JP2004242671A - Antibody and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a monoclonal antibody capable of quantitatively determining human ZAQL-1 sensitively and specifically, a method of detection/determination of the human ZAQL-1 using the antibody and a diagnostic agent using these. <P>SOLUTION: The antibody specifically reacts with the human ZAQL-1. The antibody is used in the method of detection/determination of the human ZAQL-1. These are used in the diagnostic agent. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a novel antibody having binding specificity for 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 the development of a diagnostic and prophylactic / therapeutic agent for a disease involving the polypeptide or a salt thereof utilizing a neutralizing activity, etc. About.

Human ZAQ ligand-1 (a 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 for ZAQ receptor, It is a peptide having an ileal contraction effect (Patent Document 1 WO 02/06483). In addition, a mature human-type Bv8 peptide (hereinafter sometimes abbreviated as human ZAQL-2), which is a mammalian type peptide of frog Bv8 having a structure similar to human ZAQ ligand-1, is composed of MAP kinase and PI-3. Activates kinase and has a neuroprotective effect (Eur. J. Neuroscience 13, 1694, 2001). Subsequently, these peptides were also reported as novel 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 involved in the formation of a highly permeable endothelial structure (fenestrate) that characterizes endocrine tissue, as an endocrine tissue-specific growth factor (EG-VEGF). It is reported that the transcription control region of the ZAQL-1 gene contains a recognition site for hypoxia-inducible factor-1 (HIF-1), which is involved in the induction of expression under hypoxia, and induces gene expression under hypoxia. (Non-Patent Document 1 Nature. 412, 877, 2001).
WO 02/06483 Nature. 412, 877, 2001

  In order to further elucidate the physiological function of human ZAQL-1, a measurement system for detecting and quantifying human ZAQL-1 simply and with high sensitivity has been desired.

  The present inventors have conducted intensive studies in order to solve the above problems, and as a result, using human ZAQL-1 as an immunogen, produced a plurality of monoclonal antibodies, and by combining these, human ZAQL-1 with high sensitivity An immunoassay that can be detected specifically and specifically was developed. This makes it possible to easily and highly sensitively measure the fluctuation of human ZAQL-1 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 (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 to 9, 11, 15, 17, 21, 23, 25 to 28, 30, 34, 36 to 37, 39 to 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 78-86. 1) the monoclonal antibody according to
(4) the monoclonal antibody according to (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) above,
(6) the monoclonal antibody according to the above (1), designated by ZL1-107a, which can be produced from the hybridoma cell designated by ZL1-107 (FERM BP-8256);
(7) The monoclonal antibody according to the above (1), designated by ZL1-234a, which can be produced from the hybridoma cell designated by ZL1-234 (FERM BP-8257).
(8) The monoclonal antibody according to (1), which has a neutralizing activity against a peptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
(9) a medicine comprising the monoclonal antibody according to (1),
(10) The medicament according to the above (9), which is an agent for preventing or treating endometrial cancer, endometriosis or ovulation disorder.
(11) a diagnostic agent comprising the monoclonal antibody according to (1) above,
(12) The diagnostic agent according to the above (11), 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, comprising using the monoclonal antibody according to (1) above,
(15) A method for diagnosing 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, comprising using the monoclonal antibody according to (1) above,
(16) The diagnostic method according to (15), wherein the disease is endometrial cancer, endometriosis or ovulation disorder.
(17) The monoclonal antibody described in (1) above is competitively reacted with a test solution and a labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or 2 or a salt thereof. 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. Method for quantifying a polypeptide or a salt thereof,
(17a) reacting the monoclonal antibody according to the above (1) insolubilized on the carrier, the labeled monoclonal antibody according to the above (1) (an antibody different from the monoclonal antibody insolubilized on the carrier), and a test solution; Measuring the activity of the labeling agent, and then quantifying 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,
(18) (a) After reacting the monoclonal antibody according to the above (6), the labeled monoclonal antibody according to the above (7) and a test solution, which are insolubilized on a carrier, measure the activity of the labeling agent. Or
(B) reacting the monoclonal antibody according to the above (7), the labeled monoclonal antibody according to the above (6), and a test solution which are insolubilized on a carrier, 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 test solution,
(19) a hybridoma cell producing the monoclonal antibody according to (1),
(20) The hybridoma cell according to the above (19), which is represented by ZL1-107 (FERM BP-8256) or ZL1-234 (FERM BP-8257),
(21) The method according to (6), wherein the hybridoma cell according to (19) is cultured in vivo or in vitro, and the monoclonal antibody according to (6) or (7) is collected from the body fluid or culture. ) Or the method for producing a monoclonal antibody according to (7),
(22) a method for preventing or treating endometrial cancer, endometriosis or ovulation disorder, which comprises administering to a mammal an effective amount of the monoclonal antibody according to (1);
(23) Use of the monoclonal antibody according to (1) above for producing a prophylactic / therapeutic agent for endometrial cancer, endometriosis or ovulation disorder is provided.

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 has an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is capable of neutralizing the intracellular [Ca ²⁺ ] elevating activity of the polypeptide or the salt thereof. By inhibiting the action of the peptide or its salt, for example, gastrointestinal diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.), diseases associated with angiogenesis [eg, cancer (eg, thyroid cancer, testicular cancer) , Adrenal gland tumor, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, etc.) , Polycystic ovary syndrome, ovarian hyperstimulation Group), pregnancy-related diseases (eg, preeclampsia, placental growth retardation, imminent abortion, endometriosis, infertility, ovulation disorders, etc.), eating disorders (eg, anorexia nervosa, bulimia, etc.), Sleep disorders [eg, primary insomnia, circadian rhythm disorders (eg, physical shifts due to three shifts, time band shift syndrome (jet lag), etc.)], seasonal depression, reproductive dysfunction, endocrine disorders, senile dementia , Alzheimer's disease, aging disorders, cerebral circulatory disorders (eg, stroke), 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 syndrome, glaucoma, cancer, AIDS, diabetes and the like. Preferably, it is a prophylactic / therapeutic agent for a disease associated with angiogenesis, a disease related to pregnancy, and more preferably a prophylactic / therapeutic agent for an endometrial cancer, endometriosis, ovulation disorder and the like. In addition, a cancer expressing the 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. By the immunoassay by the sandwich method using the monoclonal antibody of the present invention, the 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 specificity. Therefore, the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof is useful for elucidating the physiological function and disease state. 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 and the like can be diagnosed. Further, the antibody of the present invention can also be used for immunohistological staining of the polypeptide.

In the protein (polypeptide) in this specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. The protein used in the present invention, including the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, may have a carboxyl group at the C-terminus, a carboxylate, an amide or an ester.
Examples of the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 include, for example, the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the number (1 to 5) , An amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and an amino acid sequence obtained by inserting a number (1 to 5) of amino acids into the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2; For example, a polypeptide having an amino acid sequence in which several (1 to 5) amino acids in the amino acid sequence are substituted with another amino acid 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 and organic acids) and bases (eg, alkali metal salts) And the like, and especially preferred are physiologically acceptable acid addition salts. 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, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

Examples of a monoclonal antibody that specifically reacts with the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof (hereinafter, may be referred to as the antibody of the present invention) include, for example, the sequence A monoclonal antibody specifically reacting with a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, and preferably a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 Or a monoclonal antibody specifically reacting with a salt thereof.
The antibody of the present invention is preferably an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence of amino acids 8 to 9, 11, 15, 17, 21, 23, 25 to 28, 30, 34, 36. To 37, 39 to 40, 44 to 46, 48, 52 to 53, 55, 64, 66, 68, 70 to 73, 75 to 76 and at least one of amino acids 78 to 86 (preferably about 3 to ~ 6) specifically. In addition, it does not recognize the polypeptide having the amino acid sequence represented by SEQ ID NO: 3.
More preferred is an antibody that neutralizes the activity of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof.
Specific examples include the monoclonal antibody represented by ZL1-107a or ZL1-234a, and the like.

The method for preparing the antigen of the antibody of the present invention and the method for producing the antibody are described below.
(1) Preparation of Antigen The antigen used for preparing the antibody of the present invention is, for example, the same as the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof. Any peptide such as a (synthetic) peptide having one or more antigenic determinants can be used (hereinafter, these may be simply referred to as human ZAQL-1 antigen).
The 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 further comprises (a ) For example, prepared from tissues or cells of mammals such as humans, monkeys, rats, and mice using known methods or modifications thereof, and (b) chemically synthesized by known peptide synthesis methods using peptide synthesizers and the like. , (C) also by culturing a transformant containing a DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof.
(A) When preparing human ZAQL-1 antigen from the mammalian tissue or cells, the tissue or cells are 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, and affinity chromatography.
(B) When preparing human ZAQL-1 antigen chemically, the synthetic peptide may be, for example, one having the same structure as the above-mentioned natural purified human ZAQL-1 antigen, SEQ ID NO: 1 or SEQ ID NO: In the amino acid sequence represented by 2, a peptide containing one or more of the same amino acid sequence as the amino acid sequence at an arbitrary position consisting of three or more, preferably six 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 the DNA, the DNA is prepared by a known cloning method [ For example, it can be prepared according to the method described in Molecular Cloning (2nd ed .; J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). The cloning method includes the steps of (1) using a DNA probe or a 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, using a cDNA library A method for obtaining a transformant containing a DNA encoding the polypeptide having 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 the 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. Methods for obtaining a transformant containing a DNA encoding the polypeptide or a salt thereof are described. It is.

The peptide as a human ZAQL-1 antigen can be prepared by (1) 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 using an appropriate peptidase. It can also be manufactured by cutting.
As a method for synthesizing the peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can constitute the peptide with the remaining portion, and removing the protective group when the product has a protective group. Known condensation methods and elimination of protecting groups include, for example, 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)
After the reaction, the peptide can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and when it is obtained by a salt, it can be converted to 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 a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethylphenyl. Acetamide methyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like can be given. . 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 in accordance with the sequence of the target peptide according to various known condensation methods. 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 desired peptide. Alternatively, a partially protected peptide can be taken out using a chlorotrityl resin, an oxime resin, a 4-hydroxybenzoic acid-based resin, or the like, and the protecting group can be removed by a conventional means to obtain the desired peptide.

  For the condensation of the protected amino acids described above, 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. Activation by these involves adding the protected amino acid directly to the resin with a racemization inhibitor additive (eg, HOBt, HOOBt, etc.), or the amino acid previously protected as a symmetric acid anhydride or HOBt ester or HOOBt ester. Can be added to the resin after activation. The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for the peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and sulfoxides such as dimethyl sulfoxide. And 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 the peptide bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to about 50 ° C. The activated amino acid derivative is usually used in about 1.5 to about 4-fold excess. 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 cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole so that the subsequent reaction is not affected.

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

As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane Examples include acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, and the like, and reduction with sodium in liquid ammonia. The elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of -20 ° C to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, It is effective to add a cation scavenger such as 2,2-ethanedithiol. Also, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an 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 is also removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.
The protection and protection of the functional group which should not be involved in the reaction of the raw materials, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a peptide, first, after amidating the α-carboxyl group of the carboxyl-terminal amino acid, extending the peptide chain to a desired chain length on the amino group side, and then N-terminal of the peptide chain To produce a peptide (or amino acid) from which only the α-amino group protecting group has been removed and a peptide (or amino acid) from which only the C-terminal carboxyl group protecting group has been removed, and condense these peptides in a mixed solvent as described above. . 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-mentioned method to obtain a desired crude peptide. This crude peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired peptide.
In order to obtain an ester of the 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 amide of the peptide.

The human ZAQL-1 antigen can also be directly immunized with the insolubilized one. Alternatively, a complex obtained by binding or adsorbing a human ZAQL-1 antigen to a suitable carrier may be immunized. The mixing ratio between the carrier (carrier) and the human ZAQL-1 antigen (hapten) is determined by the ratio of what kind of antibody to human ZAQL-1 antigen bound or adsorbed to the carrier, if the antibody can be efficiently produced. May be bound or adsorbed, and a natural or synthetic polymer carrier commonly used in the production of an antibody against a hapten antigen is bound or adsorbed at a ratio of 0.1 to 100 to hapten 1 by weight. Can be used. Examples of the natural polymer carrier include serum albumin of mammals such as bovine, rabbit and human, and thyroglobulin of mammal such as bovine and rabbit, for example, hemoglobin of mammalian such as bovine, rabbit, human and sheep, and key. Whole limpet hemocyanin or the like is used. As the synthetic polymer carrier, for example, various latexes such as a polymer such as polyamino acids, polystyrenes, polyacryls, polyvinyls, and polypropylenes or a copolymer can be used.
Various coupling agents can be used for coupling the hapten and the carrier. For example, diazonium compounds such as bisdiazotized benzidine that crosslink tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldetite that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and crosslinks between thiol groups A dimaleimide compound such as N, N'-o-phenylenedimaleimide, a maleimide active ester compound that bridges an amino group and a thiol group, and a carbodiimide compound that bridges an amino group and a carboxyl group are conveniently used. Also, when bridging amino groups, an active ester reagent having a dithiopyridyl group at one amino group (eg, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), etc.) was reacted. After the reduction, a thiol group can be introduced, and a maleimide group can be introduced into the other amino group with a maleimide active ester reagent, and then the two can be reacted.

(2) Preparation of Monoclonal Antibody Human ZAQL-1 antigen can be used alone or in a carrier at a site where antibody production can be performed on a warm-blooded animal by an administration method such as intraperitoneal injection, intravenous injection, or subcutaneous injection. Administered with a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chickens and the like, and mice are preferably used for preparing monoclonal antibodies.
When preparing a monoclonal antibody, a warm-blooded animal immunized with human ZAQL-1 antigen, for example, an individual having an antibody titer was selected from a mouse, and the spleen or lymph node was collected 2 to 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 measurement of the anti-human ZAQL-1 antibody titer in the serum is performed, for example, by reacting the labeled human ZAQL-1 described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, and the like, and P3U1 and the like are preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of bone marrow cells used is usually about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be carried out efficiently by incubating at usually 20 to 40 ° C, preferably 30 to 37 ° C, usually for 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 directly or A hybridoma culture supernatant is added to a solid phase (eg, a microplate) adsorbed together with a carrier, and then an anti-immunoglobulin antibody labeled with a radioactive substance, an enzyme, or the like (if the cell used for cell fusion is a mouse, A method of detecting the antibody of the present invention bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, Amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 labeled with a substance, an enzyme, or the like 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 an animal cell culture medium (eg, RPMI1640) containing 10 to 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 measurement of the antibody titer of the antibody of the present invention in the antiserum.
The antibody of the present invention can be separated and purified by a method for separating and purifying immunoglobulins [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger ( Example: DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-bound solid phase or specific purification in which only antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody Etc.].
As described above, the antibody of the present invention can be produced by culturing the hybridoma cells in vivo or in vitro of a warm-blooded animal and collecting the antibody from the body fluid or culture.
In addition, (a) a hybridoma that produces an 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-mentioned polypeptide Screening of a hybridoma that produces an antibody of the present invention that reacts but does not react with a partial region thereof is performed, for example, by measuring the binding between a peptide corresponding to the partial region and an antibody produced by the hybridoma. be able to.

Hereinafter, 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 (immunoassay) will be described in more detail.
By using the antibody of the present invention, the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 can be measured or detected by tissue staining or the like. 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, the antigen, or the antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of human ZAQL-1) in the test solution is determined. Any measurement method may be used as long as it is detected by chemical or physical means and 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 nephelometry and the like are used, but a sandwich method and a competitive method described later are particularly preferable in terms of sensitivity and specificity, and a sandwich method is particularly preferable.

(1) Sandwich method After reacting the antibody of the present invention insolubilized on a carrier, the 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 by measuring the activity of. Preferably,
(I) reacting the monoclonal antibody represented by ZL1-107a, the labeled monoclonal antibody represented by ZL1-234a and the test solution, which have been insolubilized on a carrier, and 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 test solution,
(Ii) reacting the monoclonal antibody represented by ZL1-234a, the labeled monoclonal antibody represented by ZL1-107a and the test solution, which have been insolubilized on the carrier, and 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 test sample, which is a characteristic feature, may be mentioned.

  In the sandwich method, a test solution is reacted with an insolubilized antibody of the present invention (primary reaction), and a labeled antibody of the present invention is further reacted (secondary reaction). By measuring the activity of the polypeptide, 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 can be determined. it can. The primary reaction and the secondary reaction may be performed at the same time or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with 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 kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving the measurement sensitivity and the like. You may. In the measurement method by the sandwich method, for example, when the antibody used in the primary reaction is the monoclonal antibody represented by ZL1-234a, the antibody used in the secondary reaction is the monoclonal antibody represented by ZL1-107a. Preferably, when the antibody used in the primary reaction is the monoclonal antibody represented by ZL1-107a, the antibody used in the secondary reaction is the monoclonal antibody represented 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 having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof are competitively reacted with each other. By measuring the ratio of the labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or the salt thereof bound to the antibody, the sequence of SEQ ID NO: 1 or the sequence in the test solution is determined. 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 an immobilized 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, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody of the present invention, and then the solid phase and the liquid phase are separated. Alternatively, the antigen in the test solution is allowed to react with an excessive amount of the labeled antibody of the present invention, and then the immobilized antigen is added, and the unreacted labeled antibody of the present invention is immobilized on the solid phase. After binding, the solid and liquid phases are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
(4) Nephrometry In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

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, e.g., ^[125 I], ^[131 I], ^[3 H], ^[14 C] and the like, as the enzymes, large preferably stable and specific activity, for example β- galactosidase, beta -Glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like, as fluorescent substances, for example, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Biosciences), etc.), Examples of luminescent substances include fluorescamine and fluorescein isothiocyanate, and examples of the luminescent substance include luminol, luminol derivatives, luciferin, and lucigenin. Further, a biotin-avidin system can be used for binding the antibody to the labeling agent.
For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, and synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass.

In applying these individual immunological measurement methods to the method of the present invention, no special conditions, operations, and the like need to be set. The measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books, etc. [For example, “Radio Immunoassay” edited by Hiro Irie (Kodansha, published in 1974), “Radio Immunoassay” edited by Hiro Irie (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Medical Publishing, published in 1978), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical Publishing, Showa 57) Ed., Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical Publishing, 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: Monooclonal) Antibodies and General Immunoassay Methods)), Ibid. Vol. 121 (see Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press)]. Therefore, when constructing a measurement system using the sandwich immunoassay of the present invention, the method is not limited to the examples described later.
As described above, the antibody of the present invention can quantify a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof with high sensitivity. Is useful for further elucidation of the above and diagnosis of diseases involving the polypeptide. 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 a polypeptide thereof By measuring the amount of salt, for example, gastrointestinal diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.), diseases associated 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, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, etc.), polycyst Ovarian syndrome, ovarian hyperstimulation syndrome, etc.), diseases related to pregnancy (eg, preeclampsia, placental growth retardation, imminent abortion, endometriosis, infertility, ovulation disorders, etc.), eating disorders (eg, anorexia) Disorders, bulimia, etc.), sleep disorders [eg, primary insomnia, circadian rhythm disorders (eg, Modulation of physical condition due to three shift work, time band change syndrome (jet lag, etc.)), seasonal depression, reproductive dysfunction, endocrine disease, senile dementia, Alzheimer's disease, various disorders associated with aging, cerebral circulation disorders (eg, Stroke), head trauma, spinal cord injury, epilepsy, anxiety, depression, manic depression, schizophrenia, alcoholism, Parkinson's disease, hypertension, arteriosclerosis, arrhythmia, premenstrual syndrome, glaucoma, cancer, AIDS, diabetes and the like (preferably, diseases associated with angiogenesis, diseases related to pregnancy, and more preferably endometrial cancer, endometriosis, ovulation disorders, etc.) can be diagnosed. For example, in the diagnosis of polycystic ovary syndrome, the polypeptide in body fluid is quantified, and when the amount of the polypeptide is larger than that of a healthy person, for example, the blood concentration is about 3 fmol / ml or more, preferably about 3 fmol / ml or more. If it is 10 fmol / ml or more, diagnose as polycystic ovary syndrome.

In addition, the antibody of the present invention may be 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, for example, a gastrointestinal disease (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, Gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, etc.), polycystic ovary syndrome, ovarian hyperstimulation syndrome, etc.), diseases related to pregnancy (eg, preeclampsia, Placental dysgenesis, impending abortion, endometriosis, infertility, ovulation disorders, etc., eating disorders (eg, anorexia nervosa, bulimia, etc.), sleep disorders [eg, primary insomnia, circadian rhythm disorders (eg, Modulation of physical condition due to three shifts, time band change syndrome (jet lag), etc. ], Seasonal depression, reproductive dysfunction, endocrine disease, senile dementia, Alzheimer's disease, various disorders associated with aging, cerebral circulation disorders (eg, stroke), head trauma, 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 syndrome, glaucoma, cancer, AIDS, diabetes and the like. Preferably, it is a prophylactic / therapeutic agent for a disease associated with angiogenesis, a disease related to pregnancy, and more preferably a prophylactic / therapeutic agent for an endometrial cancer, endometriosis, ovulation disorder and the like.
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, rabbit, sheep, pig, Cattle, cats, dogs, monkeys, etc.) orally or parenterally. The dose varies depending on the administration target, target disease, symptoms, administration route, and the like.For example, when used for treatment of adult polycystic ovary syndrome, 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, It is convenient to administer orally. In the case of oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in 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 and the like). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, etc. Is included. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, 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 is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually about 5 to 500 mg per dosage unit dosage form, especially about 5 to 500 mg for injections. It is preferred that 100 mg, in other dosage forms, about 10 to 250 mg of the above antibody is contained.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the antibody.

In the specification of the present invention, when amino acids and the like are indicated by abbreviations, they are based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When amino acids may have optical isomers, L-form is indicated 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: glutamate Asp: aspartic acid Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine SPDP: 3- (2-Pyridyldithio) propionate N-succinimidyl GMBS: N- (4-maleimide Oxy) succinimide BSA: Bovine serum albumin BTG: Bovine thyroglobulin EIA: Enzyme immunoassay HPLC: Reversed-phase high-performance liquid chromatography HRP: Horseradish peroxidase FBS: fetal bovine serum d-FBS: dialyzed fetal bovine serum TMB: 3,3 ', 5,5'-tetramethylbenzidine H / HBSS: Hepes buffered Hanks balanced solution

The SEQ ID NOs used herein represent the amino acid sequences of the following peptides.
[SEQ ID NO: 1]
2 shows the amino acid sequence of human ZAQL-1.
[SEQ ID NO: 2]
2 shows the amino acid sequence of human ZAQL-1. Val at position 48 of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with Ile.
[SEQ ID NO: 3]
2 shows the amino acid sequence of human ZAQL-2.

The hybridoma cell ZL1-107 obtained in Example 1 described below has been used since December 9, 2002 at 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki Prefecture, Chuo No. 6 (zip code 305-8566). Deposited with the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary under the deposit number FERM BP-8256.
The hybridoma cell ZL1-234 obtained in Example 1 to be described later was an independent administrative corporation of 1-1, Higashi 1-1, Tsukuba City, Ibaraki Prefecture, Chuo No. 6 (zip code 305-8566) from December 9, 2002 (2002). Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM BP-8257.
In addition, the antibody obtained from each hybridoma cell is indicated by adding "a" to the cell name.

Hereinafter, the present invention will be described in more detail 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 a 0.02 M phosphate buffer (pH 6.8) containing 0.1 M sodium chloride, mixed with 100 μl of a DMF solution containing 1.86 mg of SPDP, and reacted at room temperature for 60 minutes. Furthermore, 0.19 ml of 0.1 M acetate buffer (pH 4.5) containing 160 μmol of dithiothreitol was added, and the mixture was reacted at room temperature for 30 minutes. After centrifugation at 13,000 rpm for 1 minute, the supernatant was separated with Sephadex G-25. Separation was performed using a column (eluent, 0.1 M phosphate buffer containing 2 mM EDTA, pH 6.0) to obtain BTG into which SH groups had been introduced. 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 by a Sephadex G-25 column (eluent, 0.1 M phosphate buffer, pH 6.7) to obtain human ZAQL-1 having a maleimide group introduced. Next, 6.5 mg of the BTG into which the SH group was introduced and 1.2 mg of the antibody fraction into which the maleimide group was introduced were mixed and reacted at 4 ° C. for 24 hours. After the reaction, the solution was dialyzed against physiological saline at 4 ° C. for 3 days.

(2) Immunity
BALB / C female mice aged 6 to 8 weeks were immunized subcutaneously with the human ZAQL-1-BTG complex obtained in the above (1) together with complete Freund's adjuvant at a concentration of about 20 μg / animal. Thereafter, the same amount of immunogen was boosted twice or three times with incomplete Freund's adjuvant every three 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). Body.
8.5 mg (213 nmol) of HRP was dissolved in 0.95 ml of 0.02 M phosphate buffer (pH 6.8) containing 0.1 M sodium chloride, mixed with 50 μl of DMF solution containing 1.99 mg of SPDP, and reacted at room temperature for 60 minutes. . Further, 0.5 ml of 0.1 M acetate buffer (pH 4.5) containing 9.25 mg of dithiothreitol was added, and the mixture was reacted at room temperature for 30 minutes. Then, a Sephadex G-25 column (eluent, 0.1 M containing 2 mM EDTA) was added. Separation with a phosphate buffer (pH 6.0) yielded HRP with SH groups introduced. 2 mg of human ZAQL-1 was 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 reacted with Sephadex G- Separation was performed on 25 columns (eluent, 0.1 M phosphate buffer, pH 6.8) to obtain human ZAQL-1 having a maleimide group introduced therein. The thus-prepared 1.67 mg (41.4 nmol) of the HRP having the SH group introduced thereinto and 1.2 mg (124 nmol) of the human ZAQL-1 having the maleimide group introduced thereinto were reacted at 4 ° C. for 1 day. . After the reaction, fractionation was performed using 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 three-week intervals, and one week later, blood was collected from the fundus. Performed and blood was collected. The blood was further centrifuged at 12,000 rpm for 15 minutes at 4 ° C., and the supernatant was recovered to obtain an antiserum. The antibody titer in the antiserum was measured by the following method. First, a 0.1 M carbonate buffer (pH 9.6) solution containing 100 μg / ml of an anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel) was added to a 96-well microplate to prepare an anti-mouse immunoglobulin antibody-bound microplate. , And left at 4 ° C for 24 hours. Next, the plate is washed with phosphate buffered saline (PBS, pH 7.4), and then 300 μl of PBS containing 25% Block Ace (Snow Brand Milk Products Co., Ltd.) is dispensed to cover the excess binding site of the well. 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 obtained anti-mouse immunoglobulin antibody-bound microplate. Dihydrate (Ethylenediamine-N, N, N ', N'-tetraacetic acid, disodium salt, dihydrate), DOJINDO] containing 0.02 M phosphate buffer, pH 7.0], and 50 µl of buffer C. 100 µl of antiserum to the complex was added and reacted at 4 ° C for 16 hours. Next, after the plate was washed with PBS, 100 μl of the HRP-labeled human ZAQL-1 (diluted 300-fold with buffer C) prepared in the above (4) was added, and reacted at room temperature for 1 day. Next, after the plate was washed with PBS, the enzymatic activity on the solid phase was measured by adding 100 μl of TMB microwell peroxidase substrate system (KIRKEGAARD & PERRY LAB, INC, Funakoshi Chemicals) and reacting at room temperature for 10 minutes. After stopping the reaction by adding 100 μl of 1M phosphoric acid, the absorbance at 450 nm was measured with a plate reader (BICHROMATIC, manufactured by Dainippon Pharmaceutical Co., Ltd.).
The results are shown in FIG.
Increased antibody titers to human ZAQL-1 were observed in antisera to human ZAQL-1-BTG conjugate in all eight immunized mice.

(5) Preparation of anti-human ZAQL-1 monoclonal antibody A mouse having a relatively high antibody titer was finally injected intravenously with 50 to 100 μg 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 Eagle's Minimum Essential Medium (MEM) to obtain a spleen cell suspension. As a cell used for cell fusion, BALB / C mouse-derived myeloma cell P3-X63.Ag8.U1 (P3U1) was used (Current Topics in Microbiology and Imnology, Vol. 81, page 1, 1978).
Cell fusion was carried out according to the original method (Nature, 256, 495, 1975).
The spleen cells and P3U1 were each washed three times with serum-free MEM, mixed with spleen cells and P3U1 at a ratio of 5: 1, and centrifuged at 800 rpm for 15 minutes to precipitate the cells. After sufficiently removing the supernatant, the precipitate was loosened gently, 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 warm water bath for 7 minutes to perform 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. The cell sediment was suspended in GIT medium (Wako Pure Chemical) (GIT-10% FCS) containing 10% fetal bovine serum so that P3U1 became 2 × 10 ⁵ per 1 ml, and a 24-well multi-dishes ( (Limbro) was seeded in 192 wells at 1 ml / well. After seeding, cells were cultured at 37 ° C. in a 5% CO 2 incubator. After 24 hours, 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 The HAT selection culture was started by adding each of the HATs. The HAT selective culture was continued by discarding 1 ml of the old solution 3 days, 6 and 9 days after the start of the culture, and then adding 1 ml of HAT medium. Hybridoma growth was observed 9 to 14 days after the cell fusion. When the culture broth turned yellow (about 1 × 10 ⁶ cells / ml), the supernatant was collected and the method described in Example 1 (4) was used. The antibody titer was measured according to the following.
As an example of selecting a mouse-derived hybridoma antibody-producing cell line immunized with human ZAQL-1-BTG, a hybridoma antibody obtained by performing cell fusion using mice No. 2 and No. 3 (see FIG. 1) The production state is shown in FIGS. From the obtained antibody-producing hybridomas, the following four types of hybridomas were selected [Table 1].

Next, the hybridoma obtained above was subjected to cloning by the limiting dilution method. At the time of cloning, thymocytes of BALB / C mice were added as feeder cells at 5 × 10 ⁵ cells / well. After cloning, the hybridoma was intraperitoneally administered to mice (BALB / C) previously administered with 0.5 ml of mineral oil intraperitoneally at 1 to 3 × 10 ⁶ cells / animal, and antibody-containing ascites was collected 6 to 20 days later did.
The monoclonal antibody was purified from the obtained ascites by a protein-A column. An equal volume of binding buffer ascites 6~20ml [3.5 M NaCl, 0.05% 1.5M glycine (pH 9.0) containing NaN _3] After dilution with, a recombinant protein -A- agarose equilibrated in advance with binding buffer (Repligen) column, and the specific antibody was eluted with an elution buffer [0.1 M citrate buffer containing 0.05% NaN ₃ (pH 3.0)]. The eluate was dialyzed against PBS at 4 ° C. for 2 days, sterilized and filtered through a 0.22 μm filter (Millipore), and stored at 4 ° C. or −80 ° C.
In determining the class and subclass of the monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA) using a solid phase bound with the purified monoclonal antibody was performed. That is, a 0.1 M carbonate buffer (pH 9.6) solution containing 2 μg / ml of the antibody was dispensed in 100 μl portions into a 96-well microplate and left at 4 ° C. for 24 hours. According to the methods described above, examined after plugged redundant binding sites in the wells with Block Ace, the immobilized antibody by ELISA using isotype typing kit (manufactured by Mouse-Typer ^TM Sub-Isotyping Kit Bio-Rad) class, subclass Was. In ZL1-222a, the H chain was IgG2b and the L chain was κ, and the remaining three types were IgG1 for the H chain and κ for the L chain.

Example 2
Competition 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) above. The antibody concentration that resulted in 50% was determined (about 30-50 ng / ml). Next, (i) 50 μl of an anti-human ZAQL-1 antibody solution in which each monoclonal antibody was diluted to 50 ng / ml with buffer C was added to the anti-mouse immunoglobulin antibody-binding microplate described in Example 1- (4) above. (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 the anti-mouse immunoglobulin antibody-bound microplate and reacted at 4 ° C. for 16 hours. After the reaction, the plate was washed with PBS and the enzyme activity on the anti-mouse immunoglobulin antibody-bound microplate was measured by the method described in Example 1- (4) above.
Table 1 shows the results.
It can be seen that all the antibodies have reactivity with human ZAQL-1, but have no reactivity with human ZAQL-2.
As an example, the results of the competition method-EIA of the monoclonal antibodies ZL1-107a and ZL1-234a showing the highest reactivity with human ZAQL-1 among them are shown in FIGS.
From ZL1-107a and standard curve of human ZAQL-1 of ZL1-234a, human ZAQL-1 concentration giving a ratio (B / B ₀₎ = 0.5 for the maximum reaction was found to be respectively a 0.8nM and 1.2nM . From these results, it is considered that ZL1-107a and ZL1-234a show high reactivity to human ZAQL-1.

Example 3
Preparation 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 ZL1-234a purified fraction, and reacted at room temperature for 40 minutes. The reaction solution was separated on 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, to 1.14 ml of 0.02 M phosphate buffer (including 0.15 M NaCl) (pH 6.8) containing 18.3 mg (457 nmol) of HRP, 60 μl of DMF containing 6.85 μmol of SPDP was added, 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 added. (M phosphate buffer, pH 6.0) to obtain 9.8 mg of HRP into which SH groups were introduced. Next, 8 mg of the HRP into which the SH group was introduced and 3 mg of the antibody fraction into which the maleimide group was introduced were mixed, concentrated to about 0.5 ml with a Collodion bag (manufactured by Sartorius), and then left at 4 ° C. for 16 hours. did. The reaction solution was applied to a SephacrylS-300HR column (Pharmacia) equilibrated with 0.1 M phosphate buffer (pH 6.5) to purify the ZL1-234a-HRP complex fraction.

Example 4
Sandwich method-EIA
A 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 in 100 μl portions 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 a human ZAQL-1 or human ZAQL-2 standard solution diluted with a 0.02 M 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 (diluted 10,000-fold 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 according to the method described in Example 1 (4) (enzyme reaction for 20 minutes).
FIG. 7 shows the results.
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 very selectively.

Example 5
Neutralization of biological activity of human ZAQL-1 by ZL1-107a and ZL1-234a
The neutralizing activity of human ZAQL-1 by ZL1-107a and ZL1-234a was measured 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.
ZAQ-expressing CHO cells were adjusted to a concentration of 1.2 × 10 ⁵ cells / ml with Dulbecco's modified Eagle medium (DMEM) medium (Nissui Pharmaceutical Co., Ltd.) containing dialyzed fetal bovine serum (dFBS) (JRH BIOSCIENCES). Suspended in 10% dFBS-DMEM) and seeded in a 96-well plate for FLIPR (Black plate clear bottom, Coster) using a pipettor, 200 μl per well (4 × 10 ⁴ cells / 200 μl / well). It was used after culturing overnight at 37 ° C. in a 5% CO ₂ incubator (hereinafter referred to as a cell plate). FLIPR assay buffer [Nissui Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, pH 7.4 with 6M sodium hydroxide solution, fill up to 1 L, filter sterilize 20 ml), 200 μl of 250 mM Probenecid (SIGMA), and 210 μl of fetal bovine serum (FBS). Also, two vials (50 μg) of Fluo 3-AM (Dojindo 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 solution]. Hanks Balance Solution (Nissui Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, adjusted to pH 7.4 with sodium hydroxide solution, filter sterilized) 20 ml, 250 mM 200 μl of Probenecid and 200 μl of fetal bovine serum (FBS) were added to the H / HBSS-Probenecid-FBS solution, and after mixing, 100 μl of each well was dispensed into a cell plate from which the culture solution was removed using an 8-tube pipette. Incubated for 1 hour at 37 ° C. in a 5% CO ₂ incubator (dye loading). ZL1-107a and ZL1-234a and an anti-PrRP monoclonal antibody (P2L-1Ta) having the same IgG subclass structure (IgG1, κ) as ZL1-107a and ZL1-234a as a control antibody (Biochem. Biophys. Res. Commun., 257) Volume, pp. 264-268 (1998)), diluted with 120 μl of Hanks' / HBSS containing 2.5 mM Probenecid and 0.2% BSA, and human ZAQL-1 (1 × 10 ⁻⁸ M) for 1 hour at 37 ° C. 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 the dye loading on the cell plate was completed, the cell plate was washed four times with a washing buffer obtained by adding 2.5 mM Probenecid to Hanks' / HBSS using a plate washer (Molecular Devices), and 100 μl of the washing buffer was left after washing. . The cell plate and the sample plate were set on the FLIPR, and the measurement was performed (the FLIPR transfers 50 μl of the sample from the sample plate to the cell plate).
FIG. 8 shows the results.
Thus, the activity of human ZAQL-1 (3.3 × 10 ⁻¹⁰ M) was about 97% at 3.3 × 10 ⁻¹⁰ M for ZL1-107a, and about 97% at 3.3 × 10 ⁻⁹ M at a 10-fold molar concentration. It turns out that it suppressed. ZL1-234a exhibits about 87% of the activity of human ZAQL-1 (3.3 × 10 ⁻¹⁰ M) at an equimolar concentration of 3.3 × 10 ⁻¹⁰ M, and at about 3.3 × 10 ⁻⁹ M at a 10-fold higher molar concentration. It turns out that 98% was suppressed. On the other hand, in the control antibody P2L-1Ta, suppression 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 is clear 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 volume of buffer C, and human ZAQL-1 was quantified by the sandwich method-EIA described in Example 4 above.
Table 2 shows the results.

Example 7
Detection of human ZAQL-1 in human plasma by RP-HPLC To identify the human ZAQL-1 immunoreactivity contained in human plasma described in Example 6, after adding 20 ml of acetonitrile to 10 ml of human plasma and mixing. Then, centrifugation (15,000 rpm, 5 minutes) was performed to remove proteins. After the supernatant was lyophilized, the fraction was concentrated and then fractionated by reverse-phase HPLC using a column (ODS-80 ^™ ).
Column conditions:
Column: ODS-80 ^TM (4.6 x 250 mm)
Eluent: Solution A (5% acetonitrile containing 0.05% trifluoroacetic acid)
Solution B (60% acetonitrile containing 0.05% trifluoroacetic acid)
Elution method: Raise acetonitrile concentration from 5% to 30% in the first 5 minutes,
It was then increased linearly to 30-40% over 30 minutes.
Flow rate: 1.0 ml / min Fractionation: 0.5 ml / tube
After the eluted fraction was lyophilized, it was dissolved in 250 μl of buffer C and subjected to the sandwich method-EIA described in Example 4 above.
FIG. 9 shows the results.
Since the immunological activity of human ZAQL-1 in plasma was almost detected at the elution position of human ZAQL-1 (recovery rate: 102%), the sandwich method-EIA must have detected human ZAQL-1 Was confirmed.
Thus, this measurement system is an important tool when 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
Determination of Human ZAQL-1 in Pregnant Woman Plasma Pregnant woman plasma obtained at each lactation period was diluted 2-fold with the same volume of buffer C, and human ZAQL-1 was quantified by the sandwich method-EIA described in Example 4 above. did. Pregnant woman plasma was purchased from DCP and had informed consent.
Table 3 shows the results.

From Table 3, it is considered that the blood human ZAQL-1 concentration increases in the early pregnancy (up to 13 weeks of pregnancy), then decreases with the progress of pregnancy, and returns to normal levels after childbirth. Compared to non-pregnant women (1.51 ± 0.35 fmol / ml), they increased about 4.0 times in early pregnancy (4-13 weeks) and about 2.8 times in late pregnancy (28-40 weeks).
This indicates that production or secretion of human ZAQL-1 increases during pregnancy. Therefore, the blood human ZAQL-1 concentration can be an indicator of pregnancy, and thus 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, centrifugation (15,000 rpm, 5 minutes) was performed to remove proteins. After the supernatant was freeze-dried, the fraction was concentrated and then fractionated by reverse phase HPLC using a column (ODS-80 ^™ ) under the same conditions as in Example 7.
After the eluted fraction was lyophilized, it was 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.
The immunological activity of human ZAQL-1 in the plasma of pregnant women was almost detected at the elution position of human ZAQL-1 (recovery rate 70%). 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 volume of buffer C, and human ZAQL-1 was quantified by the sandwich method-EIA described in Example 4 above. Follicular fluid was provided by a joint study with Assistant Professor Usuki of the Department of Obstetrics and Gynecology at the University of Tsukuba, and was informed consent. Also approved by the Ethics Committee of the University of Tsukuba and Takeda Pharmaceutical Company Limited.
Table 4 shows the results.

From the above results, in the follicular fluid of patients with concurrent endometriosis and ovulation disorders, human ZAQL-1 showed a high level, human ZAQL-1 endometriosis and endometriosis Involvement was suggested.

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 freeze-dried, the fraction was concentrated and then fractionated by reverse phase HPLC using a column (ODS-80 ^™ ) under the same conditions as in Example 7.
After the eluted fraction was lyophilized, it was 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 follicular fluid was almost detected at the elution position of human ZAQL-1 (recovery rate 70%), the sandwich method-EIA showed that human ZAQL-1 It was confirmed that it was detected.

4 shows the measurement results of antibody titers in antisera of mice immunized with the human ZAQL-1-BTG complex. In the figure,-◇-is mouse No. 1,-□-is mouse No. 2,-△-is mouse No. 3,-○-is mouse No. 4,-◆-is mouse No. 5,-■. -Indicates mouse No. 6,-▲-indicates mouse No. 7, and-●-indicates mouse No. 8. The figure shows a state in which a hybridoma derived from a mouse immunized with the human ZAQL-1-BTG complex is producing an antibody (result of absorption analysis). The figure shows a state in which a hybridoma derived from a mouse immunized with the human ZAQL-1-BTG complex is producing an antibody (result of absorption analysis). The figure shows a state in which a hybridoma derived from a mouse immunized with the human ZAQL-1-BTG complex is producing an antibody (result of absorption analysis). The figure shows a state in which a hybridoma derived from a mouse immunized with the human ZAQL-1-BTG complex is producing an antibody (result of absorption analysis). FIG. 9 shows the results of the competition method-EIA of ZL1-107a and ZL1-234a. In the figure,-●-indicates the reactivity between ZL1-107a and ZAQL-1,-○-indicates the reactivity between ZL1-107a and ZAQL-2, and-■-indicates the reactivity between ZL1-234a and ZAQL-1. Reactivity and-□-represent the reactivity between ZL1-234a and ZAQL-2. 3 shows the results of a sandwich method-EIA using ZL1-107a and ZL1-234a. In the figure,-●-indicates the reactivity of ZAQL-1, and-○-indicates the reactivity of ZAQL-2. FIG. 9 shows the neutralizing effect on the intracellular Ca ²⁺ ion concentration increasing activity using ZAQL-1 ZAQ-expressing CHO cells (ZAQC-B1) in the presence of ZL1-107, ZL1-234a, or P2L-1Ta. In the figure, the black bar represents the ratio of the activity of increasing the intracellular Ca ²⁺ ion concentration in ZAQC-B1 cells when ZL1-107a coexists with ZAQL-1 to the control (when no antibody was added), and the hatched bar represents ZL1-107a. The white bar shows the ratio of P2L-1Ta to ZAQL-1 when 234a was added to ZAQL-1 and the ratio of intracellular Ca ²⁺ ion concentration increasing activity in ZAQC-B1 cells to the control (when no antibody was added). The ratio of the activity of increasing the intracellular Ca ²⁺ ion concentration in the ZAQC-B1 cells to the control (when no antibody was added) when the antibody was added is shown. FIG. 9 shows the elution position of human ZAQL-1 immunoreactivity in human plasma fractionated using reverse phase HPLC. FIG. 3 shows the elution position of human ZAQL-1 immunoreactivity in plasma of pregnant women fractionated using reverse-phase HPLC. FIG. 9 shows the elution position of human ZAQL-1 immunoreactivity in follicular fluid fractionated using reverse-phase HPLC.

Claims (23)

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. The monoclonal antibody according to claim 1, which specifically reacts with a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. Nos. 8 to 9, 11, 15, 17, 21, 23, 25 to 28, 30, 34, 36 to 37, 39 to 40, 44 to 44 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 The method according to claim 1, which specifically reacts with a peptide containing at least one selected from the amino acids at positions 46, 48, 52 to 53, 55, 64, 66, 68, 70 to 73, 75 to 76, and 78 to 86. Monoclonal antibody. The monoclonal antibody according to claim 1, which does not recognize the polypeptide having the amino acid sequence represented by SEQ ID NO: 3. The labeled monoclonal antibody according to claim 1. The monoclonal antibody according to claim 1, which is designated as ZL1-107a, which can be produced from the hybridoma cell designated as ZL1-107 (FERM BP-8256). The monoclonal antibody according to claim 1, which is designated as ZL1-234a, which can be produced from the hybridoma cell designated as ZL1-234 (FERM BP-8257). The monoclonal antibody according to claim 1, which has a neutralizing activity against a peptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. A pharmaceutical comprising the monoclonal antibody according to claim 1. The medicament according to claim 9, which is an agent for preventing or treating endometrial cancer, endometriosis or ovulation disorder. A diagnostic agent comprising the monoclonal antibody according to claim 1. The diagnostic agent according to claim 11, which is a diagnostic agent for endometrial cancer, endometriosis or ovulation disorder. A diagnostic agent comprising the monoclonal antibody according to claim 1. A method for quantifying a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, comprising using the monoclonal antibody according to claim 1. A method for diagnosing a disease involving a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, comprising using the monoclonal antibody according to claim 1. The diagnostic method according to claim 15, wherein the disease is endometrial cancer, endometriosis or ovulation disorder. The monoclonal antibody according to claim 1, which is reacted with a test solution and a labeled polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a salt thereof, and then the antibody is reacted. Measuring the ratio of the labeled polypeptide or a salt thereof bound to the polypeptide or the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in a test solution; Determination of the salt. (A) measuring the activity of the labeling agent after reacting the monoclonal antibody according to claim 6 insoluble on a carrier, the labeled monoclonal antibody according to claim 7 and a test solution, or
(B) reacting the monoclonal antibody according to claim 7 insoluble on a carrier, the labeled monoclonal antibody according to claim 6 and a 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 test solution. A hybridoma cell that produces the monoclonal antibody according to claim 1. 20. The hybridoma cell according to claim 19, which is designated as ZL1-107 (FERM BP-8256) or ZL1-234 (FERM BP-8257). The hybridoma cell according to claim 19 is cultured in vivo or in vitro, and the monoclonal antibody according to claim 6 or 7 is collected from the body fluid or culture. Production method of monoclonal antibody. A method for preventing or treating endometrial cancer, endometriosis or ovulation disorder, comprising administering an effective amount of the monoclonal antibody according to claim 1 to a mammal. Use of the monoclonal antibody according to claim 1 for producing a prophylactic / therapeutic agent for endometrial cancer, endometriosis or ovulation disorder.

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