JP2003038173A - Method for selecting antibody, hybridoma, monoclonal antibody and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inhibiting the generation of an abnormal value in an immunity measuring method by selecting an antibody hardly affected by an inhibitor of an antigen-antibody reaction which has higher possibility of the coexistence in the sample when carrying out the analysis and the measurement by the immunity measuring method. SOLUTION: This method for selecting the antibody comprises immobilizing an antibody of a measuring object on a carrier, bringing an antigen-enzyme complex (a labeled antigen), the measuring object (the antigen) and a reaction inhibitor into contact with the immobilized antibody, adding an enzyme substrate to develop the color, calculating the amount of the antigen from a calibration curve, and selecting the antibody resistant to the antigen-antibody reaction inhibitor by using the recovery.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody which has a property of reacting with an antigen in the presence of an antigen-antibody reaction-interfering substance (hereinafter sometimes referred to as an interfering substance) without being influenced by the substance, that is, an interfering substance. The present invention relates to a substance resistant antibody, a method for selecting the antibody, a hybridoma producing the antibody, and use of the antibody.

[0002]

2. Description of the Related Art In recent years, an immunoassay (immunoassay, hereinafter abbreviated as IA) has been required because no expensive equipment is required and no operation proficiency is required in the field of environmental measurement. Is used frequently. However, I
Since the antibody specifically acting on the substance to be measured used in A is a high molecular weight protein, the activity of the antibody is affected by various substances that affect the target protein, and as a result, the substance to be measured is Occasionally, the measured value of a substance may be inaccurate. On the other hand, ppb (part per billion, 1 part per billion) and ppt (part per billion,
In order to measure a very small amount of a substance to be measured, such as the order of one trillionth, a high-magnification concentration operation may be required. In such a case, concentration using solid phase extraction method ( Hereinafter, the measurement target substance is measured using a sample concentrated by an operation such as solid phase concentration) and concentration by solvent extraction (hereinafter sometimes referred to as solvent concentration). However, since various miscellaneous compounds are mixed in the sample sample in addition to the substance to be measured, when the concentration operation by solid phase concentration or solvent concentration is performed, the compound that affects the antibody, that is, the substance that interferes with the antigen-antibody reaction, is detected. As a result of being concentrated, the activity of the antibody may be affected and the measured value of the substance to be measured may be abnormal.

[0003]

One of the problems to be solved by the present invention is to select an antibody which is less likely to be affected by an antigen-antibody reaction interfering substance which is likely to be present in the sample when the antigen is measured by IA. Thus, it is to provide a method for suppressing the occurrence of an abnormal value in IA. Another object of the present invention is to provide an IA kit that causes less abnormal values by using the antibody selected in the present invention.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to establish a method for detecting and measuring various substances by IA without being affected by contaminating reaction-interfering substances as much as possible. As a result, when selecting an antibody to be used in IA, the antigen and the labeled antigen are reacted with the antibody in the presence of a reaction interfering substance such as an environmental pollutant or its degradation product, a sterilizing / disinfecting agent, or a solvent, and the reaction rate From this, it was found that by selecting the target antibody having high resistance to the reaction interfering substance, the target substance can be measured even in a sample concentrated at a high magnification because the amount of the target substance to be measured is small. As a result of further research based on such findings, the present invention has been completed.

That is, the present invention provides (1) when selecting an antibody against a substance to be measured using an antigen-antibody reaction,
A method for selecting an antibody for selecting a target antibody in the presence of an antigen-antibody reaction-interfering substance, (2) a method for selecting an antibody according to (1), wherein the measurement target substance is an environmental pollutant, and (3) a hormone for the measurement target substance. There is a method of selecting an antibody according to (1), (4) a method of selecting an antibody according to (1) in which the antibody is a monoclonal antibody, and (5) an antigen-antibody reaction-interfering substance is an environmental pollutant or a decomposed product thereof, and sterilization. The method for selecting an antibody according to (1), which is an agent or a solvent, (6) the method for selecting an antibody according to (5), wherein the environmental pollutant is a surfactant, environmental water or a concentrate thereof, or a humic substance. ) The method for selecting an antibody according to (6), wherein the surfactant is an anionic surfactant, a cationic surfactant or a nonionic surfactant, (8) environmental water is river water, lake water, sea water, or Water treatment process water or sewage treatment process water (6) selection method according antibodies,
(9) The method of selecting an antibody according to (5), wherein the germicidal disinfectant is a chlorine agent, (10) the solvent is an alcohol, a nitrile,
(5) The method for selecting an antibody which is a ketone or an ester, (11) a polyclonal antibody obtained from an animal immunized with a substance to be measured or a complex of a substance to be measured and a hapten and a protein as an antigen, or a substance to be measured A substance to be measured or a hapten of the substance to be measured and a protein thereof obtained by fusing spleen cells or lymph node cells of an animal immunized with a complex of a hapten of the substance or the substance to be measured and a protein as an antigen Monoclonal antibody obtained by culturing a hybridoma producing a monoclonal antibody against the complex of, in the presence of an antigen-antibody reaction-interfering substance, an antigen-enzyme complex (labeled antigen), a measurement target or a hapten of the measurement target and its A complex (antigen) with a protein is reacted, and an antigen-antibody reaction interfering substance resistant antibody is selected based on the reaction rate (1 Screening method of the antibodies described, (12)
A hybridoma producing an interfering substance-resistant monoclonal antibody selected by the method for selecting an antibody according to (11),
(13) The hybridoma is a mouse hybridoma EE
2-227 (FERMBP-7567), mouse hybridoma E1-420 (FERM BP-7568) or mouse hybridoma E2-73 (FERM BP-).
7569) and the hybridoma according to (12),
4) Antigen-antibody reaction-interfering substance resistant antibody selected by the antibody selection method according to (11), (15) Antigen-antibody reaction-interfering substance resistant monoclonal antibody produced by the hybridoma according to (12), (16) (14) ) Or (15)
A kit for immunological analysis of a substance to be measured, which comprises an antibody resistant to an antigen-antibody reaction interfering substance as an essential component, and the antibody resistant to an antigen-antibody reaction interfering substance according to (17), (14) or (15) A kit for immunological concentration of a substance to be measured, which is included as an essential component.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for selecting an antibody which reacts with an interfering substance in an antigen-antibody reaction and an antibody against the substance to be measured is coexisted in an aqueous medium to select a reaction-interfering substance resistant antibody by IA.

An example of a more specific antibody selection method belonging to the present invention is as follows. That is, a solution containing an antibody against a substance to be measured is immobilized as it is or in contact with a species-specific antibody previously immobilized on a carrier, and reacted at an appropriate time and temperature to immobilize the antibody in the antibody-containing solution on the carrier. Turn into. After the antibody in the antibody-containing liquid that has not been immobilized is removed by washing with a washing liquid, the antigen-enzyme complex (labeled antigen), the substance to be measured (antigen) and the reaction-interfering substance are brought into contact with the immobilized antibody to react. After washing away unreacted substances, an enzyme substrate is added to develop color. After the color development or after the color development is stopped, the absorbance or fluorescence is measured, and the measured concentrations in the presence and absence of the interfering substance are compared from the standard curve to calculate the degree of the interfering substance resistance. As a result of the calculation, an antibody having a high degree of resistance to a reaction interfering substance is selected. When the antibody is a monoclonal antibody, the hybridoma producing the antibody is specified. The hybridoma is cultured, and an antibody highly resistant to a reaction interfering substance is obtained from the culture supernatant. The original substance to be measured is analyzed by IA using these antibodies.

The antibodies to be selected according to the present invention include detection,
Any antibody used for measurement is included. Antibodies include polyclonal and monoclonal antibodies.

The substances to be measured in the IA of the present invention include all substances to be detected and measured by using the antibody, for example, all the substances to be measured in measuring environmental pollutants. Specific examples of the substance to be measured include various hormones, plant hormones, environmental pollutants, synthetic surfactants, pesticides, mold poisons, toxins, drugs, allergens, microorganisms, etc. .

Among various hormones, as animal hormones, female hormones such as estrogen, estradiol (E2), estrone (E1) and estriol (E3), androgens, testosterone, dehydroandrosterone, androstenedione, etc. Androgens, thyroxine (T3), triiodothyronine (T
4) and other thyroid hormones, ethinylestradiol, diethylstilbestrol, raloxifene, tamoxifen, moxestrol, allylestrenol, mestranol, linenestrenol, chlormadinone acetate, dydrogesterone, medroxyprogesterone, norethisterone, norgestrel, levonorgestrel, Examples include synthetic hormones such as pregnanediol and clomiphene, and hormones such as progesterone, zeranol, trenbolone, and clenbuterol (β agonist). In addition, conjugates (eg, glucuronide conjugates, sulfate conjugates) that are metabolites thereof are included. (Including coalescing) and their decomposition products are also included in these.

The plant hormones include isoliquiritigenin, phloretin, coumestrol, hesperetin,
Naringenin, apigenin, baicalein, chrysin,
Luteolin, galangin, kaempferol, quercetin, equol, biocanin A, daidzein, formonenetin, genistein, betulin and the like are included, and their metabolites and degradation products are also included.

Environmental pollutants to be measured include benzyl butyl phthalate, diethyl phthalate, di-n-butyl phthalate, diisobutyl phthalate, di-n-propyl phthalate, di-n-phthalate. Pentyl, di-n-hexyl phthalate, dicyclohexyl phthalate, di (2-ethylhexyl) phthalate, dioctyl phthalate, mono (2-ethylhexyl) phthalate, diisononyl phthalate, diisodecyl phthalate, di-n-phthalate
Phthalates such as octyl and ditridecyl phthalate, adipates such as di (2-ethylhexyl) adipate, 4-ethylphenol, 3-t-butylphenol, 4-s-butylphenol, 4-t-
Butylphenoel, 4-propylphenol, 4-isopentylphenol, 4-t-pentylphenol,
Octylphenol, 4-octylphenol, 4-t
-Alkylphenols such as octylphenol, 4- (1,1,3,3, -tetramethylbutylphenol), 4-nonylphenol (straight chain), 4-nonylphenol (branched), nonylphenol (mixed isomer), bisphenol A, tetra Bromobisphenol A
Such as diphenylalkanes, PBBs such as polybrominated biphenyls, polybrominated biphenyl ethers, styrene, styrene dimers, styrene trimers, 2-chlorophenol, 2,4-dichlorophenol, 2,4, 6-
Chlorophenols such as trichlorophenol and pentachlorophenol, t-butylated hydroxyanisole (BHA), n-butylbenzene, benzophenone,
Examples thereof include endocrine disrupting chemicals such as 6-bromo-2-naphthol, dibromoacetic acid, 2-bromopropane, 4-nitrotoluene, and octachlorostyrene, and their metabolites and degradation products are also included.

Furthermore, dioxins such as 2,3,7,8-tetrachlorodioxin (2,3,7,8-TCDD) (Poly
chlorinated dibenzodioxins (PCDDs)), 2, 3, 7,
Dibenzofurans (Polychlorinated dibenzofurans) such as 8-tetrachlorodibenzofuran (2,3,7,8-TCDF)
(PCDFs)), Aroclor 1016, Aroclor 1221, Aroclor 1232, Aroclor 1242, Aroclor 124
PCBs such as 8, Aroclor 1254, Aroclor 1260, Aroclor 1262, Aroclor 1268, Bifenox, Halowax 1000, Halowax 1051, Halowax 1099, etc. (Polychlorinated Biphenyls (PCBs)), acenaphthene, acenaphthylene, anthracene, benzo [a]
Anthracene, benzo [a] pyrene, benzo [b] fluorane, benzo [ghi] perylene, benzo [k] fluorane, biphenyl, chrysene, creosote, 1,2-dichlorobenzene, 2-ethyltoluene, 4-ethyltoluene, Hexachlorobenzene, dibenzo [a, h] anthracene, fluoranthene, fluorene, naphthalene, 1-
Methylnaphthalene, 2-methylnaphthalene, 1-chloronaphthalene, o-cresol, phenanthrene, n-propylbenzene, pyrene, 1,2,4-trimethylbenzene, 1,3,5-trimethylbenzene, gasoline, kerosene, jet A Fuel (Jet A fuel), JP-4, JP-5, Fu
el oil # 1, Fuel oil # 2, Fuel oil # 4, Fuel oil # 6,
Polynuclear Aromatic Hydrocarbons such as heating fuel, diesel oil, turbine oil, etc.
(PAHs)), cyclic polycyclic aromatic hydrocarbons (C-PAH), total petroleum hydrocarbons (TPHs)
s)), BTEX (benzene, toluene, ethyl benzene, and xylen
s), benzene, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-
Dinitroaniline, 1,3-dinitrobenzene, 2,4
-Dinitrophenol, 2-nitrotoluene, 3-nitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, picric acid, methyl-2,4,6-trinitrophenylnitramine, 1,3,5-trinitrobenzene , Trinitrotoluene (TNT), octahydro-
1,3,5,7-Tetranitro-1,3,5,7-tetrazocine, nitroglycerin, nitroguanidine, pentaerythritol tetranitrate, RDX explosive (RDX Explosive
s) Also included are gold trichloride, silver nitrate, mercury, trihalomethane, trichlorethylene, tetrachloroethylene and the like.

Examples of synthetic surfactants include anionic surfactants such as linear alkylbenzene sulfonic acid and salts thereof, nonionic surfactants such as nonylphenol ethoxylate and octylphenol ethoxylate, and the like. , These metabolites and degradation products are also included in this.

Examples of pesticides include carpropamide, thiocarbamate compounds, chlorfenapyr, pretilachlor, propizzamide, chlormecote, malathion, fenitrothion, imidacloprid, carbaryl, inabenfide, butamiphos, probenazole, isoprothiolane, bentazone, tolclofos-methyl, fensulfothion, bendaiocalybone, benzulfiobuone. , Iprodione, isoxathion, pirimicarb, oxamyl, daminozide, phoxime, imazalil, microbutanyl, triflumizole, diazinon, propiconazole, bitertanol, phenoxyacetic acid compounds, acid amide compounds, 2,4-
D, 2,4-DNT, 2,4-D butyl butyrate, acetanilide, acetochlor, alachlor, sulfonate sulfonate,
Aldicarb, aldicarb sulfone, aldicarb sulfoxide, aldrin, ametrine, atrazine, azinphos, benomir, α-BHC, δ-B
HC, γ-BHC (lindane), biolesmethrin, captan, carbaryl, carbendazim, carbofuran, chlordane, chlorothalonil, chlorpyrifos, chlorpyrifos-methyl, chlorosulfuron, cyanazine, cyclodiene, DDD, DDE, DDT, dicamba, dichlorprop, dieldrin, Diquat, Dorsban, Endosulfan, Endrin, Ethylated Atrazine, Fenitrothion, Heptachlor, Hexazinone, Hydroxyatrazine, Imazaquin, Imazapyr, Isoproturon, Metulfuron, Metalaxyl, Mesomil, Mesoprene, Metrachlor, Metribuzin, Molinate, Paraquat, Parathion, Pichloram, Pythram, Pythramine, Pythram Procymidone, Promethon, Promethrin, Lerdan, Sylvex, Sylvex 2,4,5-T P, simazine, thiabendazole, toxaphene, triasulfuron, triazine, trichloropyridinol, triclopyr, trifluralin, urea herbicides and the like are also included, and metabolites and decomposition products thereof are also included therein.

Mold venoms include aflatoxin, T2
Examples include toxin, ochratoxin, zearalenone, DON (deoxynivalenol, vomitoxin), fumonisin, and the like, and their metabolites and degradation products are also included therein. Examples of the toxins include paralytic shellfish poison, Staphylococcus aureus enterotoxin (A, B, C, D, E), and their metabolites and degradation products are also included therein.

Examples of the drugs include β-lactam antibiotics having a skeleton such as penicillin, cephalosporin, penem, monobactam and clavulanic acid, polyether antibiotics such as monensin, salinomycin, and enjuracidin, kanamycin. Such as aminoglycoside antibiotics, new quinolone synthetic antibacterial agents such as enrofloxane, sulfamethazine, sulfadimethoxine and other sulfa drugs, chloramphenicol, gentamicin and other antibiotics, digoxin, theophylline, phenobarbital, trenbolone and other drugs. Moreover, these metabolites and degradation products are also included in this.

Examples of allergens are house dust, mites, feline epithelium, dog epithelium, cedar, cypress, alder tree, cypress, cypress, thornweed, ragweed, mugwort, aspergillus, candida, alternaria, egg white, milk, wheat, rice, Examples include allergens such as soybean, cod, tuna, crab, shrimp, penicillium, cladosporium, cheddar cheese, beef, chicken and salmon, and their metabolites and decomposition products are also included.

The microorganisms include pathogenic bacteria such as Salmonella, Listeria, Escherichia coli, Cryptosporidium, Giardia, Campylobacter, Staphylococcus aureus, Yersinia, Vibrio parahaemolyticus, and fungi, and the constituents thereof are also included therein.

On the other hand, examples of the antigen-antibody reaction interfering substance include substances that affect proteins such as antibodies and enzymes, for example, environmental pollutants, degradation products thereof, bactericidal disinfectants, solvents and the like. As environmental pollutants and their decomposition products,
Surfactants, environmental water and its concentrates, humic substances and the like can be mentioned.

Examples of the surfactants include carboxylic acid types such as fats and oils, fatty acids, sulfuric acid ester types such as alkyl sulfates and alkyl ether sulfates, and / or sulfonic acid types such as alkyl (allyl) sulfonates and (alkyl) naphthalic acids. Anionic surfactants such as phosphoric acid ester type such as alkyl phosphoric acid ester, 4
Cationic surfactants such as quaternary ammonium type, ether type such as polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether and other ethers, ester ether type such as polyhydric alcohol ester / ether and other ester ethers , Nonionic surfactants such as polyhydric alcohols and esters. Examples of humic substances include fulvic acid, humic acid, humic acid and / or salts thereof.

Examples of the environmental water and its concentrate include river water, lake water, sea water, tap water treatment process water, sewage treatment process water, and concentrates thereof. Examples of sterilizing agents include chlorine gas, which is a chlorine agent, and hypochlorous acid and its salts.

Examples of the solvent include alcohols, nitriles, ketones and esters, and examples of the alcohols include methanol, ethanol, propanol,
Butanol and nitriles include acetonitrile, ketones include acetone and methyl isobutyl ketone (MIBK), and esters include ethyl acetate.

Production of immunoglobulin (Ig), which is an antibody against the substance to be measured, and production of an antibody against the Ig (that is, anti-Ig antibody), are known per se, for example, enzyme immunoassay, pages 46 to 71, and The method described in pages 85 to 110 (P.TIJSSEN, translated by Eiji Ishikawa, Tokyo Kagaku Dojin (1989)) or a method analogous thereto.

In these methods, the substance to be measured which can be an immunogen itself is left as it is, and the substance to be measured which has no immunogenicity is prepared by forming its hapten and then forming a complex (immunogen) with a carrier protein. Inoculate the animals. Carrier proteins used for immunization by forming a complex include, for example, bovine serum albumin (hereinafter abbreviated as BSA), ovalbumin (hereinafter abbreviated as OVA), keyhole limpet hemocyanin (hereinafter abbreviated as KLH), bovine. Examples thereof include thyroglobulin (hereinafter abbreviated as BTG).

The formation of the complex between the substance to be measured and the carrier protein can be carried out, for example, by the formula (1) AR (1) (wherein R is COOH, NH ₂ or SH, and A is R).
Indicates the group that becomes the measurement target substance when the group is eliminated. The compound (hapten) represented by () can be fused with a carrier protein by a method known per se. For example, a compound represented by the formula (1) in which R is COOH and A is polyoxyethylene alkylphenyl ether can be produced by dehydration condensation (half ester) of polyoxyalkylphenyl ether and succinic anhydride [ Cancer Biochem. Biophys., 7,175
(1984)]. The compound represented by the formula (1) in which R is NH ₂ and A is polyoxyethylene alkylphenyl ether is obtained by oxidizing the hydroxyl group of polyoxyalkylphenyl ether with thionyl chloride [Journal of American Chemical・ Society (J. Am. Che
m. soc.), 60, 2540 (1938)], and can be produced by treating with ammonia [Organic Functional Group Preparations (Organi
c Functional Group Preparations), Volume 1, 382
page〕.

The compound represented by the formula (1) in which R is SH and A is polyoxyethylene alkyl phenyl ether is obtained by converting the hydroxyl group of polyoxyalkyl phenyl ether to hydrochloric acid with thionyl chloride [Journal of American・ Chemical Society (J. Am. Chem. So
c.), 60, 2540 (1938)], and can be produced by reacting with sodium hydroxide [Journal of American Chemical Society (J. Am. Che.
m. Soc.), 72, 1843, (1950)].

The antibody to the substance to be measured in the present invention is obtained by a method known per se, that is, from the substance to be measured or from an animal immunized with the complex (immunogen) of the hapten of the substance to be measured and a carrier protein. It can be produced by producing a hybridoma producing a monoclonal antibody obtained by fusing the polyclonal antibody obtained or the spleen cells or lymph node cells of the immunized animal with myeloma cells. To immunize an animal, the substance to be measured or the complex of the hapten obtained above and a carrier protein is inoculated into the animal. The animals to be inoculated include, for example, goat, sheep, rabbit, rat, mouse,
Although guinea pigs, chickens, etc. are used, when a monoclonal antibody against the substance to be measured is desired, mice are particularly preferably used. The method of inoculation may be a method that is usually carried out. For example, about 1 to 100 μg, preferably 50 to 100 μg per mouse is used in equal volumes (0.1 ml) of saline and Freund's complete adjuvant or emulsified in RIBI adjuvant system ^{T M,} back, a method of inoculating 2-6 times every 2-3 weeks subcutaneously or intraperitoneally abdomen taken. When a polyclonal antibody is obtained, it is collected from the serum of the inoculated animal. The following operation is further performed to obtain a monoclonal antibody. These immunized animals, for example,
An individual with a high antibody titer is selected from mice, and 3 to 5 days after the final immunization, the spleen or lymph node is collected, and the antibody-producing cells contained therein are fused with myeloma cells. As a method for immunization, a known in vitro immunization method (in vitro immuni
zation) and Mouse Foot Pad method
The antibody titer increases in a shorter period of time by using such as.

The fusion operation can be carried out according to known methods,
As the fusion accelerator, polyethylene glycol (hereinafter,
(Abbreviated as PEG), Sendai virus and the like, but PEG is preferably used. It can also be carried out by a method using a known electric pulse (Pulse Electric Fusion). NS-1, P3U as myeloma cells
1, Sp2 / O, etc. are used, and P3U1 is particularly preferable. For example, a preferred ratio of splenocytes to myeloma cells is about 1: 1 to 10: 1 with a molecular weight of about 1,000-6,6.
000 PEG is added at a concentration of about 10 to 80%, and it is good to incubate at about 20 to 37 ° C, preferably 30 to 37 ° C for about 3 to 10 minutes. The production and purification of the monoclonal antibody by the hybridoma can also be performed by a method known per se. The obtained monoclonal antibody becomes an antibody against the substance to be measured and the compound represented by the formula (1).

Specific examples of antibody production and purification methods include:
For example, the aforementioned "enzyme immunoassay" No. 46-
71, pp. 85-110, salting out (Na ₂ S
O ₄ , (NH ₄ ) ₂ SO ₄ , and ion exchanger (DEA
E, QAE, CM / cellulose, Sephadex, Sepharose, etc.), gel filtration (Sephadex G-200, Bio
-gel P-300, etc.), electrophoresis (zone electrophoresis with agarose gel, isoelectric focusing, isokinetic electrophoresis, etc.),
Ultracentrifugation (sucrose density gradient centrifugation), affinity chromatography (immobilized protein A (Protein A Seph
arose, Protein A Superose, etc.) Immobilized protein G
(Protein-G Sepharose, etc.)) and the like.

The antibody-immobilizing carrier used in the present invention (hereinafter,
It may also be called a carrier. As the), those usually used in the immunoassay can be used. Examples thereof include microplates (eg, 96-well microplate, 24-well microplate, 19-well microplate,
2-well microplate, 384-well microplate, etc.), test tube (eg, glass test tube, plastic test tube), glass particle, polystyrene particle, modified polystyrene particle, polyvinyl particle, latex (eg polystyrene latex), nitrocellulose Membrane, cyanogen bromide activated filter paper, DBM activated filter paper, granular solid phase (eg, sepharose, sephadex, agarose, cellulose,
Sephacryl), iron-containing polycarbonate film, magnet-containing beads and the like. For carrying an antibody on a carrier, a method known per se [eg, "Enzyme Immunoassay" pp. 268 to 296, "Affinity Chromatography Handbook" (Amersham Pharmacia Biotech Co., Ltd. (December 20, 1998))
Issued daily)] etc.

The antibody-containing liquid for obtaining the antibody resistant to the antigen-antibody reaction interfering substance includes serum of an immunized animal, HAT
Culture supernatant of wells in which colonies of fused cells (hybridomas) were confirmed by screening, culture supernatant of hybridoma during cloning, culture supernatant of hybridoma purified by cloning, ascites fluid of purified hybridoma grown in mouse ascites, Alternatively, an antibody-containing solution at any stage of the antibody acquisition process such as an antibody solution purified from mouse ascites fluid may be used. The antibody-containing solution may be diluted and added depending on the antibody concentration of the antibody-containing solution.
This dilution factor varies depending on the antibody concentration and assay conditions, but in some cases it may be necessary to dilute and measure from several times to several hundreds of thousands times. The antigen-enzyme complex (labeled antigen) may have any structure as long as it reacts with the antibody being obtained. Further, the enzyme and its substrate may be any enzyme and its substrate used in a usual enzyme immunoassay method, such as alkaline phosphatase, alcohol dehydrogenase, β-D-galactosidase, glucose-6. -Phosphate dehydrogenase, horseradish peroxidase, xanthine oxidase, glucose oxidase, invertase, acetate kinase and its substrate are preferable, but horseradish peroxidase and its substrate are preferable from the viewpoint of measurement sensitivity.

The addition concentration of the antigen-enzyme complex (labeled antigen) is adjusted with a phosphate buffer, physiological saline or the like so that the absorbance becomes 1 to 2 when blank without addition of the substance to be measured (antigen) and the interfering substance. It is better to dilute. The target substance to be added should be diluted with a buffer solution or 10% methanol so that the inhibition rate when no interfering substance is added is approximately 50% .The final concentration of interfering substance is 1 to 1000 mg / L. Dilute serially with buffer solution or 10% methanol, etc. For interfering substances such as environmental water and its concentrate, use the stock solution or its concentrate. The concentrated solution of environmental water may be prepared by any known method such as solvent concentration, solid phase concentration or concentration by evaporative drying. The concentration rate varies depending on the type of environmental water used, but it does not solidify. Any concentration ratio may be used as long as the solution cannot be prepared with a buffer solution or a solvent. When using a solvent as an interfering substance, add it in the range of less than 100%.
Antigen-enzyme complex (labeled antigen), substance to be measured (antigen),
After the interfering substance is mixed, it is reacted with the immobilized target substance antibody or the target substance antibody bound to the immobilized species-specific antibody, but the reaction temperature and reaction time do not denature the target substance antibody or species-specific antibody. Although any conditions may be used as long as the temperature and the time, it is preferable that the temperature is 4 ° C. overnight (16 to 24 hours), and the room temperature is 0.1 to 4 hours. The washing liquid for unreacted substances is
Although any solution may be used as long as it is a washing solution that does not denature the antibody, phosphate buffered saline (PBS), T-PBS containing Tween-20 and the like are preferable in order to suppress pH fluctuation.

The enzyme substrate for color development may be any substrate as long as it is a substrate for the enzyme used. For example, in the case of alkaline phosphatase, p-nitrophenyl phosphate, phenyl phosphate / 4-aminoantipyrine,
In the fluorescence method, 4-methylumbelliferyl phosphate, o-
Methyl fluorescein acid, flavone-3-diphosphoric acid,
For chemiluminescence, oxydelectase / ethanol / AD
H / NADH, Isoluminol / Microperoxida
Ze, ascorbic acid-2-phosphoric acid / lucigenin / OH ^-, B
CIP (5-bromo-4-chloro-3-indoxysilyl)
Phosphoric acid) / isoluminol / microperoxida
Ze, AMPPD (3- (2'-spiroadamantane) -4-
Methoxy- (3'-phosphoryloxy) phenyl 1,2
-Dioxetane), CSPD (3- (2-chlorinal adamantane) -4-methoxy- (3'-phosphoryloxy) phenyl 1,2-dioxetane), and bioluminescence methods such as D-luciferin phosphate / luciferase,
In the case of peroxidase, 5-aminosalicylic acid / hydrogen peroxide, ABTS (2,2'-azinodi (3
-Ethylbenzthiazoline) -6-sulfonic acid) / hydrogen peroxide, tetramethylbenzidine / hydrogen peroxide, o-phenylenediamine / hydrogen peroxide, homovanillic acid / hydrogen peroxide, tyramine / hydrogen peroxide in the fluorescence method , P-hydroxyphenylpropionic acid / hydrogen peroxide, luminol / hydrogen peroxide, luminol / hydrogen peroxide / p-iodophenol in the chemiluminescence method, and glucose / western in the case of glucose oxidase in the colorimetric method. Wasabi peroxidase / ABTS (2,2'-azinodi (3-ethylbenzthiazoline) -6-sulphonic acid), fluorescent method: glucose / horseradish peroxidase / p-hydroxyphenylpropionic acid, chemiluminescent method , Glucose / luminol / Fe (CN) ₆ ³⁺ , glucose / TCPO
(Bis (2,4,6-trichlorophenyl) oxalate) / ANS (8-anilinonaphthalene sulfonic acid, glucose / isoluminol / microperoxidase,
Glucose / lucigenin / OH ^- / copper chloride, etc., β-D-
In the case of galactosidase, o-nitrophenyl β-D-galactopyranoside was used in the colorimetric method, and 4-o in the fluorescence method.
Methylumbelliferyl β-D-galactoside, lactose / glucose oxidase / isoluminol / microperoxidase, lactose /
Glucose oxidase / TCPO (bis (2,4,6-trichlorophenyl) oxalate) / ANS (8-anilinonaphthalene sulfonic acid), AMPGD (3- (4-methoxyspiro (1,2-deoxydioxytan-3) , 2'-
Tridecane-4-phenyl), o-NPGal (o-nitro-
β-D- is lactoside) / GalDH (galactose-dehydrogenase) / NAD ⁺ / NADH, o-NPGal by bioluminescence method
When (o-nitro-β-D- is lactoside) / GalDH (galactose-dehydrogenase) / NAD ⁺ / NADH is glucose-6-phosphate dehydrogenase, glucose- 6-phosphate / NADP ⁺ , in the chemiluminescence method, glucose-6-phosphate / NAD (P) ⁺ / NAD (P) H, etc.
Many substrates are known, and any substrate can be used as long as it is a substrate for the enzyme used.

After color development, measurement is carried out as it is or after addition of a reaction stop solution at a predetermined wavelength. For example, in the case of alkaline phosphatase, the colorimetric method is 405 nm for the p-nitrophenyl phosphate substrate, the fluorescence method is for the 4-methylumbelliferyl phosphate substrate, the chemiluminescence method is for AMPPD and CSPD, and the peroxidase case is for peroxidase. Is a colorimetric method 492 nm for the 5-aminosalicylic acid substrate, 340 nm or 414 nm (oxide) for the ABTS substrate and 492 nm (pH 1.0) or 445 nm (pH 5.0) for the o-phenylenediamine substrate. ), The colorimetric method is 655 nm or 450 nm for the tetramethylbenzidine substrate (when the reaction is stopped with hydrochloric acid or sulfuric acid), the fluorescence method is used for p-hydroxyphenylpropionic acid, and the chemiluminescence method is used for the luminol / hydrogen peroxide substrate. . After the measurement, a commercially available data processing software (for example, Delta Soft) is used, and the concentrations of the substances with and without addition of the interfering substance are calculated. The obtained concentrations are compared by the recovery rates with and without interfering substances as shown below, and the recovery rate is close to 100% even when a higher concentration of interfering substances is added (50
~ 200%) are selected as antibodies with high resistance to interferents. The recovery rate is calculated by the following formula. Recovery rate (%) = concentration of calculated target substance / concentration of added target substance × 100 Various methods can be used for screening the desired antibody-producing hybridoma. For example, OVA to which the hapten of the target substance is bound is adsorbed. The hybridoma culture supernatant was added to the microplate, and then a horseradish peroxidase (hereinafter, abbreviated as HRP) -labeled anti-mouse immunoglobulin antibody was added to detect the antibody bound to the plate solid phase. To be Hybridomas positive for antibody activity are immediately subjected to cloning, which is usually easily performed by the well-known limiting dilution method or the like.

The antibody titer of the cloned hybridoma supernatant is measured by the above-mentioned method, and the hybridoma producing a stable antibody having a high titer is selected to obtain the hybridoma producing the target monoclonal antibody. You can Examples of hybridomas obtained by such a method include mouse-hybridomas EE2-227, E1-420, and E2-73 obtained in Example 1 described later. These are deposited in the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology, on April 25, 2001, under the deposit number FERM-BP756.
7, FERM-BP7568 and FERM-BP75
Deposited as 69.

[0037]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 1. Acquisition of Monoclonal Antibody 1-1 Preparation of hapten (1) Preparation of hapten for ethinyl estradiol antibody 1.0 g of ethinyl estradiol (EE2) and 0.76 g of sodium methylate were dissolved in 35 ml of ethanol, and 0.41 g of monochloroacetic acid was further added. The mixture was heated under reflux for 22 hours, concentrated under reduced pressure, and about 100 ml of water and ethyl acetate were added to separate the layers. After washing the aqueous layer with 50 ml of ethyl acetate, the aqueous layer was acidified with concentrated hydrochloric acid (pH 1-2). After extraction with 100 ml and 50 ml of ethyl acetate, the organic layer was washed with 30 ml of saturated saline and dried over anhydrous sodium sulfate. A part of the solution was crystallized by allowing the concentrated solution under reduced pressure to stand at -20 ° C for 2 days. A part of this was collected and used as a seed crystal. The concentrated solution was dissolved in a small amount of acetone, hexane and seed crystals were added to precipitate crystals. The target product EE2-3-carboxymethyl ether (EE2-3CME) was obtained by drying the crystals collected by filtration under reduced pressure.

(2) Preparation of hapten for 17β-estradiol antibody 1.0 g of 17β-estradiol (E2) and 0.82 g of sodium methylate were dissolved in 35 ml of ethanol, and 0.45 g of monochloroacetic acid was further added, followed by heating under reflux for 2 days. After concentration under reduced pressure, about 300 ml of water and about 200 ml of ethyl acetate were added to separate the layers.
After washing the aqueous layer with 50 ml of ethyl acetate, the aqueous layer was acidified with concentrated hydrochloric acid (pH 1-2). After extracting twice with 100 ml of ethyl acetate, the organic layer was washed with 30 ml of saturated saline and dehydrated with anhydrous sodium sulfate. The concentrated solution under reduced pressure was dissolved in a small amount of acetone, and isoprovir ether (IPE) was added to precipitate crystals. The crystals collected by filtration are washed with IPE and then dried under reduced pressure to obtain the desired product E2-3-carboxymethyl ether (E2-3CME).
Got

(3) Preparation of hapten for estrone antibody Estradiol-3.17-diacetate (E2-3,17-diAce) 2
0 g is dissolved in 25 ml of acetic acid, and 4/5 of a mixed solution of 9 g of chromic anhydride, 27 ml of acetic acid and 4 ml of water is gradually added while cooling. After stirring overnight, water was added and the mixture was extracted with chloroform, washed with potassium carbonate solution, dehydrated with sodium sulfate, and concentrated under reduced pressure. After the concentrated solution was dissolved in benzene, it was subjected to column chromatography (Wako gel 2w) and eluted with hot benzene to collect crude 6-oxo-E2-3,17-diAce (remove the raw material while confirming by TLC). . This substance was dissolved in 2-methoxy-ethanol, 20% NaOH was added in the same amount, and the mixture was reacted at 100 ° C for 1 hour. After acidification with dilute hydrochloric acid, elution with ethyl acetate, dehydration with sodium sulfate, concentration under reduced pressure, and recrystallization from methanol (6oxo-E2). 6oxo-E2 300mg ethanol
Dissolve in 2 ml, add 50 mg of fine powder of potassium carbonate, add 50 μl of benzyl bromide, and stir (70 ℃, 2 hours)
The concentrate was recrystallized from methanol (6-oxo-E2-3-benzyl ether). 6-oxo-E2-3-benzyl ether was dissolved in methanol, and carboxymethoxylamine 1 / 2HCl and sodium methoxide (1.2 molar amount)
Was dissolved in a small amount of water, diluted with methanol and added. The reaction was carried out at room temperature for 1 hour, 2/3 was evaporated, acidified with water and HCl, extracted with ethyl acetate and dried over sodium sulfate. After dissolving in hexane, the product was applied to a silica gel column and eluted with methanol to purify it, and then recrystallized from methanol. 0.6 g of the recrystallized substance is dissolved in 2 ml of acetone,
Add half of the mixture of chromic anhydride 1 g, acetic acid 5 ml, water 1 ml,
After the exotherm had ended, it was extracted with ethyl acetate and evaporated to dryness (6-oxo-E1-3-benzyl ether). 6-oxo-E1-
Dissolve 400 mg of 3-benzyl ether in 5 ml of methanol,
% Pd.C 100 mg was added and hydrogen was stirred. After reacting for 1 hour, the mixture was evaporated to dryness and recrystallized from methanol to obtain the desired product 6-oxo-E1-6 carboxymethyl oxime (E1-6CMO).

1-2 Preparation of Immunogen For each of the three haptens obtained in 1-1, 0.1 mol of hapten, 0.14 mol of water-soluble carbodiimide, and 0.14 mol of N-hydroxysuccinimide were added overnight in 2 ml of dimethyl sulfoxide. Reacted to make an activated ester. Next, 10 mg of keyhole limpet hemocyanin (KLH) was dissolved in a 0.13 molar sodium bicarbonate (NaHCO ₃ ) solution, 200 μl of the activated ester was added, and the mixture was reacted overnight at 4 ° C. Unreacted reagents were removed by dialysis against Dulbecco's Phosphate buffer (PBS) and stored frozen as an immunogen.

1-3 Immunization The immunogen obtained in 1-2 was dissolved in PBS at 500 μg / ml and added to an equal amount of Freund's adjuvant or RIBI adjuvant system. After sufficient emulsification, 100 μg / mouse was subcutaneously administered to BALB / C mice (female), and booster immunization was performed at 2 week (Freund) or 3 week (RIBI) intervals. After 5 to 6 booster immunizations, the same immunogen was intravenously administered (50 μg / 0.1 ml P
(BS / mouse) The final immunization.

1-4 Spleen was excised from the mouse which was finally immunized by cell fusion 1-3 (3 days after the last immunization) to prepare spleen cells. Separately cultured mouse myeloma cells and spleen cells were contacted at a ratio of 1: 5 in the presence of polyethylene glycol (average molecular weight 4000), and cell fusion was performed to obtain fused cells (hybridomas). After suspending this hybridoma in HAT medium,
It was rolled up in a 6-well microplate and cultured in a carbon dioxide gas incubator (37 ° C, 5% CO2).

1-5 Screening of hybridoma (1) Preparation of assay plate Goat anti-mouse IgAGM antibody (ICN / Cappel, # 55461) was used in PBS.
Dissolve 50 μg / ml in 100 μl / well in a microplate.
Added in. After reacting overnight at 4 ° C, after washing with PBS containing 0.05% Tween 20 (T-PBS) (twice at 300 μl / well), Block Ace diluted 4 times with PBS (Snow Brand Milk Products Co., Ltd.), (Tokyo) was added at 200 μl / well. After reacting at 4 ° C overnight or more, it was stored refrigerated until use.

(2) Preparation of antigen-enzyme complex (labeled antigen) For each of the three haptens prepared in 1-1, 0.1 mol of hapten, 0.14 mol of water-soluble carbodiimide and 0.14 mol of N-hydroxysuccinimide were added to dimethyl sulfoxide. The activated ester was made by reacting in 2 ml overnight. Next, add horseradish peroxidase (HRP) 10 mg to 0.13
It was dissolved in 10 ml of a molar sodium bicarbonate (NaHCO ₃ ) solution, 15 μl of the activated ester was added thereto, and the mixture was reacted overnight at 4 ° C.
The unreacted reagent was removed by ultrafiltration and refrigerated at a concentration of 3 mg / ml.

(3) Primary screening (antigen-binding ability test) Regarding the microplate in which cells were involved in 1-4,
100 μl of the culture solution in the well in which cell growth was confirmed was added to the assay plate prepared in (1) (washed with PBS (or T-PBS) before use (300 μl / well twice)). After reacting for 1 hour at room temperature, wash with T-PBS (300 μl / well 3 times), 1-
The labeled antigen of the target antibody prepared in 5 (2) was diluted 5000-fold with T-PBS and added to the microplate. After reacting at room temperature for 1 hour, the plate was washed with T-PBS (300 μl / well three times), and TMB peroxidase substrate kit (Nippon Bio-Rad, Tokyo, # 172-10).
66: The following chromogenic substrate) was added at 100 μl / well. Thirty
After reacting at room temperature for 1 minute, 1N phosphoric acid was added at 100 μl / well,
The color reaction was stopped. The absorbance was read at a wavelength of 450 nm, and cells having an absorbance of more than 1 were scaled up to a 24-well microplate.

(4) Secondary screening (female hormone interference test) For cells scaled up to a 24-well microplate in 1-5 (3), a well culture medium in which sufficient cell growth was confirmed was prepared in (1). Assay plate (PBS before use
(Or T-PBS) after washing (300 μl / well twice) to 100 μ
l was added to each well. After reacting for 1 hour at room temperature, TP
Wash with BS (300 μl / well three times), and measure the female hormone (1 ng / ml in 10% methanol (hereinafter MeOH)) or 10% MeOH only (control) and the labeled antigen (TP) of the target antibody.
(5000-fold diluted with BS) were mixed in equal amounts and added to the microplate. After reacting at room temperature for 1 hour, wash with T-PBS (300 μl
chromogenic substrate was added at 100 μl / well. Thirty
After the reaction at room temperature for 1 minute, 100 μl / well of 1N phosphoric acid was added to stop the color reaction. The absorbance was read at a wavelength of 450 nm, and compared with the control, cloning was performed according to a conventional method on cells in which the decrease in absorbance was confirmed to be 20% or more due to the presence of female hormone, and the target antibody-producing hybridoma candidate strain shown below was obtained. did.

1-6 Acquisition of Purified Antibody The cell culture supernatant was fractionated with 45-50% saturated ammonium sulfate, and the mouse ascites was subjected to protein G affinity chromatography according to a conventional method to obtain and purify the antibody.

2. Selection of Interfering Substance Resistant Antibodies Using the culture supernatant of the cells obtained in 1-5 (4), an interfering substance resistant test of each antibody was carried out. As the interfering substance, a surfactant and a humic substance, which are substances present in the environment at a high concentration and possibly concentrated together with the measurement substance in the pretreatment stage of the measurement sample, were used.

According to the experimental method 1-5 (4), the dilution ratio of the culture supernatant which gives the highest sensitivity for measuring female hormone was determined. Next, after mixing the same amount of the following sample and the labeled antigen of the target antibody (diluted 5000 times with T-PBS) with the plate in which the antibody in the culture supernatant was bound to the anti-mouse IgG plate at that concentration , 100 μl was added. As samples, straight chain sodium alkylbenzene sulfonate (LAS), alkylphenol ethoxylate (APE), alkyl ethoxylate (AE) (0, 1, 10, 1 for each)
00, 1000mg / L in 10% MeOH), sodium humate (0,
A female hormone to be measured was added to 1, 10, 100 mg / L in 10% MeOH) solution so as to be 0.5 μg / L, and a female hormone was not added. ELISA measurement was performed according to 1-5 (4), and the female hormone concentration in the sample was calculated by comparison with a standard curve prepared using a standard solution of known concentration. Female hormone concentration (0.5 μg /
It was divided by L) and multiplied by 100 to calculate the recovery rate.

Result

[Table 1]

For the anti-EE2 antibody, the anti-EE2 antibody selected by this selection method was used.
-227 antibody is an anti-EE2-8 antibody obtained without using this selection method
Compared to the (control), the recovery rate was about 50-200% even when the concentration of the interfering substance was about 10 to 100 times higher. Therefore, a mouse producing an antibody highly resistant to the reaction interfering substance. Hybridoma strain EE2-227 (FEEM
BP-7567) was selected. Also, with anti-E2 antibody,
Anti-E2-73 antibody selected by this selection method was obtained by three types (E2-CC, E2-NG, E2-RB) commercial ELISA obtained without this selection method.
Even if an interfering substance (surfactant) of about 10 to 100 times higher concentration than the antibody used in the kit is added, the recovery rate is 50 to 2
Since it was 00%, E2-73 strain (FEEM BP-7569) was selected as a mouse hybridoma producing an antibody having a high degree of resistance to the reaction-interfering substance. In addition, since the anti-E1-420 antibody selected by this selection method also has high surfactant resistance comparable to that of the anti-E2-73 antibody, the E1-420 strain (FERM) as a mouse hybridoma producing the anti-E1-420 antibody. -B
P-7568) was selected.

Example 2 Preparation of EE2-ELISA kit (1) Preparation of “solid-phased plate” Goat anti-mouse IgG antibody (ICN / ICN /) dissolved in Dulbecco's PBS (-) (Wako Pure Chemical Industries Code No. 041-20211) Cappel CodeN 0.55
479) to 0.5 μg / well immobilized plate (Costa
r, EIA / RIA plate strip8, # 2592), the mixture was allowed to stand at 4 ° C. overnight, and then washed 3 times with 300 μL of a washing solution (T-PBS).
Broking liquid (1% BlockAce (Snow Brand Milk Products) + 0.05% Slaoff 72
N (Takeda Pharmaceutical Co., Ltd. in PBS) (200 μL / well) was added, and the mixture was allowed to stand overnight at 4 ° C., and then washed 3 times with 300 μL of a washing solution (T-PBS).
Next, 0.002 μg / w of ethinyl estradiol antibody (EE2-227) dissolved in PBS (containing 0.05% slough 72N + 0.1% BSA) was added.
Add each ell, leave it at 4 ℃ overnight, and wash solution (T-PBS) 300
Washed 3 times with μL. Broking Liquid (1% BlockAce (Snow Brand Milk Products) + 0.05% Slaoff 72N (Takeda) in PBS) 200
After adding μl / well and leaving it at 4 ° C. overnight, the whole amount was sucked with an aspirator and then water was removed by tapping. The dehydrated and dried solid-phased plate was enclosed in an aluminum bag, deaerated and sealed with a vacuum dryer, and stored in a refrigerator at 2 to 8 ° C.

(2) "EE2 standard stock solution" (0.1 mg EE2 / L 10% Me
Preparation of stock solution 1 (1000mgEE2 / L): Ethinyl estradiol standard product (Wako Pure Chemical Industries Code No.055-05011)
100 mg was accurately weighed with respect to 100%, placed in a 100 ml volumetric flask, and the volume was measured up with methanol. Stock solution 2 (10mgEE
2 / L): Accurately weigh 1 ml of stock solution 1 with a whole pipette, put in a 100 ml volumetric flask, and make up to volume with methanol. Preparation of standard stock solution (0.1mgEE2 / L): Precisely weigh 1ml of stock solution 2 with a whole pipette, put it in a 100ml volumetric flask, make up with methanol and distilled water, and finish.
It was prepared to be a 10% methanol solution. 4 in a suitable container
ml standard stock solution was added and stored in a refrigerator at 2-8 ° C.

(3) Preparation of "antigen-enzyme complex solution" Water-soluble carbodiimide (for WSC Dojindo Peptide synthesis)
Code No.348-03631) 26 mg and N-hydroxysuccinimide (NHSI Wako Pure Chemicals for peptide synthesis Code No.089-0403
2) 16 mg was dissolved in 2 ml of dimethyl sulfoxide (DMSO Wako Pure Chemical Industries, Ltd.). 0.5 ml of it and 10 mg of the hapten (EE2-3CME) prepared in Example 1 were dissolved in 1 ml of DMSO and reacted at room temperature overnight. 13 μL of the obtained reaction solution and 10 ml
10 mg of peroxidase (POD Berliner EIA Code No.814393) dissolved in 1.1% NaHCO3 was reacted with stirring at 4 ° C overnight, and the resulting reaction solution was filtered through an ultrafiltration membrane with a molecular weight cut off of 30,000 to obtain 0.05%. Slaof 72N final PBS 30m
By setting l, an antigen-enzyme complex solution was obtained. Dispense 200 μl of this solution into an appropriate container and cap it.
Stored in a refrigerator at ~ 8 ° C.

(4) Preparation of “antigen-enzyme complex solution” 500 μL of Tween-20 (Code No. 160-115 for Wako Pure Chemical Industries)
22) is dissolved in 4.5 ml of distilled water to make a 10% Tween-20 solution. Na2HPO4 / 12H2O 13.26 g, NaH2PO4 / 2H2O 2.
02 g, NaCl 14.61 g, 10% Tween-20 solution 10 ml, slaof 72
After dissolving 200 μl of N in 1 L of distilled water, 8 ml of the solution was dispensed into an appropriate container, capped, and stored in a refrigerator at 2 to 8 ° C.

(5) Preparation of "6-fold concentrated washing solution" 20 ml Tween-20 was dissolved in 200 ml distilled water to prepare 10% Tween-20.
A solution was made. Dulbecco's PBS (-) (for Wako Biochemistry
CodeNo.041-20211) 12 bags, Slaoff 72N 0.3ml, 10% T
After dissolving 30 ml of ween-20 in 1 L of distilled water, 50 ml each was dispensed into an appropriate container, which was then capped and stored in a refrigerator at 2 to 8 ° C.

(6) Preparation of "Chromogenic Substrate Solution-A" 13.4 mg of 5,5'-tetramethylbenzidine (TMBZ Dojindo Chemical Research Code No.346-040301) was added to dimethylformamide (DMF Wako Pure Chemicals Reagent Special Grade). Code No.045-0291
6) Dissolve in 1 ml, mix with 100 ml of 0.1 M sodium acetate buffer (pH 5.5) containing 0.1 g / L Tween 20, dispense 10 ml each in a suitable brown container, cap and then 2-8 ° C. Stored in the refrigerator.

(7) Preparation of "coloring substrate solution-B" 30% hydrogen peroxide solution (Wako reagent special grade Code No. 081-0421
Dilute 5) to 0.1 g / L with distilled water and add to a suitable container.
After dispensing each ml, the tube was capped and stored in a refrigerator at 2 to 8 ° C.

(8) Preparation of "coloring stop solution" 1N phosphoric acid solution is prepared, dispensed in 15 ml aliquots into appropriate containers, capped, and stored at room temperature. The EE2-ELISA kit was completed by packaging the kit components (1) to (8) produced as described above in a box.

Example 3 Quantification using EE2-ELISA kit The quantification method using the EE2-ELISA kit prepared in Example 2 is as follows. (1) Preparation of “ethinyl estradiol (EE2) standard solution” Using methanol and distilled water, EE2 standard stock solution (0.1 mg / L)
Dilute 10% methanol solution) to prepare the EE2 standard solution at the required concentration (eg 0, 0.05, 0.1, 0.3, 1.0, 3.0 μg / L in 10% methanol). (2) Preparation of “antigen-enzyme complex solution” 14 μL of “antigen-enzyme complex solution” was dissolved by adding 7 mL of “antigen-enzyme complex solution” to “antigen-enzyme complex solution”
To prepare. (3) Preparation of “mixed solution” Measurement sample or “EE2 standard solution” (10% methanol solution in each case) 100 μL / well and “antigen-enzyme complex solution” 100 μL / well
Mix wells on a "mixing microplate". (4) Antigen-antibody reaction (competitive reaction) (3) on "Anti-EE2 monoclonal antibody immobilized plate"
Dispense 100 μL / well of the “mixed solution” prepared in step 1, and allow to react for 60 minutes at room temperature. (5) Preparation of “washing solution” During the reaction time of antigen-antibody, “6 times concentrated washing solution” and distilled water were added.
Prepare a "washing liquid" by mixing at a ratio of 1: 5. (6) Removal of unreacted material The antigen-antibody reaction solution is discarded, and the inside of the well is washed 3 times with “washing solution” 300 μL / well. (7) Preparation of "coloring reagent""Coloring substrate solution-A" and "coloring substrate solution-B" are mixed at a ratio of 3: 1 to prepare "coloring reagent". (8) Coloring reaction / stop reaction Add 100 μL / well of the “coloring reagent” prepared in (7), react at room temperature for 30 minutes, and then add “coloring stop solution” to 100 μL / wel.
l Add and stop the reaction. (9) Colorimetric and concentration calculation Using a plate reader, the absorbance is measured at a wavelength of 450 nm, and the EE2 concentration in the sample is calculated from the standard curve.

Example 4 Preparation of Standard Curve by EE2-ELISA Kit The standard curve prepared according to Example 3 is shown in FIG. In addition,
The EE2 concentration is plotted on the vertical axis, and the ratio of the absorbance at each EE2 concentration of 0 μg / L and the absorbance at each concentration (inhibition rate, B / B0%) is plotted on the horizontal axis. From this result, the quantifiable range of EE2 is 0.05-3 μg
It was considered to be about / L.

Example 5 Cross-reactivity test A standard curve was prepared for each of the estrogens shown in Table 2 in the same manner as in Example 4, and the EE2 concentration (IC ₅₀ ) when the inhibition rate = 50% was determined. The results of cross-reactivity obtained by the following formula are shown in [Table 2]. Cross-reactivity (%) = determine estrogen IC ₅₀ / EE2 of I
C ₅₀ x 100

[0063]

[Table 2]

The anti-EE2-227 antibody had a very high specificity for EE2 and hardly reacted with other estrogens.

Example 6 LC-MS / in sewage treatment water measurement
Comparison of MS and ELISA measurement values 10L of sewage treated water was filtered through glass fiber filter paper and then 1M
The pH was adjusted to 5 with an acetate buffer (pH 5.0). Then, after passing water through a C-18 solid phase cartridge conditioned with 5 ml of methanol and 10 ml of distilled water, the cartridge was distilled water and hexane each 5 m
washed with l. EE2 was eluted from the C-18 solid phase cartridge with 5 ml of dichloromethane, the eluate was evaporated to dryness under a nitrogen stream (40 ° C.), dissolved in 10% MeOH and concentrated, and then the EE2 concentration in the sample was measured according to Example-3. Was quantified. For the ELISA measurement, a commercially available EE2 ELISA kit (R-Biopharm GmbH, German,
# 04330) was also used as a control and quantified according to the enclosed operating instructions. In addition, regarding the sample treated in the same manner, the method of Tsujimura et al. (Abstracts commemorating the 50th anniversary of the Chemicals Inspection Association and the 4th Research Presentation Abstracts, 1999, 17-2
According to 6), LC-MS / MS analysis was performed and compared with the measured value of ELISA. The results are shown in [Table 3].

[0066]

[Table 3]

The value measured by the ELISA kit using the EE2-227 antibody selectively obtained as an interfering substance resistant antibody by this selection method is L
Although it was close to the C-MS / MS measurement value, the measurement value with a commercial ELISA kit using the antibody obtained without applying this selection method was LC-
It was about 8 times that of MS / MS, which was considered to be influenced by the reaction-interfering substances present in the concentrate.

[0068]

EFFECT OF THE INVENTION Antigen-antibody reaction-interfering substance-resistant antibodies selected by the method of the present invention can be measured without being affected by the antigen-antibody reaction-interfering substance contained in the sample containing the measurement object. It is a useful antibody that can analyze and measure a target substance and can also be used for concentration of the target substance.

[Brief description of drawings]

Figure 1: EE2-227 antibody standard curve

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/531 C12N 15/00 C 33/577 5/00 B (72) Inventor Ayako Kobayashi Osaka City, Osaka Prefecture 2-17-85, 13 Sanhonmachi, Yodogawa-ku Takeda Pharmaceutical Co., Ltd. Living environment company (72) Inventor Masato Hirobe 2-17-85, 13 Sanhonmachi, Yodogawa-ku, Osaka-shi, Osaka Takeda Pharmaceutical Co., Ltd. Living environment Company F term (reference) 4B024 AA11 BA41 DA02 GA03 HA15 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA92X AB05 AC14 CA25 CA46 4H045 AA11 AA20 AA30 DA76 EA50 FA74

Claims (17)

[Claims] 1. A method for selecting an antibody, which comprises selecting a target antibody in the presence of an antigen-antibody reaction-interfering substance when selecting an antibody against a substance to be measured using an antigen-antibody reaction. 2. The method for selecting an antibody according to claim 1, wherein the substance to be measured is an environmental pollutant. 3. The method for selecting an antibody according to claim 1, wherein the substance to be measured is a hormone. 4. The antibody is a monoclonal antibody.
A method for selecting the described antibody. 5. The method for selecting antibodies according to claim 1, wherein the substance that interferes with the antigen-antibody reaction is an environmental pollutant or a decomposed product thereof, a bactericidal disinfectant, or a solvent. 6. The method for selecting an antibody according to claim 5, wherein the environmental pollutant is a surfactant, environmental water or a concentrate thereof, or a humic substance. 7. The surfactant is an anionic surfactant, a cationic surfactant or a nonionic surfactant.
A method for selecting the described antibody. 8. The method for selecting an antibody according to claim 6, wherein the environmental water is river water, lake water, seawater, water in the water treatment process or water in the sewage treatment process. 9. The method of selecting an antibody according to claim 5, wherein the germicidal disinfectant is a chlorine agent. 10. The method for selecting an antibody according to claim 5, wherein the solvent is an alcohol, a nitrile, a ketone or an ester. 11. A polyclonal antibody obtained from an animal immunized with a substance to be measured or a complex of a hapten and a protein of the substance to be measured as an antigen, or a substance to be measured or a complex of a hapten and a protein of the substance to be measured. A hybridoma producing a monoclonal antibody against a substance to be measured or a complex of the substance to be measured hapten and its protein obtained by fusing spleen cells or lymph node cells of an animal immunized as an antigen with myeloma cells is cultured. The obtained monoclonal antibody is allowed to react with an antigen-enzyme complex (labeled antigen) in the presence of a substance that interferes with an antigen-antibody reaction, a substance to be measured or a complex of the hapten of the substance to be measured and its protein (antigen), and the reaction The method for selecting an antibody according to claim 1, wherein an antibody resistant to an antigen-antibody reaction interfering substance is selected based on the rate. 12. A hybridoma producing an antigen-antibody reaction-interfering substance-resistant monoclonal antibody selected by the antibody selection method according to claim 11. 13. The hybridomas are mouse hybridoma EE2-227 (FERM BP-7567) and mouse hybridoma E1-420 (FERM BP-75).
68) or mouse hybridoma E2-73 (FERM
The hybridoma according to claim 12, which is BP-7569). 14. An antigen-antibody reaction-interfering substance-resistant antibody selected by the antibody selection method according to claim 11. 15. An antigen-antibody reaction-interfering substance-resistant monoclonal antibody produced by the hybridoma according to claim 12. 16. A kit for immunological analysis of a substance to be measured, which comprises the antigen-antibody reaction-interfering substance-resistant antibody according to claim 14 or 15 as an essential component. 17. A kit for immunological concentration of a substance to be measured, which comprises the antigen-antibody reaction-interfering substance-resistant antibody according to claim 14 or 15 as an essential component.