as glutaraldehyde or carbodiimide may be used to bind the functional group on the surface of the carrier and the antibody covalently. Alternatively, a substance capable of capturing the antibody of the present invention, for example, the second antibody, protein A, protein G, protein L, or biotinylated substance is immobilized on a carrier in advance, and captured and immobilized by its affinity. Good. The carrier shape of the ligand capture reagent is not particularly limited, but a known shape can be used, for example, 96-well ELISA plate, silica beads, polystyrene beads, membrane for immunochromatography strip, glass fiber, porous membrane, magnetic beads, latex particles, dextran Examples thereof include particles, agarose particles, gold colloids, blood cells, cells, viruses, liposomes, crystal resonators, and gold thin films. These carriers may be colored as necessary.
After binding the antibody to these carriers, a blocking step may be performed in which a solution containing other protein / surfactant, polymer / serum, etc. is brought into contact with the solid phase for the purpose of suppressing nonspecific protein adsorption. Proteins known as blocking proteins can be used as proteins used for blocking, such as bovine serum albumin, non-fat milk protein, casein, gelatin, and sericin. A 0.1 to 10% solution can be prepared and used. . As a surfactant or polymer used for blocking, a known surfactant / polymer can be used as a blocking agent, and examples thereof include sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate (Tween 20), polyvinyl alcohol, and polyvinylpyrrolidone. . These are selected according to the carrier to be used, and a 0.1 to 0.5% concentration solution can be prepared and used. As the serum used for blocking, any serum that does not contain a large amount of biotin can be used. For example, fetal bovine serum, newborn cow serum, horse serum, goat serum, rabbit serum, rat serum, guinea pig serum, pig serum, A mouse serum can be used, and a 0.5% to 100% solution can be used, and it is preferable to use a serum lot check before use to confirm that it does not affect the ligand capture ability.
The ligand capture reagent using the antibody of the present invention may be stored without being dried in a preservation solution after preparation, or may be stored in a dry state. Add ingredients selected from protein, polymer, buffer, salt, and preservative to the preservation solution as necessary to maintain the capture activity without problems in actual use when stored in the preservation solution. It is desirable to examine the storage stabilization effect of these combinations in consideration of the respective carrier used and the mode of binding with the antibody, and to set the additive substance and amount. In addition, when storing in a dry state, from known sugars, sugar alcohols, amino acids, proteins, polymer polymers, buffers, salts, so that the capture activity does not decrease by drying, and the capture performance during use is easily restored. It can be brought into contact with the stabilizing solution to which the selected component has been added and dried. Examples of the protein to be added include bovine serum albumin and casein. As sugars / sugar alcohols, known stabilizing sugars / sugar alcohols can be used, such as glucose, sucrose, sorbitol, and glycerol. As amino acids, amino acids having known stabilizing effects, such as glycine, lysine, and arginine. . These concentrations can be prepared and used in a 0.1 to 10% solution. Any drying method can be applied as long as it is a method that does not lower the ligand capturing ability by a known drying method. For example, natural drying, air drying, vacuum drying, reduced pressure drying, freeze vacuum drying, drying by infrared rays or far infrared rays, Examples include drying using high frequency waves. After drying, it is preferably stored under conditions that do not absorb moisture, and is preferably sealed and stored together with a desiccant such as silica gel.

Hereinafter, the present invention will be described in detail and specifically with reference to examples, but the present invention is not limited to these examples.
Example 1 Preparation of Anti-Biotin Monoclonal Antibody-Producing Hybridoma (1) Preparation of Biotinylated KLH to be Used as Immunogen 10 mM phosphate physiological buffer pH 7.2 (hereinafter PBS buffer solution) was prepared in a gamma-ray sterilized polypropylene sample tube with a lid. KLH (concentration 25.2 mg / ml endodoxin-free) 200 μL, 1M sodium bicarbonate solution 50 μL, autoclave sterilized distilled water (hereinafter abbreviated as sterilized water) 205 μL Mix to prepare a KLH solution. Next, 10 mg of Biotin- (AC ₅ ) ₂ -Sulfo-OSu (manufactured by Dojindo) was dissolved in 300 μL of sterilized water, and 45 μL of the rapidly dissolved solution was added to and mixed with the above KLH solution for 2 hours at 25 ° C. Reacted. In order to remove the unreacted biotin reagent, a Sephadex G25 carrier (manufactured by GE Healthcare Bioscience) was equilibrated with a sterilized PBS buffer, and a mini-column filled with 3 ml of the carrier was prepared in advance. The KLH solution after the biotinylation reaction was added to the same column, and then eluted with 1 ml of PBS buffer, and the entire eluted amount was recovered as biotinylated KLH.
(2) Immunization of mice, acquisition of antiserum, and cell fusion biotinylated KLH was diluted with physiological saline for injection to 1 mg / ml to prepare an antigen solution. 0.2 ml of the antigen solution was mixed with an equal volume of complete Freund's adjuvant (manufactured by DIFCO LABORATORIES) to make an emulsion, and 100 μL of emulsion per mouse was injected into the abdominal cavity of mice (Balb / cA, 5 weeks old, female, Claire Japan) The first immunization was performed. After 2 weeks, 4 weeks, and 7 weeks, prepare an antigen solution by diluting biotinylated KLH in a similar manner to 0.5 mg / ml, and mix incomplete adjuvant (manufactured by DIFCO LABORATORIES) and the antigen solution in equal amounts. -Emulsified and immunized again in the same manner. After 10 weeks from the initial immunization, 4 times at 1-month intervals, then 2 times at 2-week intervals, and diluted with physiological saline for injection to a concentration of 50 μg / ml of the antigen solution, 100 μl was injected into the peritoneal cavity. Four days after the final immunization, the mice were anesthetized, and mouse blood was obtained by cardiac blood sampling. After confirming blood coagulation, centrifugation was performed at 700 × g for 20 minutes, and antiserum was obtained as a supernatant, and the temperature was kept at −20 ° C. And stored frozen.
Subsequently, after dislocation of the mouse cervical spine, the spleen was aseptically removed to prepare spleen cells (2.9 × 10 ⁸ cells). On the other hand, myeloma cells Sp2 / 0-Ag14 were expanded in an e-RDF medium (Kyokuto Pharmaceutical) containing 10% (v / v) fetal calf serum at 37 ° C. under 5% carbon dioxide concentration, and then contained serum components Cell suspension in no e-RDF medium. The thus obtained Sp2 / 0-Ag14 5.7 × 10 ⁷ cells and the above spleen cells were mixed in a 50 ml centrifuge tube, centrifuged at 200 × g for 10 minutes, the supernatant was discarded, and the whole tube was tapped. The cell mass was loosened, 1 ml of 50% polyethylene glycol 1500 solution (Roche) was added and mixed with the cells in a 37 ° C. environment, and then the e-RDF medium was added in the order of 1 ml, 3 ml and 10 ml. After centrifugation at 200 × g for 10 minutes, the mixture was allowed to stand at 37 ° C. for 5 minutes, and then the supernatant was discarded. Using a Pasteur pipette, the fused cells are suspended in 5.8 ml of fetal bovine serum, 2 ml of which is 1/10 volume of selective culture medium (100 × HAT supplement (GIBCO) and 1/10 volume of CondimedH1 (Roche)), fetal bovine (E-RDF medium containing 1/10 volume of serum) was suspended in 240 ml, and aseptically dispensed into 20 wells of 20 well-coated 96-well floating cell culture plates (Sumitomo Bakelite Co., Ltd.). HAT selective culture was performed at 37 ° C. under 5% carbon dioxide. Ten days after the fusion, colonies were visually observed, and the appearance of the hybridoma was confirmed.
(3) Primary screening of antibody-producing hybridoma by ELISA: 50 μL of each well culture supernatant after selective culture is diluted with 200 μL of PBS buffer, used as a sample, and the reactivity to the ELISA plate to which biotinylated KLH is immobilized is used as an index. Screening was performed. That is, 50 μl of a biotinylated KLH solution diluted to 10 μg / ml with PBS buffer was dispensed into each well of a 96-well Eliza plate (Sumitomo Bakelite Co., Ltd.) and stirred uniformly to stand at 25 ° C. for 2 hours. Then, biotinylated KLH was immobilized on the plate. Next, the biotinylated KLH solution was removed from the ELISA plate, and after washing by repeatedly dispensing and discarding 0.3 ml of PBS buffer containing 0.05% (W / V) Tween 20 to each well, Blocking was performed by dispensing 0.3 ml of PBS buffer containing 1% (W / V) bovine serum albumin into each well and leaving at 25 ° C. for 1 hour. Next, after removing the solution and washing, 50 μL of the selective culture supernatant was dispensed and reacted with a biotinylated KLH-immobilized plate at 25 ° C. After removing the solution and washing, a peroxidase-labeled sheep anti-mouse immunoglobulin antibody (manufactured by GE Healthcare Biosciences) prepared by diluting 4000 times with PBS buffer containing 0.1% (W / V) bovine serum albumin. 50 μl was dispensed and allowed to react with the plate at 25 ° C. for 1 hour. After removing the solution and washing, a coloring solution TMB + containing tetramethylbenzidine (manufactured by Dacocytomation) was dispensed at 50 μL per well, enzymatically reacted at 25 ° C. for 10 minutes, and 50 μL of 1N sulfuric acid was dispensed to stop the reaction. Absorbance at 450 nm was measured with a plate reader. As a control, the same procedure was performed on an ELISA plate on which biotinylated KLH was not immobilized.
Coloration was confirmed in well number 0021, and strong reactivity with biotinylated KLH was observed. This well was selected as a primary screening and subsequently cloned.
(4) Cloning of hybridoma: Cloning was performed by the limiting dilution method. That is, 100 cells in each selective culture well selected by screening are aseptically sampled, and each of them is a cloning medium (e-RDF medium containing 1/10 volume of CondimedH1 (Roche) and 1/10 volume of fetal calf serum. ) 20 ml was suspended so that the final concentration was 5 / ml, and 200 μl of each 96-well floating cell culture plate with lid (manufactured by Sumitomo Bakelite) was dispensed aseptically into 96 wells. Cultivation was performed for 10 days at 37 ° C. under 5% carbon dioxide. After cloning culture in each well, 50 μl of supernatant was sampled, diluted with 200 μl of PBS buffer to make an ELISA sample, ELISA was performed in the same manner as in the primary screening, and the culture supernatant in which reactivity was observed was sampled from the well in which sampling was performed. The wells were selected and hybridoma 0021 after primary cloning was obtained. The present cell culture expanded, the Cellbanker (Ju慈field Co.) cells to a viable cell density 5x10 ⁶ cells / ml fully Ken Nigoshi, the gamma sterilized 1.8ml cryotubes (manufactured by Sumitomo Bakelite) 1 ml of the cell suspension was dispensed, and after being sealed, stored frozen in a −80 ° C. freezer. Hybridoma 0021 has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, under the accession number FERM P-20979 on August 2, 2006.

Example 2: Preparation of 0021 purified antibody (1) Preparation of 0021 culture supernatant: A tube containing the hybridoma 0021 that had been cryopreserved in liquid nitrogen was stored at −80 ° C. for 1 hour, and then quickly in 37 ° C. hot water. Thawed. The whole amount was put in a centrifuge tube containing 10 ml of e-RDF medium containing 10% (v / v) fetal calf serum, and the cells were suspended by pipetting. 150xg for 5 minutes centrifugation, after discarding the supernatant, 10% of 5ml (v / v) were suspended the cells in pipetting e-RDF medium containing fetal bovine serum, respectively in the bottom area of 25 cm ² suspension cell culture flasks The seed was seeded and cultured at 37 ° C. under 5% carbon dioxide concentration. After culturing in the same culture medium for 2 passages, the medium was changed to CD-Hybridoma Medium (Invitrogen) containing 60 μg / L gentamicin sulfate, 2 mM L-glutamine, 6 mM L-alanyl-L-glutamine, and conditioned for 3 more passages. The cells were cultured in the same medium. Thereafter, the cells are suspended in 15 ml of medium and subcultured in a flask with a bottom area of 75 cm ² and then subcultured in 45 ml of medium and subcultured in a flask with a bottom area of 225 cm ^{2 and} cultured for 7 days. Was fully secreted into the medium. The cell culture solution obtained by the culture was transferred to a 50 ml centrifuge tube and centrifuged at 700 × g for 10 minutes to obtain 43.9 ml of a hybridoma culture supernatant.
(2) Monotyping of monoclonal antibody: Using a commercially available immunochromatographic subtyping reagent (Istrip: Roche), the obtained culture supernatant was diluted 10-fold with PBS buffer, and 150 μl of diluted solution was used as a sample. analyzed. The subtype of the monoclonal antibody contained in the culture supernatant of hybridoma 0021 was IgG ₁ κ.
(3) Preparation of 0021 purified antibody: The obtained culture supernatant was dissolved and mixed by adding 7.9 g of NaCl and 2.3 ml of 1M borate buffer pH 9 and filtered through 0.8 μm filter. 1 ml of rProtein A Sepharose FastFlow (manufactured by GE Healthcare Biosciences) equilibrated with 50 mM borate buffer (pH 9: binding buffer) containing 3M NaCl in advance is packed in the column, and the total amount of the culture supernatant is 1 ml / The column was passed through in minutes. Next, the column was washed with 15 ml of binding buffer, and 0.1 M citrate buffer (pH 3) was passed at the same flow rate to elute the antibody from the column, thereby obtaining 2 ml of eluate containing the antibody. Immediately after, 0.4 ml of 1M Tris buffer (pH 9) was added, and dialysis was performed 3 times against 1 l of PBS buffer containing 0.05% (w / v) sodium azide. As a result of calculating the amount of protein by absorption at OD 280 nm, the obtained 0021 purified antibody was 5.8 mg. Next, the 0021 purified antibody was filtered through a 0.22 μm low protein adsorption membrane filter (manufactured by Nihon Millipore) sterilized by gamma ray and stored at 4 ° C. in a 15 ml tube made of gamma sterilized propylene.
(4) Gel filtration analysis of 0021 purified antibody: FPLC Director System (manufactured by GE Healthcare Bioscience), TSKGEL G3000SW (column length 30 cm, manufactured by Tosoh Corporation), eluent 50 mM phosphate buffer pH 7 containing 0.1 M sodium sulfate. Using a gel filtration analysis system with a flow rate of 0.5 ml / min using 0, the separation pattern after injection of 0021 purified antibody 1 mg / ml 0.1 ml was monitored by absorbance change at 280 nm. A pattern having an elution 16.7 minutes as the main peak was obtained, and the ratio of the main peak to the absorbance integrated value based on the baseline was 95%.
(5) Electrophoretic analysis of 0021 purified antibody: 1 μl of purified antibody 1 mg / ml was added to PastGel IEF3-9 (GE Healthcare Biosciences) using PhastSystem (GE Healthcare Biosciences). Electrophoresis was performed using a program. Simultaneously, electrophoresis was performed using pI KIT (manufactured by GE Healthcare Bioscience) as a sample. The gel after electrophoresis was stained with Coomassie to visualize the protein in the gel, and the band mobility of the purified antibody 0021 was measured from the isoelectric point and mobility of a known pI marker protein. As a result, with the 0021 purified antibody, multiple bands were observed in the range of 6.4 to 6.8 centering on pI 6.6.

Example 3: Preparation of 0021 purified antibody-immobilized ELISA plate (1) The 0021 purified antibody was diluted to 10 μg / ml with 0.1 M phosphate buffer pH 7.0 to prepare a 0021 purified antibody dilution.
(2) 50 μl of the 0021 purified antibody diluted solution was dispensed into each well of a 96-well Eliza plate (NUNC Maxi Soap), stirred uniformly and then allowed to stand at 25 ° C. for 2 hours to be immobilized on the well. Next, the liquid was removed from the ELISA plate, and 0.3 ml of PBS buffer containing 0.05% (W / V) Tween 20 was dispensed and discarded in each well three times, and then washed with 1% ( (W / V) 0.3 ml of PBS buffer solution containing bovine serum albumin was dispensed into each well and allowed to stand at 25 ° C. for 1 hour for blocking. After discarding the solution, washing was performed in the same manner, and 0.3 ml of PBS buffer containing 0.05% (W / V) sodium azide and 0.1% (W / V) bovine serum albumin was dispensed to each well. . In order to prevent the liquid from drying, the upper part of the well was sealed with an adhesive plate seal and stored at 4 ° C. The solution was discarded immediately before use, and after removing the solution remaining in the well as much as possible while hitting with a paper towel, it was used as a biotin capture reagent.

Comparative Example 1: Preparation of avidin-immobilized plate (1) Egg white-derived avidin (Nacalai Tesque) was dissolved in 0.1 M phosphate buffer pH 7.0 to 10 μg / ml to prepare a diluted avidin solution.
(2) In the procedure described in Example 1 (2), the 0021 purified antibody diluted solution was replaced with the avidin diluted solution, and the work was performed to prepare an avidin immobilized ELISA plate. In order to prevent the liquid from drying, the upper part of the well was sealed with an adhesive plate seal and stored at 4 ° C. The solution was discarded immediately before use, and after removing the solution remaining in the well as much as possible while hitting with a paper towel, it was used as a biotin capture reagent.

Comparative Example 2: Preparation of Streptavidin-immobilized Eliser Plate (1) Streptavidin (Nacalai Tesque) was dissolved in 0.1 M phosphate buffer pH 7.0 to 10 μg / ml to prepare a streptavidin dilution. .
(2) In the procedure described in Example 1 (2), the 0021 purified antibody diluted solution was replaced with the streptavidin diluted solution, and the work was performed to prepare a streptavidin-immobilized ELISA plate. In order to prevent the liquid from drying, the upper part of the well was sealed with an adhesive plate seal and stored at 4 ° C. The solution was discarded immediately before use, and after removing the solution remaining in the well as much as possible while hitting with a paper towel, it was used as a biotin capture reagent.

Comparative Example 3: Preparation of neutravidin-immobilized ELISA plate (1) Neutravidin (Pierce) was dissolved to a concentration of 10 μg / ml in 0.1 M phosphate buffer pH 7.0 to prepare a neutravidin diluted solution.
(2) In the procedure described in Example 1 (2), the 0021 purified antibody dilution was changed to the neutravidin dilution to carry out the operation to prepare a neutravidin immobilized ELISA plate. In order to prevent the liquid from drying, the upper part of the well was sealed with an adhesive plate seal and stored at 4 ° C. The solution was discarded immediately before use, and after removing the solution remaining in the well as much as possible while hitting with a paper towel, it was used as a biotin capture reagent.

Comparative Example 4: Preparation of commercially available biotin antibody-immobilized ELISA plate (1) Lyophilized anti-biotin monoclonal antibody clone 33 (Roche) was reconstituted by adding manufacturer-designated distilled water, and then 0.1 M phosphate buffer Dilute to 10 μg / ml at pH 7.0.
(2) In the procedure described in Example 1 (2), the 0021 purified antibody diluted solution was replaced with the anti-biotin monoclonal antibody diluted solution, and the work was carried out to prepare a commercially available anti-biotin antibody-immobilized ELISA plate. In order to prevent the liquid from drying, the upper part of the well was sealed with an adhesive plate seal and stored at 4 ° C. The solution was discarded immediately before use, and after removing the solution remaining in the well as much as possible while hitting with a paper towel, it was used as a biotin capture reagent.

Example 4 Comparison of Sensitivity in CEA Measurement (1) Biotinylation of anti-CEA monoclonal antibody: 1M sodium hydrogen carbonate solution was added to 0.5 ml of antibody solution containing 0.37 mg of anti-CEA monoclonal antibody clone 5909 (Medix Biochemica). 50 μl was added. Subsequently, 10 mg of Biotin-Sulfo-OSu (manufactured by Dojindo) was dissolved in 1 ml of sterilized water, and 5.5 μl of the dissolved solution was quickly added to the above antibody solution and reacted at 25 ° C. for 2 hours. To remove the unreacted biotin reagent, equilibrate the desalting column PD-10 (manufactured by GE Healthcare Biosciences) with a sterilized PBS buffer, add the entire amount of the antibody solution after the biotinylation reaction to the column, Elute with PBS buffer. The OD280 nm absorbance of the eluate was monitored, and the first eluted peak was collected as a biotinylated anti-CEA monoclonal antibody, and then concentrated with an ultrafiltration membrane. The antibody concentration was calculated with an OD280 nm per 1 mg / ml concentration of 1.4, which was 0.67 mg / ml.
(2) Peroxidase labeling of anti-CEA monoclonal antibody: 0.2 mg of anti-CEA monoclonal antibody clone 5905 (Medix Biochemica) was labeled with peroxidase labeling kit NH ₂ (manufactured by Dojindo), and 200 μl of peroxidase was labeled. An anti-CEA monoclonal antibody was obtained.
(3) Biotinylated anti-CEA antibody capture Comparison of capture ability of each biotin capture reagent by sandwich EIA: CEA standard products 30 ng / ml and 0 ng / ml were used as samples. After dispensing 25 μl of the sample into the biotin capture reagents described in Example 3 and Comparative Examples 1 to 4, 0.1% (W / V) BSA is contained so that the concentration is 0.2, 0.4, and 0.8 μg / ml. 25 μL each of biotinylated anti-CEA monoclonal antibody diluted with PBS buffer was dispensed and mixed, and the biotin capturing ability and CEA antigen-antibody reaction were simultaneously reacted at 37 ° C. for 1 hour. The plate was washed with a PBS buffer containing 0.05% (W / V) Tween 20, and the remaining water was removed as much as possible while hitting the plate with a paper towel. 50 μl each of peroxidase-labeled anti-CEA monoclonal antibody solution diluted 2000 times with PBS buffer containing 0.1% (W / V) BSA was dispensed and further reacted at 37 ° C. for 1 hour. The plate was washed with a PBS buffer containing 0.05% (W / V) Tween 20, and the remaining water was removed as much as possible while hitting the plate with a paper towel. Chromogenic solution TMB + (manufactured by Dakocytomation) containing tetramethylbenzidine was dispensed in 50 μl per well, enzymatic reaction was performed at 25 ° C. for 10 minutes, 50 μl of 1N sulfuric acid was dispensed, and the reaction was stopped with a plate reader at 450 nm. Was measured.
In the CEA 0 ng / ml sample, only a signal with an absorbance of 0.03 or less was observed. On the other hand, in the CEA 30 ng / ml sample, as shown in Table 1, the signal intensity differs for each biotin capture reagent, and the highest signal was obtained with the biotin capture reagent of Example 3 to which the 0021 purified antibody was immobilized. It was found that anti-CEA monoclonal antibody capture ability was the highest.

(4) Biotinylated anti-CEA antibody capture in the presence of biotin Comparison of capture ability of each biotin capture reagent by sandwich EIA: CEA standard product containing 2 μg / ml D-biotin as final sample at 30 ng / ml and 0 ng / ml Using. Other than that, it measured according to the procedure of the above-mentioned (3) description.
In the CEA 0 ng / ml sample, only a signal with an absorbance of 0.03 or less was observed. On the other hand, in the CEA 30 ng / ml sample, as shown in Table 2, the signal intensity was different for each biotin capture reagent, and the highest signal was obtained with the biotin capture reagent of Example 3 to which the 0021 purified antibody was immobilized. It was found that anti-CEA monoclonal antibody capture ability was the highest.

Example 5: Preparation of ligand capture reagent containing 0021 purified antibody-immobilized glass fiber (1) Production of absorbent element: Tobacco filters were cut in 11.5 mm increments.
(2) Production of container used for ligand capture reagent: Glass fiber filter paper (Advantech Toyo Co., Ltd.) having a diameter of 9.0 mm and a thickness of 1.0 mm is rounded and laminated on the absorbent element produced in (1) above. It was stored in a liquid impermeable container made of polyethylene as shown in FIG. The liquid impervious container used this time had an inner diameter of 9.0 mm and a measurement container having a length of 15 mm.
(3) Immobilization of antibody: From the opening of the container prepared in (2) above, (a) Mouse anti-biotin monoclonal antibody 0021 Purified antibody 47 μg / ml (100 mM citrate buffer: pH 3.0) 50 μl, (b ) 100% of a 5% glycerol / 1% casein solution (10 mM phosphate-saline buffer pH 7.4) was sequentially supplied after the solution supplied immediately before was aspirated, and then lyophilized. After drying, it was put together with a silica gel desiccant in an airtight plastic bag having gas barrier ability and stored at 4 ° C. until use.

The construction of the analysis unit with the same operation is an essential process for automatic analysis system construction for labor saving of the analysis man-hours, and the ligand capture reagent is used for construction of a common analysis platform. According to the method for obtaining a hybridoma described in the present invention, it is possible to obtain an antibody-producing hybridoma having excellent biotin-labeled substance capturing performance. The antibody produced by the hybridoma is expected to expand the possibility of developing a ligand capture reagent and contributing to improving the performance of a ligand capture reagent in the measurement method of a substance involved in a specific reaction based on a biological specific reaction and the measurement method. it can.

FIG. 1 shows the biotinylated antibody concentration when the biotinylated anti-CEA monoclonal antibody was captured with the biotin capture reagent prepared in Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, and CEA measurement was performed. It is the graph which showed dependent signal sensitivity fluctuation | variation. FIG. 2 is a perspective view of a ligand capture reagent container prepared as an embodiment of the present invention. 3 is a cross-sectional view of the ligand capture reagent container shown in FIG.

Explanation of symbols

1: Outer part 2: Opening part 3: Glass fiber filter paper 4: Absorbing element 5: Cup 6: Collar 7: Cap

Claims (6)

As an immunogen, chemical formula I
An antibody obtained by using a carrier protein to which the compound described above is bound The monoclonal antibody which the antibody of Claim 1 has the following properties (1) Isoelectric point 6.4-6.8
(2) Isotype IgG1 κ
(3) Reactive to carrier protein labeled with biotin     An antibody produced by a hybridoma deposited under the accession number FERM P-20979 at the National Institute of Advanced Industrial Science and Technology (AIST)     Ligand capture reagent using antibody according to claim 1 5. A ligand capture reagent, wherein the antibody according to claim 4 is a monoclonal antibody having the following properties: (1) Isoelectric point: 6.4 to 6.8
(2) Isotype IgG1 κ
(3) Reactive to carrier protein labeled with biotin Ligand capture reagent characterized by using an antibody obtained by using a hybridoma deposited under the accession number FERM P-20979 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and an antibody fragment thereof