JP4393210B2 - Antibodies and their uses - Google Patents

Description

  The present invention relates to a novel antibody having binding specificity to a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof. More specifically, a method for quantifying the polypeptide or a salt thereof based on an antigen-antibody reaction, an antibody useful for diagnosis of a disease involving the polypeptide or a salt thereof utilizing neutralizing activity, and development of a prophylactic / therapeutic agent, etc. About.

Human ZAQ ligand-1 (polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2) (hereinafter sometimes abbreviated as human ZAQL-1) is a ligand of ZAQ receptor, It is a peptide having an ileal contraction action (Patent Document 1 WO 02/06483). Further, a mature human Bv8 peptide (hereinafter sometimes abbreviated as human ZAQL-2), which is a frog Bv8 mammalian peptide having a structure similar to that of human ZAQ ligand-1, is composed of MAP kinase and PI-3. It has a neuroprotective effect by activating kinase (Eur. J. Neuroscience 13, 1694, 2001). Subsequently, these peptides were also reported as new peptides prokineticin-1 (PK-1) and prokineticin-1 (PK-2) found from the DNA database (Mol. Pharamacol. 59, 692, 2001) Year).
Human ZAQL-1 is an endothelial cell growth factor specific for endocrine tissue (EG-VEGF), and is involved in the formation of highly permeable endothelial structures that characterize endocrine tissue, and humans It has been reported that the transcriptional control region of ZAQL-1 gene has a hypoxia-inducible factor-1 (HIF-1) recognition site that is involved in induction of expression under hypoxia, and that gene expression induction occurs under hypoxia (Non-patent Document 1 Nature. 412, 877, 2001).
WO 02/06483 Publication Nature. 412, 877, 2001

  In order to further clarify the physiological functions of human ZAQL-1, a measurement system that can detect and quantify human ZAQL-1 simply and with high sensitivity has been desired.

  As a result of intensive studies to solve the above problems, the present inventors have made human ZAQL-1 highly sensitive by preparing a plurality of monoclonal antibodies using human ZAQL-1 as an immunogen and combining them. An immunoassay that can be specifically detected has been developed. This makes it possible to easily and highly sensitively measure human ZAQL-1 fluctuations in biological components such as blood, cerebrospinal fluid, and urine.

That is, the present invention
(1) a monoclonal antibody that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof,
(2) The monoclonal antibody according to the above (1), which specifically reacts with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof,
(3) Nos. 8-9, 11, 15, 17, 21, 23, 25-28, 30, 34, 36-37, 39-40 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. 44-46, 48, 52-53, 55, 64, 66, 68, 70-73, 75-76 and the peptide containing at least one selected from 78-86, specifically reacts above ( 1) The monoclonal antibody according to
(4) The monoclonal antibody according to the above (1), which does not recognize the polypeptide having the amino acid sequence represented by SEQ ID NO: 3.
(5) a labeled monoclonal antibody according to (1),
(6) The monoclonal antibody according to (1) above, which is indicated by ZL1-107a, which can be produced from a hybridoma cell indicated by ZL1-107 (FERM BP-8256),
(7) The monoclonal antibody according to (1) above, which is indicated by ZL1-234a, which can be produced from a hybridoma cell indicated by ZL1-234 (FERM BP-8257),
(8) The monoclonal antibody according to (1) above, which has neutralizing activity against a peptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2,
(9) A medicament comprising the monoclonal antibody according to (1) above,
(10) The medicament according to (9) above, which is a prophylactic / therapeutic agent for endometrial cancer, endometriosis or ovulation disorder,
(11) a diagnostic agent comprising the monoclonal antibody according to (1) above,
(12) The diagnostic agent according to (11) above, which is a diagnostic agent for endometrial cancer, endometriosis or ovulation disorder,
(13) A diagnostic agent comprising the monoclonal antibody according to (1) above,
(14) A method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof, characterized by using the monoclonal antibody according to (1) above,
(15) A diagnostic method for a disease involving a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, characterized by using the monoclonal antibody according to (1) above,
(16) The diagnostic method according to the above (15), wherein the disease is endometrial cancer, endometriosis or ovulation disorder,
(17) Competitive reaction of the monoclonal antibody according to (1) above with a test solution and a labeled polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof And containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the test solution, wherein the ratio of the labeled polypeptide or a salt thereof bound to the antibody is measured A method for quantifying a polypeptide or a salt thereof,
(17a) reacting the monoclonal antibody described in (1) above insolubilized on a carrier, the labeled monoclonal antibody described in (1) above (an antibody different from the monoclonal antibody insolubilized on the carrier) and a test solution Thereafter, the activity of the labeling agent is measured, and the method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof in a test solution,
(18) (a) After reacting the monoclonal antibody described in (6) above, the labeled monoclonal antibody described in (7) above and a test solution, the activity of the labeling agent is measured. Or
(B) characterized by measuring the activity of the labeling agent after reacting the monoclonal antibody described in (7) above, the labeled monoclonal antibody described in (6) above, and a test solution. A method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof in the test solution,
(19) A hybridoma cell that produces the monoclonal antibody according to (1) above,
(20) The hybridoma cell according to (19), which is indicated by ZL1-107 (FERM BP-8256) or ZL1-234 (FERM BP-8257),
(21) The hybridoma cell described in (19) above is cultured in vivo or in vitro, and the monoclonal antibody described in (6) or (7) above is collected from the body fluid or culture thereof (6) ) Or a method for producing the monoclonal antibody according to (7),
(22) A method for preventing / treating endometrial cancer, endometriosis or ovulation disorder, which comprises administering an effective amount of the monoclonal antibody according to (1) above to a mammal,
(23) Use of the monoclonal antibody according to (1) above for producing an agent for preventing or treating endometrial cancer, endometriosis or ovulation disorder.

The antibody of the present invention has an extremely high binding ability to a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof, and is represented by SEQ ID NO: 1 or SEQ ID NO: 2. A polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, which can neutralize the intracellular [Ca ²⁺ ] increasing activity of a polypeptide containing the amino acid sequence represented by By inhibiting the action of peptides or salts thereof, for example, digestive tract diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome), diseases associated with angiogenesis [eg, cancer (eg, thyroid cancer, testicular cancer) Adrenal tumor, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, etc.) , Polycystic ovary syndrome, ovarian hyperstimulation Group), pregnancy related diseases (eg pregnancy toxemia, placental growth failure, imminent abortion, endometriosis, infertility, ovulation disorders, etc.), eating disorders (eg, anorexia, bulimia etc.), Sleep disorders (eg, primary insomnia, circadian rhythm disorders (eg, physical condition modulation due to three shifts, time zone change syndrome, etc.), seasonal depression, reproductive dysfunction, endocrine disease, senile dementia , Alzheimer's disease, aging-related disorders, cerebral circulation disorders (eg, stroke, etc.), head injury, spinal cord injury, epilepsy, anxiety, depression, manic depression, schizophrenia, alcoholism, Parkinson's disease, hypertension, It is useful as a prophylactic / therapeutic agent for arteriosclerosis, arrhythmia, premenstrual tension syndrome, glaucoma, cancer, AIDS, diabetes and the like. Preferably, it is a prophylactic / therapeutic agent for diseases associated with neovascularization, diseases related to pregnancy, and the like, more preferably, a prophylactic / therapeutic agent for endometrial cancer, endometriosis, ovulation disorders, and the like. In addition, a cancer expressing a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof is found, and anticancer treatment by missile therapy using the antibody of the present invention is also possible. is there. A polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof can be quantified with high sensitivity and sensitivity by an immunoassay by the sandwich method using the monoclonal antibody of the present invention. Therefore, it is useful for elucidation of the physiological function and disease state of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof. Furthermore, by measuring the concentration of a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof in blood, for example, the above-mentioned diseases can be diagnosed. The antibody of the present invention can also be used for immunohistochemical staining of the above-mentioned polypeptide.

In the present specification, the protein (polypeptide) has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end in accordance with the convention of peptide designation. The protein used in the present invention including the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 may have any of a carboxyl group, a carboxylate, an amide, or an ester at the C-terminus.
Examples of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 include, for example, several (1 to 5) amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Is added to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid sequence in which several (1-5) amino acids are inserted, represented by SEQ ID NO: 1 or SEQ ID NO: 2. A polypeptide having an amino acid sequence in which several (1 to 5) amino acids in the amino acid sequence are substituted with other amino acids is used.
Examples of the salt of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 include physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts). And the like, and physiologically acceptable acid addition salts are particularly preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

As a monoclonal antibody (hereinafter also referred to as the antibody of the present invention) that specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, for example, the sequence A polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a monoclonal antibody that specifically reacts with a salt thereof, preferably a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 Or the monoclonal antibody etc. which react specifically with the salt are mentioned.
The antibody of the present invention preferably has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, 8th to 9, 11, 15, 17, 21, 23, 25 to 28, 30, 34, 36. -37, 39-40, 44-46, 48, 52-53, 55, 64, 66, 68, 70-73, 75-76 and 78-86th amino acids (preferably about 3 Reacts specifically to peptides containing ~ 6). In addition, the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 is not recognized.
More preferably, the antibody neutralizes the activity of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof.
Specific examples include a monoclonal antibody labeled with ZL1-107a or ZL1-234a.

The method for preparing the antigen of the antibody of the present invention and the method for producing the antibody will be described below.
(1) Preparation of antigen Antigen used for preparing the antibody of the present invention is the same as, for example, the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof. Any of these (synthetic) peptides having one or more antigenic determinants can be used (hereinafter, these may be simply referred to as human ZAQL-1 antigen).
A polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof can be produced according to a known method, for example, the method described in WO 02/06483, and (a ) Prepared using known methods or similar methods from mammalian tissues or cells such as humans, monkeys, rats, mice, etc., and (b) chemically synthesized by known peptide synthesis methods using peptides, synthesizers, etc. (C) It is also produced by culturing a transformant containing DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof.
(A) When preparing a human ZAQL-1 antigen from a tissue or cell of the mammal, the tissue or cell is homogenized and then extracted with an acid or alcohol, and the extract is subjected to salting out, dialysis, Purification and isolation can be performed by combining chromatography such as gel filtration, reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like.
(B) When chemically preparing human ZAQL-1 antigen, the synthetic peptide includes, for example, one having the same structure as human ZAQL-1 antigen purified from nature, SEQ ID NO: 1 or SEQ ID NO: In the amino acid sequence represented by 2, a peptide or the like containing one or more amino acid sequences identical to the amino acid sequence of any position consisting of 3 or more, preferably 6 or more amino acids, is used.
(C) When a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof is produced using a transformant containing DNA, the DNA is obtained by a known cloning method [ For example, Molecular Cloning (2nd ed .; method described in J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989)] can be prepared. The cloning method includes: (1) a cDNA library using a DNA probe or DNA primer designed based on the amino acid sequence of a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof; A method for obtaining a transformant containing a DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof by hybridization, or (2) SEQ ID NO: 1 Alternatively, using a DNA primer designed based on the amino acid sequence of a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 or a salt thereof, the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is obtained by PCR. And a method for obtaining a transformant containing a DNA encoding the polypeptide or a salt thereof. It is.

The peptide as the human ZAQL-1 antigen is (1) according to a known peptide synthesis method, or (2) a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 with an appropriate peptidase. It can also be manufactured by cutting.
As a method for synthesizing the peptide, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the peptide of interest can be produced by condensing a partial peptide or amino acid that can constitute the peptide and the remaining part, and removing the protective group when the product has a protective group. Examples of known condensation methods and protecting group elimination include the methods described below.
(I) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
Further, after the reaction, the peptide can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. .

  As the amide form of the peptide, a commercially available resin for peptide synthesis suitable for amide formation can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenyl. Examples include acetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. . Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target peptide. At the end of the reaction, the peptide is cleaved from the resin, and at the same time, various protecting groups are removed to obtain the target peptide. Alternatively, a partially protected peptide can be taken out using chlorotrityl resin, oxime resin, 4-hydroxybenzoic acid resin, and the like, and the protective group can be removed by conventional means to obtain the target peptide.

  For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOBt, etc.), or a pre-protected amino acid as a symmetric anhydride or HOBt ester or HOBt ester. Can be added to the resin after activation. The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents that are known to be usable for the peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and sulfoxides such as dimethyl sulfoxide. , Tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from a range known to be usable for peptide bond formation reaction, and is usually selected from the range of about -20 ° C to about 50 ° C. The activated amino acid derivative is usually used in an excess of about 1.5 to about 4 times. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.

Examples of the protecting group for the amino group of the raw material amino acid include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoro Acetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like can be mentioned. Examples of the protecting group for the carboxyl group include a C _1-6 alkyl group, a C _3-8 cycloalkyl group, a C _7-14 aralkyl group, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, Examples include phenacyl and benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, and trityl hydrazide.
The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, groups derived from carbonic acid such as benzyloxycarbonyl groups and ethoxycarbonyl groups. . Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl-Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, Bom, Bum, Boc, Trt, Fmoc and the like.
Examples of the activated carboxyl group of the raw material include the corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyano Methyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOBt)] and the like. Examples of the activated amino group of the raw material include the corresponding phosphoric acid amide.

Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethane. Examples include acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia. The elimination reaction by the acid treatment is generally performed at a temperature of −20 ° C. to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, 1 It is effective to add a cation scavenger such as 1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.
The protection of the functional group that should not be involved in the reaction of the raw material and the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a peptide, first, the α-carboxyl group of the carboxyl terminal amino acid is amidated, then the peptide chain is extended to the amino group side to the desired chain length, and then the N terminal of the peptide chain Of the peptide except for the protecting group of α-amino group and the peptide (or amino acid) except for the protecting group of the C-terminal carboxyl group, and condensing both peptides in a mixed solvent as described above. . The details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above method to obtain the desired crude peptide. This crude peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired peptide.
To obtain an ester of a peptide, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired peptide can be obtained in the same manner as the peptide amide.

Human ZAQL-1 antigen can also be directly immunized with insolubilized one. Alternatively, a complex in which human ZAQL-1 antigen is bound or adsorbed to an appropriate carrier may be immunized. The mixing ratio of the carrier (carrier) and human ZAQL-1 antigen (hapten) is such that what ratio can be used as long as the antibody can be efficiently produced against the human ZAQL-1 antigen bound or adsorbed to the carrier. It may be bound or adsorbed with a natural or synthetic polymer carrier commonly used in the production of antibodies against hapten antigens and bound or adsorbed at a ratio of 0.1 to 100 with respect to hapten 1 Can be used. Examples of natural polymer carriers include serum albumin of mammals such as cows, rabbits and humans, thyroglobulin of mammals such as cows and rabbits, hemoglobins of mammals such as cows, rabbits, humans and sheep, key Whole limpet hemocyanin is used. Examples of synthetic polymer carriers that can be used include various latexes such as polymers or copolymers of polyamino acids, polystyrenes, polyacryls, polyvinyls, polypropylenes, and the like.
Various coupling agents can be used for coupling the hapten and the carrier. For example, diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldehyde that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and thiol groups A dimaleimide compound such as N, N′-o-phenylene dimaleimide, a maleimide active ester compound that crosslinks an amino group and a thiol group, a carbodiimide compound that crosslinks an amino group and a carboxyl group, and the like are advantageously used. In addition, when cross-linking amino groups, an active ester reagent having a dithiopyridyl group on one of the amino groups (for example, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP)) was reacted. It is also possible to introduce a thiol group by post-reduction, introduce a maleimide group into the other amino group with a maleimide active ester reagent, and then react both.

(2) Production of monoclonal antibody Human ZAQL-1 antigen is administered to a warm-blooded animal by itself, for example, at a site where antibody production is possible or by a carrier by administration methods such as intraperitoneal injection, intravenous injection, and subcutaneous injection. Administered with a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice are preferably used for the production of monoclonal antibodies.
When producing monoclonal antibodies, select warm-blooded animals immunized with human ZAQL-1 antigen, for example, individuals with antibody titers from mice, collect spleen or lymph nodes 2-5 days after the final immunization, The antibody-producing hybridoma of the present invention can be prepared by fusing the antibody-producing cells contained in the above with myeloma cells. The anti-human ZAQL-1 antibody titer in serum is measured, for example, by reacting labeled human ZAQL-1 described below with antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc., and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to be used and the number of bone marrow cells is usually about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. In general, cell fusion can be efficiently carried out by incubating at 20 to 40 ° C., preferably 30 to 37 ° C., usually 1 to 10 minutes.

Various methods can be used for screening the antibody-producing hybridoma of the present invention. For example, a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, a salt thereof, or a partial peptide thereof can be used directly or Hybridoma culture supernatant is added to a solid phase (eg, microplate) adsorbed with a carrier, and then an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the cell used for cell fusion is a mouse, anti-mouse An immunoglobulin antibody is used) or protein A is added to detect the antibody of the present invention bound to the solid phase, the hybridoma culture supernatant is added to the solid phase adsorbed with the anti-immunoglobulin antibody or protein A, and radioactive Amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 labeled with a substance or enzyme The polypeptide containing the added, a method for detecting antibodies of the present invention bound to the solid phase. Screening and breeding of the antibody of the present invention are usually performed in a medium for animal cells (eg, RPMI1640) containing 10-20% fetal calf serum with addition of HAT (hypoxanthine, aminopterin, thymidine). The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer of the antibody of the present invention in the above antiserum.
Separation and purification of the antibody of the present invention can be carried out in the same manner as in the separation and purification of ordinary polyclonal antibodies (eg, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger ( Specific purification, such as adsorption / desorption by DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase or active adsorbent such as protein A or protein G, and dissociating the binding to obtain the antibody The law etc.].
As described above, the antibody of the present invention can be produced by culturing the hybridoma cell in vivo or ex vivo of a warm-blooded animal and collecting the antibody from the body fluid or culture.
In addition, (a) a hybridoma producing the antibody of the present invention that reacts with a partial region of a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and (b) the above polypeptide The screening of the hybridoma producing the antibody of the present invention that reacts but does not react with the partial region is performed, for example, by measuring the binding between the peptide corresponding to the partial region and the antibody produced by the hybridoma. be able to.

Hereinafter, the quantification method (immunoassay method) of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof will be described in more detail.
By using the antibody of the present invention, a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 can be detected by detection or tissue staining. For these purposes, the antibody molecule itself may be used, or F (ab ′) 2, Fab ′, or Fab fraction of the antibody molecule may be used.
The measurement method using the antibody of the present invention is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (for example, the amount of human ZAQL-1) in the solution to be measured is determined. Any measurement method may be used as long as it is a measurement method that is detected by chemical or physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, a sandwich method, a competitive method, an immunometric method, a nephrometry, and the like are used, and the sandwich method and the competitive method described later are particularly preferable in terms of sensitivity and specificity.

(1) Sandwich method After reacting an antibody of the present invention insolubilized on a carrier, a labeled antibody of the present invention (an antibody different from the antibody of the present invention insolubilized on a carrier) and a test solution, a labeling agent The polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof in the test solution is quantified. Preferably,
(I) reacting the monoclonal antibody labeled with ZL1-107a insolubilized on the carrier, the labeled monoclonal antibody labeled with ZL1-234a, and the test solution, and then measuring the activity of the labeling agent. A method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof in a characteristic test solution,
(Ii) reacting the monoclonal antibody labeled with ZL1-234a insolubilized on the carrier, the labeled monoclonal antibody labeled with ZL1-107a, and the test solution, and then measuring the activity of the labeling agent. Examples thereof include a method for quantitatively determining a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof in the characteristic test solution.

  In the sandwich method, the test solution is reacted (primary reaction) with the insolubilized antibody of the present invention, and the labeled antibody of the present invention is reacted (secondary reaction), followed by the labeling agent on the insolubilized carrier. Quantifying the amount of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof (preferably human ZAQL-1) in the test solution by measuring the activity of it can. The primary reaction and the secondary reaction may be performed at the same time or may be performed at different times. The labeling agent and the insolubilization method can be the same as those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be. In the measurement method by the sandwich method, for example, when the antibody used in the primary reaction is a monoclonal antibody indicated by ZL1-234a, the antibody used in the secondary reaction is a monoclonal antibody indicated by ZL1-107a. Preferably, when the antibody used in the primary reaction is a monoclonal antibody indicated by ZL1-107a, the antibody used in the secondary reaction is a monoclonal antibody indicated by ZL1-234a. These antibodies are preferably used after being labeled with, for example, horseradish peroxidase (HRP).

(2) Competition method The antibody of the present invention, a test solution, and a labeled polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof are competitively reacted, By measuring the ratio of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 bound to the antibody or a salt thereof, SEQ ID NO: 1 or sequence in the test solution The polypeptide containing the amino acid sequence represented by No. 2 or a salt thereof is quantified.
This reaction method is performed using, for example, a solid phase method.
As a specific example, an anti-mouse IgG antibody (manufactured by ICN / CAPPEL) is used as a solid-phase antibody, and (i) the antibody of the present invention (eg, ZL1-107a or ZL1) -234a), (ii) a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 labeled with HRP or a salt thereof, and (iii) a test solution, and after the reaction The HRP activity adsorbed on the solid phase is measured, and the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof is quantified.
(3) Immunometric method In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of the labeled antibody of the present invention, and then the solid phase and the liquid phase are separated. Or reacting the antigen in the test solution with an excess amount of the labeled antibody of the present invention, and then adding the immobilized antigen to the unreacted labeled antibody of the present invention on the solid phase. After binding, the solid phase and the liquid phase are separated. Next, the labeled amount of any phase is measured to quantify the amount of antigen in the test solution.
(4) Nephrometry In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.

In the above (1) to (4), a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a lanthanide element, or the like is used as a labeling agent used in a measurement method using a labeling substance. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are preferable as the enzyme, and those having a stable and high specific activity are preferable, for example, β-galactosidase, β -Glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., as fluorescent substances, for example, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (Amersham Biosciences, etc.)), Examples of luminescent substances include fluorescamine, fluorescein isothiocyanate, and the like, and examples thereof include luminol, luminol derivatives, luciferin, and lucigenin. Furthermore, a biotin-avidin system can also be used for the binding between the antibody and the labeling agent.
For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

In applying these individual immunological measurement methods to the method of the present invention, special conditions, operations, and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation method in each method. For the details of these general technical means, it is possible to refer to reviews, books, etc. [For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continuing radioimmunoassay” (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Medical School, published in 1978), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Second Edition) (Medical School, 1982) Published by Eiji Ishikawa et al., “Enzyme Immunoassay” (Third Edition) (Medical School, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), ibid Vol. 74 (Immunochemical Techniques (Part C)), ibid Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid Vol. 92 (Immunochemical Techniques (Part E: Monoclonal) Antibodies and General Immunoassay Methods)), ibid. Vol. 121 (see Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (published by Academic Press). Therefore, when constructing a measurement system such as the sandwich immunoassay method of the present invention, the method is not limited to the examples described later.
As described above, the antibody of the present invention can quantitate the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof with high sensitivity. It is useful for further elucidation of the above and diagnosis of diseases involving the above polypeptides. Specifically, using the antibody of the present invention, a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 contained in a body fluid (blood, plasma, serum, urine, etc.) or its By measuring the amount of salt, for example, gastrointestinal diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome), diseases with angiogenesis [eg, cancer (eg, thyroid cancer, testicular cancer, adrenal tumors) Pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, etc.), polycyst Ovarian syndrome, ovarian hyperstimulation syndrome, etc.), pregnancy related diseases (eg pregnancy toxemia, placental growth failure, imminent abortion, endometriosis, infertility, ovulation disorders, etc.), eating disorders (eg, anorexia) Dysphagia, bulimia, etc.), sleep disorders [eg, primary insomnia, circadian rhythm disorders (eg, Physical condition modulation due to three shifts, time zone change syndrome (time difference blur, etc.)), seasonal depression, reproductive dysfunction, endocrine disease, senile dementia, Alzheimer's disease, various disorders associated with aging, cerebral circulation disorders (eg, Stroke), head injury, spinal cord injury, epilepsy, anxiety, depression, manic depression, schizophrenia, alcoholism, Parkinson's disease, hypertension, arteriosclerosis, arrhythmia, premenstrual tension syndrome, glaucoma, cancer, AIDS, diabetes, etc. (preferably diseases associated with angiogenesis, diseases related to pregnancy, etc., more preferably endometrial cancer, endometriosis, ovulation disorders, etc.) can be diagnosed. For example, in the diagnosis of polycystic ovary syndrome, when the amount of the polypeptide in body fluid is quantified and the amount of the polypeptide is higher than that of a healthy person, for example, the blood concentration is about 3 fmol / ml or more, preferably about If it is 10 fmol / ml or more, the patient is diagnosed with polycystic ovary syndrome.

In addition, the antibody of the present invention is used for diseases involving a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, such as digestive diseases (eg, enteritis, diarrhea, constipation, Malabsorption syndromes), diseases with angiogenesis [eg, cancer (eg, thyroid cancer, testicular cancer, adrenal tumor, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, Stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, etc.), polycystic ovary syndrome, ovarian hyperstimulation syndrome, etc.), pregnancy related diseases (eg pregnancy toxemia, Placental growth failure, imminent abortion, endometriosis, infertility, ovulation disorders, etc., eating disorders (eg, anorexia, bulimia etc.), sleep disorders (eg, primary insomnia, circadian rhythm disorders (eg, Modulation of physical condition due to three shifts, time zone change syndrome (time difference blur), etc. ), Seasonal depression, reproductive dysfunction, endocrine disease, senile dementia, Alzheimer's disease, various disorders associated with aging, cerebral circulation disorders (eg, stroke, etc.), head injury, spinal cord injury, epilepsy, anxiety, depression, manic depression It is useful as a prophylactic / therapeutic agent for schizophrenia, alcoholism, Parkinson's disease, hypertension, arteriosclerosis, arrhythmia, premenstrual tension syndrome, glaucoma, cancer, AIDS, diabetes and the like. Preferably, it is a prophylactic / therapeutic agent for diseases associated with neovascularization, diseases related to pregnancy, and the like, more preferably, a prophylactic / therapeutic agent for endometrial cancer, endometriosis, ovulation disorders, and the like.
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, Cattle, cats, dogs, monkeys, etc.) orally or parenterally. The dose varies depending on the administration subject, target disease, symptom, administration route, and the like. For example, when used for the treatment of polycystic ovary syndrome in adults, the antibody of the present invention is used as a single dose. Usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day, Conveniently administered orally. In the case of oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the administration comprises the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration.
That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules). ), Syrups, emulsions, suspensions, and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. Is included. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-described antibody or a salt thereof with a normal suppository base.
The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually about 5 to 500 mg, especially about 5 to 5 mg for each dosage unit dosage form. It is preferable that 100 mg, other dosage forms contain about 10 to 250 mg of the antibody.
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.

In the specification of the present invention, when amino acids and the like are represented by abbreviations, those are based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. When there are optical isomers with respect to amino acids, L form is shown unless otherwise specified.
TFA: trifluoroacetic acid DMF: N, N-dimethylformamide Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: serine Thr: threonine Cys: cysteine Met: methionine Glu: glutamic acid Asp: aspartic acid Lys Lysine Arg: arginine His: histidine Phe: phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine SPDP: 3- (2-pyridyldithio) propionic acid N-succinimidyl GMBS: N- (4-maleimidobutyryl) Oxy) succinimide BSA: bovine serum albumin BTG: bovine thyroglobulin EIA: enzyme immunoassay HPLC: reverse phase high performance liquid chromatograph HRP: horseradish peroxidase FBS: fetal bovine serum d-FBS: dialyzed fetal bovine serum TMB: 3,3 ', 5,5'-tetramethylbenzidine H / HBSS: Hepes buffered Hanks balance solution

The SEQ ID NOs used in the present specification represent the amino acid sequences of the following peptides.
[SEQ ID NO: 1]
The amino acid sequence of human ZAQL-1 is shown.
[SEQ ID NO: 2]
The amino acid sequence of human ZAQL-1 is shown. The 48th Val of the amino acid sequence represented by SEQ ID NO: 1 is substituted with Ile.
[SEQ ID NO: 3]
The amino acid sequence of human ZAQL-2 is shown.

The hybridoma cell ZL1-107 obtained in Example 1 described below is an independent administrative corporation from 1 December 1st, 1st East, Tsukuba City, Ibaraki Prefecture, from December 9th, 2002 (zip code 305-8566). Deposited at the National Institute of Advanced Industrial Science and Technology as a deposit number FERM BP-8256.
The hybridoma cell ZL1-234 obtained in Example 1 described later is an independent administrative agency of 1st Central, 1st Street, Tsukuba City, Ibaraki Prefecture, from December 9, 2002. Deposited at the National Institute of Advanced Industrial Science and Technology as a deposit number FERM BP-8257.
In addition, about the antibody obtained from each hybridoma cell, it represents with the form which added "a" after the cell name.

The present invention will be described in more detail below with reference to examples, but these do not limit the scope of the present invention.
Human ZAQL-1 (SEQ ID NO: 1) used in the examples was obtained according to the method described in Reference Example 1 of WO 02/06483.
Human ZAQL-2 (SEQ ID NO: 3) used in the examples was obtained according to the method described in Reference Example 1 of WO 02/62944.

Example 1
(1) Preparation of immunogen containing human ZAQL-1 A complex of human ZAQL-1 and bovine thyroglobulin (BTG) was prepared and used as an immunogen. 10 mg of BTG was dissolved in 1 ml of 0.02 M phosphate buffer (pH 6.8) containing 0.1 M sodium chloride, mixed with 100 μl of DMF solution containing 1.86 mg of SPDP, and reacted at room temperature for 60 minutes. Further, 0.19 ml of 0.1 M acetate buffer (pH 4.5) containing 160 μmol dithiothreitol was added, reacted at room temperature for 30 minutes, centrifuged at 13,000 rpm for 1 minute, and the supernatant was separated by Sephadex G-25. Separation with a column (eluent, 0.1 M phosphate buffer containing 2 mM EDTA, pH 6.0) gave BTG introduced with SH groups. Next, 50 μl of DMF containing 2.07 μmol of GMBS was added to 0.1 M phosphate buffer (pH 6.8) containing 2 mg (207 nmol) of human ZAQL-1, and reacted at room temperature for 60 minutes. The reaction solution was separated with a Sephadex G-25 column (eluent, 0.1 M phosphate buffer, pH 6.7) to obtain human ZAQL-1 having a maleimide group introduced therein. Next, 6.5 mg of BTG introduced with SH group and 1.2 mg of antibody fraction introduced with maleimide group were mixed and reacted at 4 ° C. for 24 hours. After the reaction, it was dialyzed against physiological saline at 4 ° C. for 3 days.

(2) Immunity
Six to eight week old BALB / C female mice were immunized subcutaneously with complete Freund's adjuvant so that the human ZAQL-1-BTG complex obtained in (1) above was about 20 μg / mouse. Thereafter, the same amount of immunogen was boosted 2-3 times with incomplete Freund's adjuvant every 3 weeks.
(3) Preparation of horseradish peroxidase (HRP) labeled human ZAQL-1 Human ZAQL-1 and HRP (for enzyme immunoassay, manufactured by Boehringer Mannheim) are cross-linked and labeled with enzyme immunoassay (EIA) The body.
HRP 8.5 mg (213 nmol) was dissolved in 0.95 ml of 0.12 M sodium chloride 0.02 M phosphate buffer (pH 6.8), mixed with 50 μl of DMF solution containing 1.99 mg of SPDP, and reacted at room temperature for 60 minutes. . Furthermore, after adding 0.5 ml of 0.1 M acetate buffer (pH 4.5) containing 9.25 mg dithiothreitol and reacting at room temperature for 30 minutes, Sephadex G-25 column (eluent, 0.1 M containing 2 mM EDTA) Separation with a phosphate buffer (pH 6.0) gave HRP with an SH group introduced. 2 mg of human ZAQL-1 is dissolved in 0.1 M phosphate buffer (pH 6.7), mixed with 50 μl of DMF solution containing 0.69 mg (2.5 μmol) of GMBS, reacted at room temperature for 60 minutes, and then separated by Sephadex G- Separation with 25 columns (eluent, 0.1 M phosphate buffer, pH 6.8) gave human ZAQL-1 having a maleimide group introduced. The SH group-introduced HRP 1.67 mg (41.4 nmol) and the maleimide group-introduced human ZAQL-1 1.2 mg (124 nmol) were mixed and reacted at 4 ° C for 1 day. . After the reaction, the reaction mixture was fractionated with an Ultrogel AcA44 (manufactured by LKB-Pharmacia) column to obtain HRP-labeled human ZAQL-1.

(4) Measurement of antibody titer in antiserum of mice immunized with human ZAQL-1-BTG complex Human ZAQL-1-BTG complex was immunized twice at 3-week intervals, and one week later, blood was collected from the fundus Blood was collected. Further, the blood was centrifuged at 12,000 rpm for 15 minutes at 4 ° C., and the supernatant was recovered to obtain antiserum. The antibody titer in the antiserum was measured by the following method. To prepare an anti-mouse immunoglobulin antibody-binding microplate, first, add a 0.1 M carbonate buffer (pH 9.6) solution containing 100 μg / ml of anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel) to a 96-well microplate. 100 μl each was dispensed and left at 4 ° C. for 24 hours. Next, after washing the plate with phosphate buffered saline (PBS, pH 7.4), 300 μl of PBS containing 25% Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) was dispensed in order to block excess binding sites in the wells. And treated at 4 ° C. for at least 24 hours.
Buffer C [1% BSA, 0.4 M NaCl, 0.05% 2 mM EDTA · Na [ethylenediamine-N, N, N ′, N′-tetraacetic acid disodium salt] was added to each well of the anti-mouse immunoglobulin antibody-binding microplate. Diluted with 0.02M phosphate buffer, pH 7.0], 50 μl, and buffer C containing dihydrate (Ethylenediamine-N, N, N ′, N′-tetraacetic acid, disodium salt, dihydrate), DOJINDO) 100 μl of antiserum against the complex was added and reacted at 4 ° C. for 16 hours. Next, after the plate was washed with PBS, 100 μl of HRP-labeled human ZAQL-1 (300-fold diluted with buffer C) prepared in (4) above was added and reacted at room temperature for 1 day. Next, after the plate was washed with PBS, the enzyme activity on the solid phase was measured by adding 100 μl of TMB microwell peroxidase substrate system (KIRKEGAARD & PERRY LAB, INC, handled by Funakoshi Chemical) and reacting at room temperature for 10 minutes. After stopping the reaction by adding 100 μl of 1M phosphoric acid, absorption at 450 nm was measured with a plate reader (BICHROMATIC, manufactured by Dainippon Pharmaceutical Co., Ltd.).
The results are shown in FIG.
An increase in antibody titer against human ZAQL-1 was observed in antisera against human ZAQL-1-BTG complex in all 8 mice immunized.

(5) Preparation of anti-human ZAQL-1 monoclonal antibody A mouse having a relatively high antibody titer was inoculated intravenously with 0.2 to 100 ml of an immunogen dissolved in 0.2 ml of physiological saline. Immunization was performed. The spleen was removed from the mouse 4 days after the final immunization, pressed with a stainless mesh, filtered, and suspended in Eagles Minimum Essential Medium (MEM) to obtain a spleen cell suspension. As cells used for cell fusion, BALB / C mouse-derived myeloma cells P3-X63.Ag8.U1 (P3U1) were used (Current Topics in Microbiology and Imnology, vol. 81, page 1, 1978).
Cell fusion was performed according to the original method (Nature, 256, 495, 1975).
Spleen cells and P3U1 were washed three times with MEM without serum, mixed so that the ratio of spleen cells to P3U1 number was 5: 1, and centrifuged at 800 rpm for 15 minutes to precipitate the cells. After sufficiently removing the supernatant, the precipitate was lightly loosened, 0.3 ml of 45% polyethylene glycol (PEG) 6000 (manufactured by Kochlite) was added, and the mixture was allowed to stand in a 37 ° C. hot water bath for 7 minutes for fusion. After the fusion, MEM was added to the cells at a rate of 2 ml / min, and a total of 15 ml of MEM was added, followed by centrifugation at 600 rpm for 15 minutes to remove the supernatant. This cell precipitate was suspended in GIT medium (Wako Pure Chemicals) (GIT-10% FCS) containing 10% fetal bovine serum so that P3U1 was 2 × 10 ⁵ per ml, and a 24-well multi-dish ( 1 ml per well was seeded in 192 wells. After seeding, the cells were cultured in a 5% carbon dioxide incubator at 37 ° C. 24 hours later, 1 ml of GIT-10% FCS medium (HAT medium) containing HAT (hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 × 10 ⁻³ M) per well HAT selective culture was started by adding them one by one. The HAT selective culture was continued by discarding 1 ml of the old solution 3, 6 and 9 days after the start of the culture, and then adding 1 ml of HAT medium. Hybridoma growth was observed 9-14 days after cell fusion, and when the culture broth turned yellow (about 1 × 10 ⁶ cells / ml), the supernatant was collected and the method described in Example 1 (4) The antibody titer was measured according to
As an example of selecting an antibody-producing cell line derived from a mouse immunized with human ZAQL-1-BTG, a hybridoma antibody obtained by cell fusion using mouse No. 2 and No. 3 (see FIG. 1) The production state is shown in FIGS. The following four types of hybridomas were selected from the antibody-producing hybridomas [Table 1].

Next, the hybridoma obtained above was subjected to cloning by limiting dilution. In cloning, BALB / C mouse thymocytes were added as feeder cells at 5 × 10 ⁵ cells / well. After cloning, 1 to 3 × 10 ⁶ cells / mouse were intraperitoneally administered to mice (BALB / C) pre-peritoneally injected with 0.5 ml of mineral oil, and the antibody-containing ascites was collected 6 to 20 days later. did.
The monoclonal antibody was purified from the obtained ascites using a protein-A column. Recombinant protein-A-agarose in which 6 to 20 ml of ascites was diluted with an equal volume of binding buffer (1.5 M glycine (pH 9.0) containing 3.5 M NaCl, 0.05% NaN ₃ ) and equilibrated in advance with binding buffer The column was applied to a column (manufactured by Repligen), and the specific antibody was eluted with an elution buffer [0.1 M citrate buffer (pH 3.0) containing 0.05% NaN ₃ ]. The eluate was dialyzed against PBS at 4 ° C. for 2 days, sterilized by filtration through a 0.22 μm filter (Millipore), and stored at 4 ° C. or -80 ° C.
In determining the class / subclass of the monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA) method using a purified monoclonal antibody-bound solid phase was performed. That is, 100 μl of 0.1 M carbonate buffer (pH 9.6) solution containing 2 μg / ml of antibody was dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. According to the above method, after blocking excess binding sites in the well with Block Ace, the class and subclass of the immobilized antibody are examined by ELISA using Isotype Typing Kit (Mouse-Typer ^TM Sub-Isotyping Kit Bio-Rad). It was. In ZL1-222a, the H chain was IgG2b and the L chain was κ, and the remaining three types all had the H chain IgG1 and the L chain κ.

Example 2
Competition Method Enzyme Immunoassay The reaction specificity of a monoclonal antibody prepared using human ZAQL-1-BTG as an immunogen was examined by the following method.
First, the antibody titers of the obtained four kinds of monoclonal antibody solutions were examined by the method described in Example 1- (5), and the binding amount of the label was about the saturation binding amount as the antibody concentration used in the competition method-EIA. The antibody concentration to be 50% was determined (approximately 30-50 ng / ml). Next, to the anti-mouse immunoglobulin antibody-binding microplate described in Example 1- (4) above, (i) 50 μl of an anti-human ZAQL-1 antibody solution diluted with buffer C so that each monoclonal antibody was 50 ng / ml. (Ii) 50 μl of human ZAQL-1 solution or 50 μl of human ZAQL-2 solution diluted with buffer C, (iii) and HRP-labeled human ZAQL-1 (buffer C) obtained in Example 1- (3) above 50 μl) was added to an anti-mouse immunoglobulin antibody-coupled microplate and reacted at 4 ° C. for 16 hours. After the reaction, the plate was washed with PBS, and then the enzyme activity on the anti-mouse immunoglobulin antibody-bound microplate was measured by the method described in Example 1- (4) above.
The results are shown in Table 1.
It can be seen that both antibodies have reactivity with human ZAQL-1, but have no reactivity with human ZAQL-2.
As an example, FIG. 6 and FIG. 7 show the results of competition method-EIA of monoclonal antibodies ZL1-107a and ZL1-234a that showed the highest reactivity with human ZAQL-1 among them.
A standard curve of human ZAQL-1 for ZL1-107a and ZL1-234a showed that the human ZAQL-1 concentration giving the ratio to the maximum response (B / B ₀ ) = 0.5 was 0.8 nM and 1.2 nM, respectively. . From these results, it is considered that ZL1-107a and ZL1-234a are highly reactive to human ZAQL-1.

Example 3
Production of HRP-labeled anti-human ZAQL-1 monoclonal antibody (ZL1-234a-HRP)
50 μl of DMF containing 0.68 μmol of GMBS was added to 0.1 M phosphate buffer (pH 6.8) containing 8.51 mg (56.7 nmol) of the purified fraction of ZL1-234a and reacted at room temperature for 40 minutes. The reaction solution was separated with a Sephadex G-25 column (eluent, 0.1 M phosphate buffer, pH 6.7) to obtain 5.96 mg of an antibody fraction into which a maleimide group was introduced. Next, 60 μl of DMF containing 6.85 μmol of SPDP was added to 1.14 ml of 0.02 M phosphate buffer (containing 0.15 M NaCl) (pH 6.8) containing 18.3 mg (457 nmol) of HRP and reacted at room temperature for 40 minutes. . Next, 0.4 ml of 0.1 M acetate buffer (pH 4.5) containing 68.5 μmol of dithiothreitol was added, and the mixture was reacted at room temperature for 20 minutes. Then, a Sephadex G-25 column (eluent, 0.1 mM containing 2 mM EDTA) was used. Separation with M phosphate buffer (pH 6.0) yielded 9.8 mg of HRP into which SH groups had been introduced. Next, 8 mg of HRP introduced with SH group and 3 mg of antibody fraction introduced with maleimide group were mixed, concentrated to about 0.5 ml with collodion bag (manufactured by Sartorius), and left at 4 ° C for 16 hours. did. The reaction solution was applied to a Sephacryl S-300HR column (Pharmacia) equilibrated with 0.1 M phosphate buffer (pH 6.5), and the ZL1-234a-HRP complex fraction was purified.

Example 4
Sandwich method-EIA
100 μl each of 0.1 M carbonate buffer (pH 9.6 solution) containing 15 μg / ml of the purified monoclonal antibody ZL1-107a obtained in Example 1 was dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. Excess binding sites in the wells were inactivated by adding 400 μl of Block Ace diluted 4-fold with PBS.
100 μl of human ZAQL-1 or human ZAQL-2 standard solution diluted with 0.02M phosphate buffer (pH 7) containing buffer C was added to the prepared plate, and reacted at 4 ° C. for 24 hours. After washing with PBS, 100 μl of ZL1-234a-HRP (10,000-fold diluted with buffer C) prepared in Example 3 above was added and reacted at 4 ° C. for 24 hours. After washing with PBS, the enzyme activity on the solid phase was measured using the TMB microwell peroxidase substrate system by the method described in Example 1 (4) (enzyme reaction 20 minutes).
The results are shown in FIG.
This sandwich method-EIA was able to detect human ZAQL-1 at 0.1 fmol / mL and did not react with human ZAQL-2 up to 10001 fmol / mL. Therefore, the sandwich method-EIA using ZL1-107a as a solid phase antibody and ZL1-234a-HRP as a label can detect human ZAQL-1 with extremely high sensitivity and extremely selective.

Example 5
Neutralizing the biological activity of human ZAQL-1 by ZL1-107a and ZL1-234a
The neutralizing activity against human ZAQL-1 by ZL1-107a and ZL1-234a was determined using ZAQ-expressing CHO cells (ZAQC-B1 cells) and FLIPR (Molecular Devices) described in Example 3 (5) of WO 02/06483. ^Was used to measure the intracellular Ca ²⁺ ion concentration increasing activity as an index.
Dulbecco's modified Eagle medium (DMEM) medium (Nissui Pharmaceutical Co., Ltd.) containing dialysis-treated fetal bovine serum (dFBS) (JRH BIOSCIENCES) to ZAQ-expressing CHO cells at 1.2 × 10 ⁵ cells / ml ( Suspend in 10% dFBS-DMEM) and inoculate 200μl of each well into a 96 well plate for FLIPR (Black plate clear bottom, Coster) using a dispenser (4 × 10 ⁴ cells / 200μl / well) It was used after overnight culture at 37 ° C. in a 5% CO ₂ incubator (hereinafter referred to as cell plate). FLIPR assay buffer [Nissui Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8g, sodium bicarbonate 0.35g, HEPES 4.77g, adjusted to pH 7.4 with 6M sodium hydroxide solution, filled up to 1L, filter sterilized 20 ml, 250 mM Probenecid (SIGMA) 200 μl, fetal bovine serum (FBS) 210 μl were mixed. In addition, 2 vials (50 μg) of Fluo 3-AM (Dojin Chemical Laboratories) were dissolved in 40 μl of dimethyl sulfoxide and 40 μl of 20% Pluronic acid (Molecular Probes), and this was dissolved in H / HBSS [Hepes Buffered Hanks Balance Solution (Nissui Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium bicarbonate 0.35 g, HEPES 4.77 g, adjusted to pH 7.4 with sodium hydroxide solution, filter sterilized)] 20 ml, 250 mM Add to H / HBSS-Probenecid-FBS solution consisting of 200 μl of Probenecid and 200 μl of fetal bovine serum (FBS), and after mixing, dispense 100 μl of each well into the cell plate after removing the culture solution using an 8-unit pipette. Incubated for 1 hour at 37 ° C. in a 5% CO ₂ incubator (dye loading). ZL1-107a and ZL1-234a and anti-PrRP monoclonal antibody (P2L-1Ta) having the same IgG subclass structure (IgG1, κ) as ZL1-107a and ZL1-234a as control antibodies (Biochem. Biophys. Res. Commun., 257 , 264-268 (1998)) is diluted with 120 μl of Hanks' / HBSS containing 2.5 mM Probenecid and 0.2% BSA, and 1 hour at 37 ° C with human ZAQL-1 (1 x 10 ^-8 M) After the incubation, 5 μl of each fraction was transferred to a 96-well plate for FLIPR (V-Bottom plate, Coster) (hereinafter referred to as a sample plate). After loading the dye on the cell plate, wash the cell plate 4 times with a wash buffer with 2.5 mM Probenecid added to Hanks' / HBSS using a plate washer (Molecular Devices), leaving 100 μl of wash buffer after washing. . The cell plate and the sample plate were set in FLIPR, and measurement was performed (FLIPR transferred 50 μl of sample from the sample plate to the cell plate).
The results are shown in FIG.
From this, the activity of human ZAQL-1 (3.3 × 10 ⁻¹⁰ M) is approximately 97% for ZL1-107a at 3.3 × 10 ⁻¹⁰ M, and approximately 97% at 3.3 × 10 ⁻⁹ M, which is 10 times higher in molar concentration. It turns out that it suppressed. ZL1-234a has the activity of human ZAQL-1 (3.3 × 10 ⁻¹⁰ M) about 87% at an equimolar concentration of 3.3 × 10 ⁻¹⁰ M, and about 10% higher at a concentration of 3.3 × 10 ⁻⁹ M It can be seen that it was suppressed by 98%. On the other hand, in the control antibody P2L-1Ta, inhibition of human ZAQL-1 activity was not observed even at 3.3 × 10 ⁻⁹ M, which was 10 times higher in molar concentration than human ZAQL-1.
From the above, it has been clarified that ZL1-107a and ZL1-234a neutralize the intracellular Ca ²⁺ increasing activity of human ZAQL-1, and these antibodies can be used as neutralizing antibodies.

Example 6
Quantification of human ZAQL-1 in plasma Human plasma was diluted 2-fold with the same amount of buffer C, and human ZAQL-1 was quantified by the sandwich method-EIA described in Example 4 above.
The results are shown in Table 2.

Example 7
Detection of human ZAQL-1 in human plasma by RP-HPLC To identify human ZAQL-1 immunoreactivity contained in human plasma as described in Example 6, add 20 ml of acetonitrile to 10 ml of human plasma and mix Then, centrifugation (15,000 rpm, 5 minutes) was performed to remove proteins. After the supernatant was lyophilized, the fraction was concentrated and fractionated by reverse phase HPLC using a column (ODS-80 ^™ ).
Column conditions:
Column: ODS-80 ^TM (4.6 x 250 mm)
Eluent: Liquid A (0.05% trifluoroacetic acid containing 5% acetonitrile)
Liquid B (0.05% trifluoroacetic acid containing 60% acetonitrile)
Elution method: Increase acetonitrile concentration from 5% to 30% in the first 5 minutes,
Then it was increased linearly to 30-40% over 30 minutes.
Flow rate: 1.0 ml / min Fraction: 0.5 ml / tube
The elution fraction was freeze-dried, dissolved in 250 μl of buffer C, and subjected to the sandwich method-EIA described in Example 4 above.
The results are shown in FIG.
Since the immunoreactivity of human ZAQL-1 in plasma was mostly detected at the elution position of human ZAQL-1 (recovery rate: 102%), the sandwich method-EIA detects human ZAQL-1 Was confirmed.
Thus, this measurement system is an important means for studying the fluctuation of human ZAQL-1 in plasma.

表３から、血中ヒトZAQL-1濃度は、妊娠初期（妊娠13週まで）に上昇し、その後妊娠の経過とともに減少していき、出産後正常レベルに戻ると考えられる。女性非妊時（1.51±0.35 fmol/ml)と比較すると、妊娠初期（4-13週）で約4.0倍に上昇し、妊娠後期（28-40週）でも約2.8倍に上昇していた。
これより、ヒトZAQL-1は、妊娠期に産生または分泌が上昇することがわかる。従って、血中ヒトZAQL-1濃度は妊娠の指標となりうるため、本発明の抗体は、臨床診断薬として有用である。 Example 8
Quantification of human ZAQL-1 in plasma of pregnant women Pregnant plasma obtained at each week of life is diluted 2-fold with the same amount of buffer C, and human ZAQL-1 is quantified by the sandwich method-EIA described in Example 4 above. did. Pregnant plasma was purchased from DCP and informed consent was taken.
The results are shown in Table 3.

From Table 3, it is considered that the blood human ZAQL-1 concentration increases in the early stage of pregnancy (up to 13 weeks of pregnancy), then decreases with the progress of pregnancy, and returns to the normal level after delivery. Compared to female non-pregnancy (1.51 ± 0.35 fmol / ml), it was about 4.0 times higher in the first trimester (4-13 weeks) and about 2.8 times in the second trimester (28-40 weeks).
This shows that production or secretion of human ZAQL-1 increases during pregnancy. Therefore, since the blood human ZAQL-1 concentration can be an indicator of pregnancy, the antibody of the present invention is useful as a clinical diagnostic agent.

Example 9
Detection of human ZAQL-1 in pregnant woman plasma by RP-HPLC To identify human ZAQL-1 immunoreactivity contained in pregnant woman plasma as described in Example 8, 2 ml of acetonitrile was added to 1 ml of pregnant woman plasma at 11 weeks of gestation After mixing, the mixture was centrifuged (15,000 rpm, 5 minutes) to remove the protein. After the supernatant was lyophilized, this fraction was concentrated and fractionated under the same conditions as in Example 7 by reverse phase HPLC using a column (ODS-80 ^™ ).
The elution fraction was freeze-dried, dissolved in 250 μl of buffer C, and subjected to the sandwich method-EIA described in Example 4 above.
The results are shown in FIG.
Since the immunoreactivity of human ZAQL-1 in the plasma of pregnant women was almost detected at the elution position of human ZAQL-1 (recovery rate 70%), the sandwich method-EIA was able to detect human ZAQL-1 in the plasma of pregnant women. It was confirmed that it was detected.
From this, it was found that the concentration of human ZAQL-1 in plasma increased during pregnancy.

以上の結果から、子宮内膜症および排卵障害を併発している患者の卵胞液中において、ヒトZAQL-1が高値を示したことより、ヒトZAQL-1の子宮内膜癌および子宮内膜症への関与が示唆された。 Example 10
Quantification of human ZAQL-1 in follicular fluid Patient follicular fluid was diluted 2-fold with the same amount of buffer C, and human ZAQL-1 was quantified by the sandwich method-EIA described in Example 4 above. The follicular fluid was provided in collaboration with an assistant professor of the Department of Obstetrics and Gynecology at the University of Tsukuba, with informed consent. It was also approved by the Ethics Committee of the University of Tsukuba and Takeda Pharmaceutical Company Limited.
The results are shown in Table 4.

Based on the above results, human ZAQL-1 showed a high level in the follicular fluid of patients with endometriosis and ovulation disorder, indicating that human ZAQL-1 endometrial cancer and endometriosis It was suggested to be involved.

Example 11
Detection of human ZAQL-1 in follicular fluid by RP-HPLC To identify human ZAQL-1 immunoreactivity contained in follicular fluid as described in Example 10, 0.9 ml of acetonitrile was added to 0.45 ml of follicular fluid. After mixing, centrifugation (15,000 rpm, 5 minutes) was performed to remove proteins. After the supernatant was lyophilized, this fraction was concentrated and fractionated under the same conditions as in Example 7 by reverse phase HPLC using a column (ODS-80 ^™ ).
The elution fraction was freeze-dried, dissolved in 250 μl of buffer C, and subjected to the sandwich method-EIA described in Example 4 above.
The results are shown in FIG.
Since the immunoreactivity of human ZAQL-1 in the follicular fluid was almost detected at the elution position of human ZAQL-1 (recovery rate 70%), the sandwich method-EIA was able to detect human ZAQL-1 in the plasma of pregnant women. It was confirmed that it was detected.

The measurement result of the antibody titer in the antiserum of the mouse | mouth which immunized the human ZAQL-1-BTG complex is represented. In the figure,-◇-is mouse No. 1,-□-is mouse No. 2,-△-is mouse No. 3,-○-is mouse No. 4,-◆-is mouse No. 5,-■ -Represents mouse No. 6,-▲-represents mouse No. 7, and-●-represents mouse No. 8. The hybridoma derived from the mouse | mouth which immunized the human ZAQL-1-BTG complex represents the state (result of an absorption analysis) producing the antibody. The hybridoma derived from the mouse | mouth which immunized the human ZAQL-1-BTG complex represents the state (result of an absorption analysis) producing the antibody. The hybridoma derived from the mouse | mouth which immunized the human ZAQL-1-BTG complex represents the state (result of an absorption analysis) producing the antibody. The hybridoma derived from the mouse | mouth which immunized the human ZAQL-1-BTG complex represents the state (result of an absorption analysis) producing the antibody. The results of competition method-EIA of ZL1-107a and ZL1-234a are shown. In the figure, − ● − indicates the reactivity between ZL1-107a and ZAQL-1, − ○ − indicates the reactivity between ZL1-107a and ZAQL-2, − ■ − indicates that ZL1-234a and ZAQL-1 Reactivity,-□-represents the reactivity between ZL1-234a and ZAQL-2. The result of sandwich method-EIA using ZL1-107a and ZL1-234a is shown. In the figure,-●-represents the reactivity of ZAQL-1, and -o- represents the reactivity of ZAQL-2. The neutralization effect | action with respect to the intracellular Ca < ²⁺ > ion concentration raising activity using the ZAQ expression CHO cell (ZAQC-B1) of ZAQL-1 at the time of ZL1-107 or ZL1-234a or P2L-1Ta coexistence is represented. In the figure, the black bars indicate the ratio of the activity to increase intracellular Ca ²⁺ ion concentration in ZAQC-B1 cells when ZL1-107a coexists with ZAQL-1 (when no antibody is added), and the hatched bars indicate ZL1- The ratio of the activity to increase the intracellular Ca ²⁺ ion concentration in ZAQC-B1 cells when 234a coexists with ZAQL-1 (when no antibody is added), and the white bar indicates that P2L-1Ta coexists with ZAQL-1. Shows the ratio of the activity of increasing the intracellular Ca ²⁺ ion concentration in ZAQC-B1 cells to the control (when no antibody is added). The elution position of human ZAQL-1 immunoreactivity in human plasma fractionated using reverse phase HPLC is shown. It represents the elution position of human ZAQL-1 immunoreactivity in pregnant woman plasma fractionated using reverse phase HPLC. The elution position of human ZAQL-1 immunoreactivity in follicular fluid fractionated using reverse phase HPLC is shown.

Claims (20)

A monoclonal antibody labeled with ZL1-107a that can be produced from a hybridoma cell labeled with ZL1-107 (FERM BP-8256). A monoclonal antibody labeled with ZL1-234a that can be produced from a hybridoma cell labeled with ZL1-234 (FERM BP-8257). The monoclonal antibody according to claim 1 or 2, which specifically reacts with a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof. The monoclonal antibody according to claim 3 , which specifically reacts with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. The monoclonal antibody according to claim 3, which does not recognize the polypeptide having the amino acid sequence represented by SEQ ID NO: 3. The monoclonal antibody according to claim 3 , which is labeled. The monoclonal antibody according to claim 3, which has neutralizing activity against a peptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. A diagnostic agent comprising the monoclonal antibody according to any one of claims 1 to 7 . The diagnostic agent according to claim 8, which is a diagnostic agent for endometrial cancer, endometriosis or ovulation disorder. The diagnostic agent according to claim 8, which is a diagnostic agent for pregnancy. A method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof, wherein the monoclonal antibody according to any one of claims 1 to 7 is used. A method for detecting a disease involving a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, wherein the monoclonal antibody according to any one of claims 1 to 7 is used. . The detection method according to claim 12 , wherein the disease is endometrial cancer, endometriosis or ovulation disorder. A method for detecting pregnancy, wherein the monoclonal antibody according to any one of claims 1 to 7 is used. The monoclonal antibody according to any one of claims 1 to 7 , and a test solution and a labeled polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof are competitive. The amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the test solution, wherein the ratio of the labeled polypeptide or the salt thereof bound to the antibody is measured For quantification of a polypeptide or a salt thereof. (A) After monoclonal antibody of claim 1, wherein the insolubilized on a carrier, a monoclonal antibody and a test fluid of labeled claim 2 is reacted to measure the activity of the labeling agent, or, (b) after monoclonal antibody of claim 1, wherein the insolubilized on a carrier, a monoclonal antibody and a test fluid of labeled claim 2 is reacted, the test fluid, which comprises measuring the activity of the labeling agent A method for quantifying a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof. Hybridoma cells producing a monoclonal antibody according to any one of claims 1-7. ZL1-107 (FERM BP-8256) or ZL1-234 hybridoma cells are labeled by (FERM BP-8257). The hybridoma cell according to claim 17 or 18 is grown in vitro or cultured in vitro, and the monoclonal antibody according to any one of claims 1 to 7 is collected from the body fluid or culture thereof. A method for producing the monoclonal antibody according to any one of 1 to 7 . 19. Hybridoma cells of claim 17 or 18, wherein culturing in vitro or in vivo, to the ascites, and recovering the monoclonal antibody of claim 1 or claim 2 wherein the supernatant of blood or culture The manufacturing method described.

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