WO2007032559A1

WO2007032559A1 - Novel monoclonal antibody and use thereof

Info

Publication number: WO2007032559A1
Application number: PCT/JP2006/318677
Authority: WO
Inventors: Jun Hashimoto; Akihide Nampei; Wataru Ando; Tomofumi Kurokawa; Tsutomu Oshima
Original assignee: Osaka University; Takeda Pharmaceutical Company Limited
Priority date: 2005-09-15
Filing date: 2006-09-14
Publication date: 2007-03-22

Abstract

Disclosed is an antibody capable of specifically recognizing an amino acid sequence identical to the antigenic determinant of a murine monoclonal antibody MAB-ME-9D2.15 (FERMBP-10327). Also disclosed is a method for diagnosis of secondary osteoporosis in rheumatoid arthritis, comprising detecting an MEPE-derived molecule in a biological sample using the antibody and an anti-MEPE polyclonal antibody. Further disclosed is a kit for use in the method.

Description

Specification

New monoclonal antibodies and their applications

The invention relates to a novel antibody that recognizes a specific antigenic determinant of Matrix extracellular phosphoglycoprotein (hereinafter abbreviated as “MEPE”) and its use, specifically as a diagnostic agent for secondary osteoporosis in patients with rheumatoid arthritis, etc. Related to the use. Background art

MEPE is highly expressed in the tumors that cause neoplastic osteomalacia (hereinafter sometimes abbreviated as “0H0”), and is a gene-cloned glycoprotein (total length of 525 amino acids in humans). ; SEQ ID NO: 2) is considered one of the symptoms of Phosphatonin causing hypophosphatemia. From recent studies, (1) It is strongly expressed in bone tissue (A. Nampei et al., J. Bone Miner. Me tab., 2 2: 176-184 (2004)), (2) Bone (3) MEPE knockout mice have increased bone mass and promoted osteoblast differentiation (LC Gowen et al., J. Biol. Chem.,- 278: 1998-2007 (2003)), etc., has been obtained, suggesting that it functions as a bone formation inhibitor. Recently, however, it has been suggested that MEPE fragments have different effects from full-length proteins, and there are still many unclear points regarding the function and pathophysiology of MEPE (and its degradation products). is there.

Measuring the blood concentration of MEPE or a degradation product of MEPE is important in clarifying the causal relationship between MEPE and the disease state, and the concentration of MEPE-derived molecules in the blood is a disease state marker for bone and joint diseases. Expected to be useful. So far, A. Jain et al. (. Clin. Endocrinol. Metab., 89: 4158-4161 (2004)) Force S, anti-MEPE polyclonal antibody as primary antibody, and goat anti-rabbit antiserum as secondary antibody. By measuring the concentration of MEPE-derived molecules in normal human blood by ELISA, MEPE and serum phosphate levels, P It is reported that TH values, femoral BMD, and total hip BMD are positively correlated. Disclosure of the invention

An object of the present invention is to provide a novel anti-MEPE antibody for detecting a MEPE-derived molecule in blood, and to use the MEPE-derived molecule that can be used as a blood pathological marker for bone and joint diseases. It is to provide a method of detecting.

In order to achieve the above-mentioned object, the present inventors produced 8 kinds of anti-human MEPE mouse monoclonal antibodies and rabbit antibody polyclonal antibodies, and constructed a plurality of sandwich ELISA systems in which the combinations thereof were changed. Using these Atsy systems, we measured the concentration of MEPE-derived molecules in plasma collected from patients with rheumatoid arthritis, and investigated the relationship between the measured values and BMD and bone metabolism markers. As a result, when the monoclonal antibody MAB-ME-9D2.15 and the polyclonal antibody were used, the MEPE-derived molecular concentration surprisingly showed a significant negative correlation with BMD, and the urinary protein, a bone resorption marker. We found a significant positive correlation with xypyridinoline (DPD). Furthermore, the present inventors examined the region of MEPE recognized by the monoclonal antibody MAB-ME-9D2.15 and found that the region exists on the N-terminal side of MEPE. As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention

[1] The following antibody (a) or (b):

(a) Mouse monoclonal antibody MAB- ME-9D2. 15 (FERM BP-10327)

(b) an antibody capable of specifically recognizing the same amino acid sequence as the antigenic determinant of the antibody of (a) above

[2] The antibody according to [1] above, wherein the antigenic determinant is present in the amino acid sequence represented by amino acid numbers 17-240 in the amino acid sequence represented by SEQ ID NO: 2;

[3] A method for diagnosing secondary osteoporosis in rheumatoid arthritis, comprising using the antibody according to [1] above and a polyclonal antibody against MEPE;

[4] The above-mentioned [3], characterized by the use of the Imno-Atsusy by the sandwich method Described method;

[5] The method according to [3] above, wherein blood, serum or plasma is used as a test sample; and

[6] A diagnostic kit for secondary osteoporosis in rheumatoid arthritis, comprising the antibody according to [1] above and a polyclonal antibody against MEPE;

Etc.

The two anti-MEPE antibodies of the present invention can be used to measure MEPE-derived molecules in a biological sample, thereby diagnosing the pathological condition of rheumatoid arthritis in the animal from which the sample was collected, particularly secondary osteoporosis. There is an advantageous effect of being able to. Brief Description of Drawings

Figure 1 shows the structure of the mature MEPE plasmid for E. coli expression (pTCII-mPHOS-2). In the figure, mPHOS is the mature MEPE coding sequence, T7P and T7T are the T7 promoter and T7 terminator, ori is the E. coli replication origin, and Tet is the tetracycline resistance gene. ''.

FIG. 2 shows an SDS-polyacrylamide gel electrophoresis image of E. coli-expressed mature MEPE obtained in Example 3 under reducing and non-reducing conditions. Multi gel 10/20 was used for electrophoresis, and electrophoresis under reducing conditions was performed by treatment with 100 mM DTT at 100 ° C for 2 minutes. Each lane 1 was applied. -Figure 3 shows the results of N-terminal amino acid sequence analysis of E. coli expression mature MEPE obtained in Example 3. The numerical value indicated above the arrow indicates the recovery rate of free amino acid obtained in each analytical step.

Figure 4 shows the structure of the plasmid for animal cell expression of phosphatonin (MEPE) -FLAG adduct. In the figure, Phosphatonin-FLAG represents the coding sequence of phosphatonin (MEPE) -FLAG adduct, T7P represents the T7 promoter, fl ori represents the fl replication origin, Neo represents the neomycin resistance gene, and Ampr represents the ampicillin resistance gene. . Fig. 5 shows the SDS-polyatalyl of the insect cell expression MEPE-FLAG obtained in Example 6. A gel electrophoresis image is shown. The electrophoresis was performed under reducing conditions using Multi gel 4/20 and treated with 100 mM DTT at 100 ° C. for 2 minutes. Lane 1: Insect cell culture supernatant; Lane 2: Purified MEPE-FLAG preparation (1 μ _§ )

FIG. 6 shows the binding affinity of the eight anti-mouse mouse monoclonal antibodies obtained in Example 7 to solid phase MEPE-FLAG. The vertical axis represents absorbance at 450 nm, and the horizontal axis represents antibody concentration (ng / ml). --: ME-2D11.9;-▲-: ME-4A2. 2; -Garden-: ME-9D2.15; _ ○-: ME-9H6.8;-氺-; ME-10E4.2; △-: ME-12C9.2; -Mouth-: ME-12E2. 1;-♦-: ME-16F4. 3

FIG. 7 shows a calibration curve of the enzyme immunoassay for measuring the plasma MEPE-derived antigen concentration described in Example 8. The vertical axis represents the absorbance at 450 nra, and the horizontal axis represents the antigen concentration (ng / ml).

FIG. 8 shows the plasma stability of MEPE-derived antigens quantified by the enzyme immunoassay described in Example 8. Each plasma sample (K, F, A, H, N, S) is left at room temperature for 0 (white), 2 (upward ^ I · line) and 4 (black) hours, and 1 day (upward left diagonal line). After that, a quantitative measurement was performed. ·

FIG. 9 shows the cryopreservation stability of a sample containing a MEPE-derived antigen quantified by the enzyme immunoassay described in Example 8. Each plasma sample (F and A) was quantified immediately after sampling (black) and after freezing at -80 ° C for 1 month (white). Fig. 10 shows plasma MEPE-derived antigen concentrations, femur proximal bone density (A), and distal tibia bone density (B) in female patients with rheumatoid arthritis who are not taking bisphosphonate and taking vitamin D And a positive correlation (C) with urinary doxypyridinoline (DPD).

Figure 11 shows the anti-MEPE rabbit polyclonal antibody obtained in Example 4 (Figure 11 A, left panel) and one anti-MEPE mouse monoclonal antibody obtained in Example 7 (MAB). -ME-9D2. 15) (Fig. 11 A, right panel) shows the binding specificity (ME) and various MEPE C-terminal deletion mutins (Western plot). In the figure, WT represents wild type MEPE-FLAG (full length of mature MEPE + FLAG tag), Δ241-525, Δ331-525 and Δ 507-525 represents a C-terminal deleted mutin from which positions 241-525, 331-525 and 507-525 of mature MEPE-FLAG are deleted. In addition, in FIG. 11B, “+” indicates binding. BEST MODE FOR CARRYING OUT THE INVENTION

The first anti-MEPE antibody of the present invention (hereinafter sometimes referred to as “the first antibody of the present invention”) is anti-human MEPE mouse monoclonal antibody MAB-ME-9D2.15 (hereinafter simply referred to as “MAB-ME”). -9D2.15J), or an antibody capable of specifically recognizing the same amino acid sequence as the antigenic determinant of the antibody (Epitope 1; E 1). The antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody, although the isotype of the antibody is not particularly limited, but is preferably IgG, IgM or IgA, IgG is particularly preferred, and there are no particular restrictions on immunogens and animals, for example, non-human mammals (eg, monkeys, chimpanzees, pigs, horses, horses, horses, cats, cats) Mouse, mouse, The region corresponding to E1 in MEPE orsolog derived from latte small, magpie, etc. (E1 '; for example, in the case of power quiz, Ε is an amino acid registered with GenBank with registration number BAB 17 010. 1) An antibody obtained using an immunogen of the amino acid sequence represented by amino acid numbers 17-271 in the sequence) or a non-human warm-blooded animal (eg, Usagi, Goat, Usushi, Chicken, Mouse, Rat, Hedges, pigs, horses, cats, dogs, monkeys, chimpanzees, etc.) antibodies obtained by immunizing human cultured cells with MEPE or fragments thereof, or non-human warm-blooded animals (eg, rabbits, Antibodies such as phage display using the antibody gene library of goats, rabbits, chickens, mice, rats, hidges, pigs, horses, cats, dogs, sals, chimpanzees, etc.) Antibodies obtained by the display method can also be included in the first antibody of the present invention.

The antigenic determinant (E1) of MAB-ME-9D2.15 is present in the amino acid sequence represented by amino acid numbers 17-240 in the amino acid sequence represented by SEQ ID NO: 2. An anti-MEPE antibody Can recognize the same amino acid sequence as El ', for example, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow and David Lane ed.

(1998) can be assayed by examining the inhibition of binding of MEB-ME-9D2.15 to MEPE using a conventional cross-blocking assay. Alternatively, it is possible to test more directly by investigating whether or not it binds to a peptide containing E1 in the same manner as MAB-ME-9D2.15 by epitope pinning using a peptide array or phage display. You can also. MAB-ME-9D2. 15 was started on April 26, 2005, from National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (IP0D) 6) is deposited with the FERM BP-10327 accession number.

The second anti-MEPE antibody of the present invention (hereinafter sometimes referred to as “the second antibody of the present invention” ′) is an anti-human MEPE polyclonal antibody. The antibody recognizes at least an antigenic determinant present in the amino acid sequence represented by amino acid numbers 17 to 240 in the amino acid sequence represented by SEQ ID NO: 2. The isotype of the antibody is not particularly limited, and preferably IgG, IgM or IgA, and particularly preferably IgG. There are no particular restrictions on the immunogen or immunized animal. For example, non-human mammals (eg, monkeys, chimpanzees, pigs, tusks, horses, hidges, cats, dogs, mice, rats, rabbits) Etc.) immunized with a MEPE ortholog derived from it or a fragment thereof (which preferably contains a region corresponding to the amino acid sequence shown by amino acid numbers 17-240 in the amino acid sequence shown in SEQ ID NO: 2). Antibodies obtained from the original, non-human warm-blooded animals (eg, rabbits, goats, rabbits, chickens, mice, rats, hidge, puta, horses, cats, dogs, monkeys, chimpanzees, etc.) Antibodies obtained by immunizing cells with MEPE or fragments thereof, or non-human warm-blooded animals (eg, rabbits, goats, tusks, chickens, mice, rats, hidges, pigs, horses) Cat, I null, monkey, also including the antibody obtained by chimpanzees) vulgar bets antibody gene libraries antibody display methods phage di spraying method using, may be included in the second antibody of the present invention. As long as the first antibody of the present invention has at least a complementarity determining region (CDR) for specifically recognizing and binding to its antigenic determinant E1 (or E1 ′), it is particularly limited to a molecular form. In addition to complete antibody molecules, for example, fragments such as Fab, Fab \ F (ab ') ₂ , genetically engineered conjugate molecules such as scFv, scFv- Fc, myebody, and diapody, or polyethylene glycol It may be a derivative thereof modified with a molecule having a protein stabilizing action such as (PEG).

'When the first antibody of the present invention is a monoclonal antibody, for example, it can be prepared by the following method.

(1) Preparation of immunogen

Immunogens include human MEPE or fragments containing E1, or MEPE orthologs from other mammals or fragments containing the E1 region (El,), or synthetic peptides containing E1 or E1 '. Can be used.

In the present specification, the peptide or protein represented by the amino acid sequence is the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide labeling. The synthetic peptide containing the amino acid sequence of the antigenic determinant recognized by MEPE or a fragment thereof or the antibody of the present invention in the present invention (hereinafter, collectively referred to as “antigenic peptide”) has a C-terminal carboxyl group, It may be any of amide, ester or ester. In addition, when the antigenic peptide has a carboxyl group (or carboxylate) other than the C terminal, the carboxyl group may be amidated or esterified. Furthermore, antigen peptides include amino acid residues in which the N-terminal amino acid residue is substituted with, for example, formyl group, acetyl group, etc., N-terminal glutamine residue is pyroglutamine oxidized, Substituents on acid side chains (eg -0H, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) substituted with other substituents (eg formyl group, acetyl group, etc.) It may be. The antigen peptide may be a salt with an acid or a base, and an acid addition salt is particularly preferable. Examples of such salts include 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, Salts with succinic acid, tartaric acid, citrate, malic acid, succinic acid, benzoic acid, methane sulfonic acid, benzene sulfonic acid, etc. are used.

The antigenic peptide can be isolated and purified (a) from antigen-producing tissue or cells of mammals such as humans, monkeys, rats, mice, etc. using a method known per se or a method equivalent thereto, (b) Peptide Chemically synthesized by a peptide synthesis method known per se using a synthesizer, etc. (c) cultivating a transformant containing DNA encoding the antigen peptide, or (d) a nucleic acid encoding the antigen peptide. It can be obtained by biochemical synthesis using a cell-free transcription / translation system as a saddle type. .

(a) When preparing an antigen from a mammalian tissue or cell, after homogenizing the tissue or cell, extraction with an acid or alcohol is performed, and the extract is used for protein separation technology known per se (eg: It can be isolated and purified by subjecting to salting-out, dialysis, gel filtration, reverse phase chromatography, ion exchange chromatography, chromatographies such as affinity mouthmatography, etc.). The obtained antigen peptide can be used as an immunogen as it is, or a partial peptide can be prepared by limited degradation using a peptidase or the like and used as an immunogen.

(b) In the case of chemically synthesizing an antigen peptide, the synthetic peptide is, for example, one having the same structure as the above-mentioned antigen peptide purified from nature, specifically, the amino acid sequence of the natural antigen peptide In the above, a peptide containing the same amino acid sequence as that of E1 (or (), which is the antigenic determinant of the first antibody of the present invention, is used.

(c) When an antigenic peptide is produced using a transformant containing DNA, the DNA can be obtained by known cloning methods [for example, Molecular Cloning, 2nd ed .; J. Sarabrook et al., Cold Spring Harbor Lab According to the method described in Press (1989)] It can be produced. Examples of the cloning method include: (i) a gene sequence encoding an antigen peptide (for example, in the case of human MEPE, a sequence containing a base sequence encoding E1 includes a base represented by SEQ ID NO: 1). The DNA encoding the antigen is isolated from the cDNA library by the hybridization method, using a DNA probe designed based on nucleotide numbers 49 to 720 in the sequence) (ii ) Using a DNA primer designed based on the gene sequence encoding the antigenic peptide, prepare the DNA encoding the antigenic peptide by PCR using the cDNA as a saddle, and place the DNA in an expression vector compatible with the host. And a method of culturing a transformant obtained by transforming a host with the expression vector in an appropriate medium.

(d) When a cell-free transcription / translation system is used, an expression vector inserted with a DNA encoding an antigenic peptide prepared by the same method as in (c) above (for example, the DNA controls T7, SP6 promoter, etc. The expression vector shown below is a saddle type, and mRNA is synthesized using a transcription reaction solution containing RNA polymerase and substrates (NTPs) compatible with the promoter, and then the mRNA is known as a saddle type. A cell-free translation system (eg, an extract of Escherichia coli, rabbit reticulocytes, wheat germ, etc.) is used. By appropriately adjusting the salt concentration, etc., the transcription reaction and the translation reaction can be carried out in the same reaction solution.

When using a polypeptide such as MEPE full-length protein as an immunogen (for example, 21 amino acid residues or more), select by cross-blocking assay using the binding ability to E1 or E2 as an index. It will be.

On the other hand, when an oligopeptide of 20 amino acids or less is used as an immunogen, for example, 3 or more, preferably 4 or more, more preferably 5 or more, more preferably 6 containing the amino acid sequence of E1 (or E1 ′). The thing which consists of the above continuous amino acid residue is mentioned. Alternatively, the oligopeptide may be, for example, 20 or less, preferably 18 or less, more preferably 15 or less, and even more preferably 12 or less consecutive containing the amino acid sequence of E1 (or E1 ′). From amino acid residues Rumo-no.

Such oligopeptides are produced by cleaving the antigen peptide prepared by the methods (b) to (d) above or the methods (a) to (d) above with an appropriate peptidase or the like. However, it is preferable to use a known peptide synthesis method.

As a peptide synthesis method, 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 removal include the methods described in 1) or 2) below.

1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience Publis hers, New York (1966)

2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965) .After the reaction, combine ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. The peptide can be purified and isolated. If the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method. Conversely, if it is obtained as a salt, the free form or other salt can be obtained by a known method. Can be converted to

As the peptide amide, a commercially available peptide synthesis resin 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. Acetamide methyl resin, polyacrylamide resin, 4- (2,4'-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2,4,4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin And so on. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected, in accordance with the sequence of the target peptide, according to various known condensation methods, Condensation on resin. At the end of the reaction, the peptide is excised from the resin, and at the same time, various protecting groups are removed to obtain the desired peptide. Alternatively, using chlorotrityl resin, oxime resin, 4-hydroxybenzoic acid resin, etc., the partially protected peptide can be taken out, and the protective group can be removed by conventional means to obtain the desired peptide. .

For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for peptide synthesis can be used, and in particular, calpositimides are preferred. Examples of carbodiimides include DCC, Ν, Ν'-disopropyl carbodiimide ', and Ν-ethyl-Ν'-(3-dimethylaminoprolyl) carbodiimide. For these activations, a protected amino acid together with a racemization inhibitor (eg, H0Bt, HOOBt, etc.) can be added directly to the resin, or it can be pre-protected as a symmetric anhydride or HOBt ester or HOOBt ester. It can be added to the resin after activation of the amino acids. The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents that are known to be usable in peptide condensation reactions. For example, N, N-dimethylformamide, Ν, Ν-dimethylacetamide, acid amides such as Ν-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohol such as trifluoroethanol , Sulfoxides such as dimethyl sulfoxide, tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propiotril, esters such as methyl acetate and ethyl acetate, or these An appropriate mixture of these is used. 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. Activated amino acid derivatives are usually used in about 1.5 or about 4-fold excess. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acids are acetylated using acetic anhydride or acetyl imidazole. Thus, it is possible to prevent the subsequent reaction from being affected.

The protection of the functional group that should not be involved in the reaction of the raw material and the protection 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. .

Examples of protecting groups for amino acid amino groups include Z, Boc, Tertiary pentinore talent / Repoenore, Isoponoleni / Leoxycanoleponyl, 4-Methoxybenzyloxycarbonyl, CI-Z, Br-Z, Examples include adamantyloxycarbonyl, trifluoroacetyl, phthaloidol, honoleminole, 2-to-2 phenenolesnorefenenore, diphenylphosphinochioyl, and Fmoc. Is a protecting group of carboxyl group, for example _ ₆ alkyl group (e.g., methyl, Echiru, n- propyl, isopropyl, n- heptyl), C ₃ - (carboxymethyl Honoré example, cyclopentyl, cyclohexylene) ₈ cycloalkyl group , C _{7 —} ₁₄ aralkyl groups (eg, benzyl, phenethyl, naphthylmethyl), 2-adamantyl, 4-nitrobenzene, 4-methoxybenzenole, 4-chlorobenzene, phenacyl and benzyloxy Canoleponinole hydrazide, tertiary butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherolation. Examples of groups appropriately used for the esterification, for example, § cell lower, such as methyl group (- ₆₎ Arukanoiru group, Aroiru group such Benzoiru group, Baie Nji Ruo alkoxycarbonyl group, such as carbonate husk derived by groups such as ethoxycarbonyl group Is mentioned. Examples of groups suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a tertiary butyl group.

Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl_Bzl, 2-nitrobenzyl, Br-Z, and tertiary butyl.

Examples of the protecting group for imidazole 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, Aji, Do, Active Estenole [Alcohol (eg, Pentac, Mouth, Phenolic, 2, 4, 5- 卜, Black, Mouth, Phenol, 2, 4-Dinitrophenol, Cyanomethyl Alcohol, Paraditrophenol, H0NB, N-H Droxy succinide, N-hydroxyphthalimide, ester with HOBt). Examples of the activated amino group of the raw material include the corresponding phosphoric acid amide.

Methods for removing (eliminating) protecting groups 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, trifluoro. Examples include acid treatment with romethanesulfonic 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 carried out at a temperature of -20 ° C to 40 ° C. In the acid treatment, anisol, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1 It is effective to add a cation scavenger such as 1,4-butanedithiol or 1,2-ethanedithiol. In addition, the 2,4-ditrophenyl group used as an imidazole protecting group for histidine was removed by thiophenol treatment, and the formyl group used as an indole protecting group for tryptophan was the 1,2-ethanedithiol described above. In addition to deprotection by acid treatment in the presence of butanedithiol, it can also be removed by alkali treatment with dilute sodium hydroxide, dilute ammonia, etc. .

As another method for obtaining a peptide amide, first, the α-force lpoxyl group of the carboxyl terminal amino acid is amidated, and then the peptide chain is extended to the desired chain length on the amino group side, A peptide with only the α-amino group protecting group at the Ν-terminal of the peptide chain removed and a peptide (or amino acid) with only the C-terminal carboxyl protecting group removed were prepared. The condensation is performed in such a mixed solvent. 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-described method to obtain the desired crude peptide. This crude peptide is purified using various known purification methods. The amide of the desired peptide can be obtained by freeze-drying the fraction.

To obtain an ester of a peptide, the α-carboxyl group of the carboxy terminal amino acid is condensed with the desired alcohol to form an amino acid ester, and then the desired peptide ester is obtained in the same manner as the peptide amide. be able to.

An antigenic peptide can be directly immunized if it has immunogenicity, but it has a low molecular weight (for example, a molecular weight of about 3,000 or less) having only one or several antigenic determinants in the molecule. ) Antigens (for example, the above-mentioned oligopeptides), these antigenic peptides are usually hapten molecules with low immunogenicity, so that they are bound or adsorbed to a suitable carrier (carrier). Can be immunized as. A natural or synthetic polymer can be used as the carrier. Natural polymers include, for example, serum albumin from mammals such as rabbits, rabbits, and humans, and thyroglobulin from mammals such as rabbits and rabbits, such as ovoalbumins from turkeys, such as rabbits, rabbits, humans, and hidges. Mammal hemoglobin, keyhole limpet hemosyanine (KLH), etc. are used. Examples of the synthetic polymer include various latus statuses such as polymers or copolymers of polyamino acids, polystyrenes, polyataryls, polybules, and polypropylenes. As long as the antibody against the antigen bound to or adsorbed to the carrier is efficiently produced, any carrier may be bound or adsorbed at any ratio. It is possible to use the above-mentioned natural or synthetic polymer carrier, which is commonly used for production of antibodies against haptens, bound or adsorbed at a ratio of 0.1 to 100 with respect to hapten 1 by weight.

Various condensing agents can be used for force pulling of hapten and carrier proteins. For example, diazonium compounds such as bisdiazobenzidine that crosslinks tyrosine, histidine, and triftophan, dialdehyde compounds such as dartaldehyde that crosslinks amino groups, disoyanate compounds such as toluene-2,4-diisocyanate, and thiol groups. Crosslinking Ν, Ν '-0-phenylene dimaleimide and other dimaleimide compounds, maleimide activity cross-linking amino and thiol groups An ester compound, a calpositimide compound that crosslinks an amino group and a carboxyl group, and the like are conveniently used. Also, when amino groups are cross-linked, an active ester reagent having a dithiopyridyl group on one amino group (for example, SPDP) is reacted and then reduced to introduce a thiol group, and the other amino group is introduced. After introducing a maleimide group into the group with a maleimide active ester reagent, both can be reacted. It is also possible to add a cysteine residue to the N-terminus or C-terminus of the hapten (peptide), introduce a maleimide group into the amino protein of the carrier protein using a maleimide active I · raw ester reagent, and then react both. '

(2) Production of monoclonal antibodies

The antigenic peptide can be administered to a warm-blooded animal alone or together with a carrier or diluent at a site where antibody production is possible by administration such as intraperitoneal injection, intravenous injection, subcutaneous injection, or intradermal injection. Be administered. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production at the time of administration. Administration is usually once every 1 to 6 weeks, for a total of 2 to 10 times. Examples of warm-blooded animals include rabbits, goats, rabbits, chickens, mice, rats, hamsters, sheep, pigs, horses, camels, cats, dogs, monkeys, chimpanzees, etc. In general, mice and rats are preferably used. When preparing monoclonal antibody-producing cells, select a mammal that has been immunized with an antigen, for example, an individual with a serum antibody titer from a mouse, and remove the spleen or lymph nodes 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells contained in the cells with myeloma cells. The antibody titer in the antiserum is measured by, for example, reacting a solid phased antigen peptide with antisera, and then immunizing the animal with an antigen peptide-specific antibody bound to the solid phase labeled with a radioactive substance or enzyme. It can be carried out by detecting with an antibody against the species antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein {Nature, 256: 495 (1975)]. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus. However, it is preferable to use PEG.

Examples of myeloma cells include NS-UP3U1, SP2 / 0, etc., and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG600 0) is at a concentration of about 10 to 80%. It is added, about 20 to 40 ° C, preferably carried out efficient cell fusion by Inkyubeto about ^{³ 0~ 3} 7 ° C in about 1 to 10 minutes.

Selection of fused cells (hypridoma) can be carried out according to a method known per se or a method analogous thereto, but can usually be carried out in a medium for animal cells to which HAT (hypoxanthine, tenopopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as it can grow a hyperprideoma. For example, 1-20%, preferably 10-20 ° /. RPMI 1640 medium containing urine fetal serum, 1 to: GIT medium (Waijun Pharmaceutical Co., Ltd.) containing L0% urine fetal serum or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Cultivation can usually be performed in 5% carbon dioxide gas. '' Various methods can be used for screening of monoclonal antibody-producing hybridomas. For example, the supernatant of a high-pridoma culture can be added to a solid phase (eg, a microplate) on which an antigen peptide is adsorbed directly or with a carrier. Next, add anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the antibody-producing cell used for cell fusion is a mouse, anti-mouse immunoglobulin antibody is used) or protein A, and then bind to the solid phase. A method for detecting the monoclonal antibody added, adding the supernatant of the hybridoma culture to a solid phase adsorbed with anti-immunoglobin antibody or protein A, adding an antigen peptide labeled with a radioactive substance or enzyme, etc. Examples thereof include a method for detecting the bound monoclonal antibody.

(3) Purification of monoclonal antibodies

The separation and purification of monoclonal antibodies is the same as the separation and purification of ordinary polyclonal antibodies. Separation and purification of immunoglobulins [Example: Salting out method, Alcohol precipitation method, Isoelectric point precipitation method, Electrophoresis method, Adsorption / desorption method by ion exchanger (Example: DEAE), Ultracentrifugation method, Gel filtration method Specific antibodies can be obtained by collecting only antibodies using an antigen-binding solid phase or an active adsorbent such as protein A or protein G and dissociating the binding to obtain antibodies.

[Preparation of polyclonal antibody]

The polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, a complex of a peptide consisting of the above antigenic determinant (E1 (or Ε)) and a carrier protein is prepared, and a warm-blooded animal is immunized in the same manner as the above-described monoclonal antibody production method. It can be produced by collecting an antibody-containing product against a hapten and separating and purifying the antibody. Regarding the complex of hapten and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten are as follows. Any one can be cross-linked at any ratio as long as it is efficient. For example, .usi serum albumin, cythyroglobulin, KLH, etc. are about 0.1 to 20 by weight ratio to hapten 1. A method of bonding at a ratio of about 1 to 5 is preferably used.

For force pulling of the hapten and the carrier protein, various alkylating agents that can use various condensing agents, active ester reagents containing maleimide active ester, thiol group, and dithiobilidyl group are used. The condensation product is administered to warm-blooded animals at the site where antibody production is possible, or with a carrier or diluent. In order to enhance antibody production at the time of administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration can usually be done about once every 2 to 6 weeks, about 3 to 10 times in total.

Polyclonal antibodies can be collected from blood, ascites, breast milk, eggs, etc. of warm-blooded animals immunized by the above method.

The measurement of polyclonal antibody titer in antiserum is the same as the measurement of antibody titer in serum above. Can be measured in the same way. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as that of the monoclonal antibody described above.

By using the first antibody of the present invention obtained as described above and the second antibody of the present invention, test animals (for example, humans, horses, dogs, cats, monkeys, mice, pigs, Diagnosis of rheumatoid arthritis, particularly secondary osteoporosis in Hedge, Goat, Usagi, Mouse, Rat, Noomster, Monoremot, Chicken, etc.). More specifically, the present invention relates to measuring a substance (that is, MEPE-derived molecule) recognized by the first and second antibodies of the present invention in a biological sample of a test animal suffering from rheumatoid arthritis. A method for diagnosing secondary osteoporosis in the animal is provided. Here, “diagnosis” not only determines the presence or absence of an onset, but also predicts whether or not there is a high probability of developing in the future. It is used to include the evaluation of degree. Since the 'MEPE-derived molecule is recognized by the first antibody of the present invention, it contains at least the antigenic determinant El (E 1,) of the antibody of the present invention.

The biological sample used in the method of the present invention may be any biological sample derived from a test animal, such as a body fluid (eg, blood, plasma, serum, lymph fluid, joint fluid, semen, urine). , Sweat, tears, etc.), various cells, tissue fragments (eg, articular cartilage, etc.), etc. 'Clot, plasma, serum, etc. are preferred because they are less invasive to the test animal.

The method of the present invention is characterized in that the MEPE-derived molecule is measured by a sandwich method. The sandwich method in the present invention includes any method including sandwiching a target MEPE-derived molecule with the first antibody and the second antibody of the present invention to form a first antibody-MEPE-derived molecule-second antibody complex. It is used in the meaning including the method. Therefore, the sandwich method of the present invention is as follows: (1) A biological sample is reacted with the first antibody of the present invention immobilized on a carrier and the free second antibody of the present invention simultaneously or successively. The amount of the first antibody-MEPE-derived molecule-second antibody complex formed on the solid support And (2) a first antibody formed in a solution by reacting a biological sample and the first and second antibodies of the present invention simultaneously or continuously in a solution. -Method for measuring the amount of MEPE-derived molecule-second antibody complex (homogeneous method).

In the heterogeneous method (the sandwich method in the narrow sense), usually a free second antibody (hereinafter sometimes referred to as a liquid phase antibody) is labeled with an appropriate labeling agent. As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. Examples of the radioisotope include [ ¹²⁵ 1], [ ¹³¹ 1], [], [ ¹⁴ C] and the like. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluores; ¾ min, fluoretsen isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, norrecigenin, etc. are used. Furthermore, a biotin-avidin system can be used for the binding between the liquid phase antibody and the labeling agent.

For the immobilization of the antibody, physical adsorption may be used, and a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, silicon, and glass are used.

In the sandwich method, a biological sample is reacted with the immobilized first antibody of the present invention (hereinafter sometimes referred to as solid phase antibody) (primary reaction), and the labeled second antibody of the present invention (liquid) Phase antibody) (secondary reaction), then the solid phase and liquid phase are separated (B / F separation) to remove unreacted labeled liquid phase antibody, etc. By measuring, soot-derived molecules in biological samples can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. Sandwich complex can be detected by surface plasmon resonance (SPR) using a BIAC0RE (registered trademark) chip as a carrier, or by mass spectrometry using a protein chip (manufactured by Cyphergen). In these cases, it is not necessary to label the free antibody.

On the other hand, as a homogenous method, for example, (i) one of the first and second antibodies of the present invention is labeled with a fluorescent substance, and the other is labeled with a quenching substance, and a biological sample and these labels are labeled. A method of measuring a decrease in fluorescence emitted from the fluorescent substance by reacting with a conjugated antibody in a liquid phase, or (ii) one of the first and second antibodies of the present invention is treated as a first fluorescent substance (Excitation light wavelength λ .; labeled with fluorescence wavelength λ, and labeled with the second fluorescent substance (excitation light wavelength λ ,; fluorescence wavelength ₂ ) to react the biological sample with these labeled antibodies in the liquid phase. And a method of detecting the fluorescence of wavelength λ ₂ by exciting the reaction solution with light of wavelength ₀ .

Examples of the fluorescent material used in the above method (i) include FAM and VIC, and examples of the quenching material include TAMRA, DABCYL, DABSYL, and SYBR (registered trademark) Green I.

The first and second fluorescent substance pairs in the above method (ii) include, for example, fluorescamine and dichlorotriazinin fluorescein, fluorescein and rhodamine B, R-phycotriene and aloficocyannin, and fluoreoresin. And dammin X, fluorescein and Texas red or malachite green.

In applying these individual immunoassays to the quantification of MEPE-derived molecules, no special conditions and procedures are required. A measurement system for MEPE-derived molecules may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures for each method. For details on these general technical means, you can refer to reviews, books, etc. [For example, Hiroshi Irie “Radio Imno Atssey” (Kodansha, issued in 1974), Hiroshi Irie “ Sequel Radio Im Nosey j (Kodansha, published in 1979), edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (Medical School, published in 1978), Sakae Ishikawa "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1987), Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, published in 1987), Methods in ENZY MOLOGY, Vol. 70 (Immunochemical Techniques (Part A)), ibid.Vol. 73 (Imrau nocheraical Techniques (Part B)), ibid.Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques ( Part D: Selected Im munoassays)), Vol. 92 (Immunochemical Techniques (Part E: Monoclona 1 Antibodies and General Immunoassay Methods)), | B], Vol. 121 (Iramunoc hemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodi es)) (See above, published by Academic Press).

As described above, the MEPE-derived molecule can be quantified with high sensitivity by using the first and second antibodies of the present invention. '

When the first and second antibodies of the present invention are used, the MEPE-derived molecular concentration is determined by the bone density (BMD) (more specifically, the bone density t-score and the distal radius of the femur It shows a significant negative correlation with (bone density) and a significant positive correlation with urinary doxypyridinoline (PPD), a bone resorption marker.

Therefore, it can be determined that the test animal in which the increase in the MEPE-derived molecule concentration is detected by the method of the present invention develops secondary osteoporosis or is likely to develop in the future. It is also possible to evaluate the degree of progression of osteoporosis by monitoring changes in MEPE-derived molecular concentrations in test animals that have already been diagnosed as having secondary osteoporosis to rheumatoid arthritis.

The present invention also provides a kit for diagnosing secondary osteoporosis in rheumatoid arthritis, comprising the first and second antibodies of the present invention. The kit may further include other components preferable for carrying out the above-described method of the present invention, such as a reaction buffer solution, a washing solution, an insolubilizing carrier, a labeling agent, and a MEPE preparation. .

In the present specification and drawings, when bases, amino acids, etc. are indicated by abbreviations, they are based on abbreviations by IUPA C-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field. Examples are as follows. Also regarding amino acids When there are possible optical isomers, the L form is indicated unless otherwise specified.

DNA: Deoxyribonucleic acid

cDNA: complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanin

C: cytosine

RNA: Ribonucleic acid

mRNA: Messenger liponucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: ethylenediamine tetraacetic acid

SDS: Sodium dodecyl sulfate

Gly: Glycine

Ala: Alanine

Val.

Leu: Leucine

lie: Isoroisin

Ser ： Serine

Thr: Thread Nin

Cys: Sistine

Met: Methionine

Glu: Gnoretamic acid

Asp: Aspartic acid Ly's

Arg

His histidine

Phe: Phenylenolanine

Tyr: Tyrosine

Trp: Tribute fan

Pro: Proline

Asn

Gin: Glutamine

pGlu: pyroglutamic acid

Me: Mechi / Le

Et: Ethyl group

Bu ： Puchinore group

Ph: Hueninore group

TC: Thiazolidine_4 (R) -carboxamide group.

In addition, the substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Tos: p—Tonoreens Nore Foninore '

CHO-: Formyl.

Bzl

Cl ₂ Bzl: 2, 6-jik mouth

Bora: Benzyloxymethyl

Z: Benzinore talent

CI-Z: 2-black mouth penzino reoxy cannole repo

Br-Z: 2-Bromopentinoreoxycanoleboninole

Boc: t-butoxy

DNP ： Jet Mouth Trt: Trityl

Bum: t-Butoxymethyl

Fmoc: N-9_fluorenylmethoxypoel

HOBt: 1-Hydroxybenztriazonore

HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine H0NB: 1-hydroxy-5-norbornene-2,3-dicarboxyimide

DCC: Ν, Ν'-dicyclohexylcarbodiimide

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention. The gene recombination operation using E. coli was performed according to the method described in Molecular cloning, 2nd ed., J. Sambrook et al., Cold spring Harbor Lab. Press (1989).

In addition, the anti-MEPE antibody-producing hybridoma obtained in the examples described below was founded on April 26, 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (IP0D); 1-1- 1 Deposited at the center number 6 with the following deposit number. ,

Hypri-Doma cell

ME-9D2. 15: FERM BP-10327 Example 1 Cloning of cDNA encoding Matrix Extracel lular Phospho-glycoprotein (MEPE)

One cDNA library derived from 0H0 patient tumor was prepared as follows. That is, total RNA was obtained from sinusoidal tumor tissue of a 0H0 patient according to FNO-LC, mouth-form, form-isoamyl alcohol, LiCl precipitation PLANT CELL PHYSIOLOGY 36 '. 85-93 (1995)). Extracted total RNA about 480 Myu§ forceゝal, 01 igotex (dT) ₃₀ - using Super (Takara Shuzo) was obtained, poly (A) + RNA of about 6 mu _§.

A cDNA library was prepared according to the method of Gubler and Hoffman. Oligo (dT) ₁₈ — linker primer ((GA) ₁₀ ACGC containing poly (A) + RN A (2.5 μg), Xho I site First strand DNA was synthesized using GTCGACTCGAGCGGCCGCGGACCG (T) ₁₈ ) (SEQ ID NO: 4), 5-methyldCTP, dATP, dGTP, dTTP, RAV-2 reverse transcriptase and Superscript II reverse transcriptase. This was reacted with RNase H, DNA polymerase I, and dNTP mixture to synthesize second strand DNA. After blunting the ends of the second strand DNA, a coR 1-Not l_BanR 1 adapter (Takara Shuzo Co., Ltd.) was ligated using T4 DNA ligase. This was cleaved with the restriction enzyme ^ o I, the low molecular weight DNA was removed using a spin column, and λ ZAPI I (EcoR l-Xho I cleaved) (STRATAGENE) was used for vector ligation using T4 DNA ligase. . The phage vector thus obtained was incorporated into a λ phage precursor protein using an in vitro packaging kit (STRATAGENE) to prepare a λ phage library. The titer of the library was measured using E. coli XL1 Blue MF, as the host. The titer of primary λ phage library was 1.0 × 10 ⁶ pfu / ml.

The primary phage library was amplified overnight at 4 ° C using E. coli XL1 Blue MRF 'as the host. Approximately 5 × 10 ⁴ plaques were formed per sheet (diameter 150 mra), and this was overlaid with 10 ml of SM buffer. Dimethyl sulfoxide was added to the collected SM buffer to a final concentration of 7% and stored at -80 ° C. The titer of the amplified λ phage library was 1.0 × 10 ⁹ pfu / tnl or more.

Cloning of MEPE 'cDNA was performed by PCR. The phosphatonin gene sequence described in the international patent application (TO 99/60017) of the international patent application for Rpwe PSN does not contain the N-terminal methionine residue secretory signal sequence, and is a complete phosphatonin (MEPE) sequence. Considering that no long cDNA was obtained, the λ phage library derived from the 0H0 patient tumor prepared above was used as a saddle, and as a primer, the host vector used in the cDNA library, pBluescript SK (+/-) Synthetic oligo DNA of SEQ ID NO: 5 on the 5th side of the Jung site, forward primer, and stop codon of the phosphatonin gene sequence described in International Publication (TO 99/60017) of Rowe PSN Synthesis of SEQ ID NO: 6 on the 3 'side of Rigo 'DNA was used as a reverse primer.

5'-GGAAACAGCTATGACGATG-3 '(SEQ ID NO: 5)

5'-TCAGGTGGCTCTCCTCTACATCAACTCACA-3 '(SEQ ID NO: 6)

For the PCR reaction, TaKaRa ExTaq DNA polymerase (Takara Shuzo Co., Ltd.) is added to the above mold and primers, and 95 ° C for 3 minutes using a thermal cycler (GeneAmp PCR System, PE Applied Biosystems). 1 cycle, 95. C, 45 seconds, 58 ° C, 45 seconds, 72 ° C, 3 minutes for 30 cycles, 72 ° C, 5 minutes for 1 cycle, 4 ° C. '

The obtained amplified fragment was subjected to 1% agarose gel electrophoresis, the main band was extracted and purified, and then inserted into the pCRI I-T0P0 vector (Invitrogen) using T0P0 TA Cloning Kit (Invitrogen), and E. coli T0P10 Introduced into the stock. Plasmid DNA is extracted from the resulting transformant, PCR is performed using the Big Dye Terminator Cycle Sequence Ready Reaction Kit (PE Applied Biosystems), and ABI PRISM ™ 377 DNA Sequencer (PE The base sequence of the cDNA fragment was determined by Applied Biosystems.

The insert sequence of the plasmid DNA retained in the obtained clone # 1 was derived from the phosphine gene sequence Val (first) described in International Publication (W0 99/60017) of Rowe PSN's international patent application. (4 ³ 0 th) has a 1, 713 pieces of nucleotide sequence containing a to 1, ²⁹ 0 of base sequence essentially the same nucleotide sequence encoding the MEPE protein consisting of 5 amino acids The phosphatonin gene sequence described in the international publication (W0 99/60017) of the international patent application of Rowe PSN and the insert sequence of the plasmid DNA held by other clones obtained at the same time A few bases were different. In order to determine the true MEPE gene sequence, PCR was performed using Pyrobest DNA polymerase, which has higher fidelity. The λ phage library derived from the 0H0 patient tumor prepared above is used as a cage, and as a primer, the synthetic oligo DNA of SEQ ID NO: 7 starting from the 5th side of the start codon ATG in the previous clone # 1 is used as a forward primer, Synthetic oligo of SEQ ID NO: 6 DNA was used as a reverse primer.

5'-CTCAAAGATGCGAGTTTTCTGTGTGGGA-3 '(SEQ ID NO: 7)

PCR is performed using a thermal cycler (GeneAmp PGR System, PE Applied Biosystems) 98. C, 10 seconds, 56 ° C, 45 seconds, 72 ° C, 3 minutes were performed for 30 cycles at 4 ° C.

The resulting amplified fragment was 1 ° /. After manufactured extracted seminal the main band was subjected to Agarosugeru electrophoresis, and inserted into _P CR-Blunt vector (Invitrogen) using Zero Blunt PCR Cloning Kit (Invitrogen) was introduced into T0P10 E. coli strain combination competent cell.

Plasmid DNA is extracted from the resulting transformant, PCR is performed using Big Dye Terminator Cycle Sequence Ready Reaction Kit 1, (PE Applied Biosystems), and ABI PRISM ™ 377 DNA Sequencer ( The base sequence of the cDNA fragment was determined by PE Applied Biosystems. Thus, a transformant containing the plasmid pCR-PHOS carrying the DNA encoding the MEPE full-length protein was obtained: Escherichia coli. T0P10 / pCR-PHOS (clone # 9). '' The plasmid DNA retained in the acquired TOPlO / pCR-PH0S (clone # 9) is 1,662 represented by SEQ ID NO: 3, which includes 1,575 nucleotide sequences represented by SEQ ID NO: 1. And encoded a phosphatonin (MEPE) protein consisting of 525 amino acids represented by SEQ ID NO: 2. The molecular weight of the protein portion (including the signal peptide) of the protein of the present invention deduced from the amino acid sequence was 58.4 kDa.

The nucleotide sequence represented by SEQ ID NO: 1 encodes the phosphatonin Val (1st) to Asp (430th) described in the international publication (W0 99/60017) of the international patent application of Rowe PSN. Contains a nucleotide sequence that is essentially the same as 1,290 nucleotide sequences, and also contains a new nucleotide sequence consisting of 2 85 bases that encodes an amino acid sequence consisting of 95 residues starting from Met in the 5 'region. Was. SEQ ID NO: 1 nucleotide sequence And the phosphatonin base sequence described in the international publication (TO 99/60017) of the international patent application of Rowe PSN, only the 286th base is different (Rowe PSN publication: G ¹ —SEQ ID NO: 1: C ²⁸⁶ ), and accordingly, the amino acid sequence represented by SEQ ID NO: 1 was also different by one residue (Rowe PSN Publication: Val ¹ —SEQ ID NO: 1: Leu ⁹⁶ ). The amino acid sequence of the protein consisting of 525 amino acids represented by SEQ ID NO: 2 and the base sequence represented by SEQ ID NO: 1 encoding it are the international publications of the international patent application of Rowe PSN (W0 The amino acid sequence of Phosphaten Met (1st) to Asp (525th) described in 01/32878) and the 1,575 base sequences encoding it were completely consistent. Example 2 E. coli expression of recombinant MEPE

A plasmid for the expression of mature MEPE excluding the signal sequence (Met and Ala ¹⁶ ) was constructed, and E. coli expression was attempted. First, Nde I site and start codon ATG are added to the terminal region sequence of mature MEPE. The synthetic oligo DNA of SEQ ID NO: 8 is the forward primer, and the synthetic oligo DNA of SEQ ID NO: ⁹ has the DNA sequence on the 3rd side of the stop codon. As a reverse primer, the plasmid pCR-PHOS (clone # 9) for animal cell expression (Example 1) is used as a saddle, and PCR using a pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) (98 ° CX for 5 seconds, 56 ° CX) 45 seconds, 72 ° CX 3 minutes, 30 cycles).

5, -CATATGGCACCAACATTTCAACCACAGA-3 '(SEQ ID NO: 8),

5, -CTCTCGTCGACATCAACTCACA-3 '(SEQ ID NO: 9)

The obtained PCR product was purified by 1% agarose gel electrophoresis, ligated with pCR Blant vector (Invitrogen ™ life technologies), and transformed into T0P10 competent cells. Plasmid DNA (pCR-mPHOS) was prepared from 12 positive strains obtained by Koguchini PCR, and three vectors with inserts in the reverse direction were selected by 5¾c I digestion. All of these had the correct DNA sequence. Using the ^ ¾Λ? Η I site of the host vector on the 5th side of the insert, these three types of plasmid DNA were digested with Ndel / BanR I and subjected to 1% agarose gel electrophoresis. The product was purified. The pTCI 1 GWe 1 / a / »HI) arm prepared in the same manner and these inserts were ligated and transformed into JM109 combi- ent cells. Plasmid DNA was prepared from the resulting transformant (JM109 / pTCI I-mPHOS-2) and the presence of the insert was confirmed by digestion with Nd "/ BasH I. pTCI I-mPHOS-2 (Fig. 1) ) Had a DNA sequence encoding the correct mature MEPE.

Next, pTCI I-tnPH0S-2 was transfected into an E. coli strain for expression carrying the T7 RNA polymerase gene (under the control of the lac promoter) (應²⁹⁴ (DE3)) and transformed into E. coli MM294 (DE3) / pTCI I. -Obtained tnPHOS-2. '

This transformed strain was cultured at 37 ° C in a 2 L flask containing 1 L of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 10 g / ml tetracycline. Cultured with shaking for 8 hours. The main culture medium containing 5 _g / ml of tetracycline (1.68% sodium monohydrogen phosphate, 0.3% phosphorous dihydrogen, 0.1% ammonium chloride, 0.05% Sodium chloride, 0.05% magnesium sulfate, 0.02% antifoam, 0.00025% ferrous sulfate, 0.005% thiamine hydrochloride, 1.5% glucose, 1.5% strength zamino acid) 1 9 It was transplanted into a 50 L fermentor charged with L, and aeration and agitation culture was started at 3'7 ° C. When the turbidity of the culture solution reached about 500 kret units, the final concentration of 70 μΜ isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the cultivation was continued for another 3.5 hours (1 , 530 kret). Finally, the culture broth was centrifuged to obtain about 300 g of wet cells, which were stored frozen at _80 ° C. .

The expression level of MEPE in this cell is about 15 mg / g wet cell (300 mg / L) based on the staining of the ⁶⁰ kDa band indicated by MEPE. Estimated. In addition, these cells were sequentially sonicated in 20 mM EDTA (H 6) and 20 mM Tris-HC1 / 8 M Urea (pH 8), and the centrifugation supernatant was subjected to SDS-PAGE. The mature MEPE was found to be expressed in the soluble fraction. Example 3 E. coli expression Purification of recombinant MEPE

To 50 g of the microbial cells obtained in Example 2, 50 mM 2- (N-morpholino) ethanesulfonic acid (MES) / NaOH, 1 raM p-amidinophenylmethanesulfonyl chloride hydrochloride (APMSF hydrochloride) ( _PH 6.0) Add 2 L and ultrasonic crusher (Sofer 450) (Branson ( )) Was repeated 3 times at 4 ° C for 10 minutes, followed by centrifugation (8,000 rpm, 30 minutes) to obtain a supernatant. Concentrate this supernatant with an ultrafiltration membrane (regenerated cellulose membrane with molecular weight cut off 10 kDa, membrane area 0.1 ra ² X 2) (Zaritrius Co., Ltd.), 50 raM ME S / NaOH (pH 6. Subsequent to 0), centrifugation (6,000 rpm, 20 minutes) was performed to obtain 1 L of the supernatant. After adsorbing 0.5 ml of this supernatant to SP—TOPAL 550C (5 cm ID X 20 cm) (Tosohichi Co., Ltd.) equilibrated with 50 mM MES / NaOH (pH 6.0) Wash with 50 mM MES / NaOH (pH 6.0) containing 0.5 M NaCl at a flow rate of 20 ml / min, and add MEPE with 50 mM MES / NaOH (pH 6.0) containing 0.8 M NaCl. Elute. This operation was carried out by dividing the supernatant by half. The eluate was concentrated with the ultrafiltration membrane described above and replaced with 50 raM MES / NaOH (pH 6.0), followed by CM-equilibration with 50 mM MES / NaOH (pH 6.0). After adsorbing to 5PW (21.5 mm ID X 150 瞧, 13 μηι) (Tosohichi Co., Ltd.), 0 to 50% Β (1.5 raCl containing 50 raM MES / NaOH (pH 6. Elution was carried out with a step gradient of 0)) for 40 minutes at a flow rate of 5 ml / min and the MEP E fractions were pooled. The eluate was concentrated with an ultrafiltration membrane (Vivapine 20: fractional molecular weight 10 kDa) (Zaritrius Co., Ltd.). Finally, it was applied to Superde x200 (10 mm ID X 30 mm, 10 μιη) (Amersham Biosciences) equilibrated with PBS and eluted at a flow rate of 0.5 ml / min. The MEPE elution fractions were pooled to obtain about 30 mg of a highly pure sample. .

In order to measure the purity of the MEPE sample thus obtained, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. This sample was mixed with sample buffer [Laemml i, Nature, 227: 680 (1979)] supplemented with 100 raM DTT, heated at 95 ° C for 1 minute, and then applied to Multigel 10/20 (Daiichi Kagaku). Electrophoresis was performed. The gel after electrophoresis was stained with Coomassie 'Brilliant' blue, and a single band protein was observed at about 60 kDa. From this, it was found that this MEPE specimen is a single, extremely high purity specimen (Fig. 2).

The N-terminal amino acid sequence of this MEPE purified sample is the gas phase protein sequencer (a It was determined using the pride biosystem model 492). As a result, this sample was consistent with the N-terminal amino acid sequence of MEPE deduced from the nucleotide sequence, except that this sample was a mixture of the N-terminal and Met adducts not added (Fig. 3). Example 4 Production of Anti-MEPE Usagi Polyclonal Antibody

A rabbit polyclonal antibody was prepared using the E. coli-expressed recombinant MEPE protein prepared in Example 3 as an immunogen. Emulsion prepared by mixing equal volumes of PBS solution of the protein and Freund's complete adjuvant. Rabbits (Japanese white, female, 3 kg) Immunized with 3 mg subcutaneously and intradermally on the back of 3 wings. did. For the second and subsequent immunizations, protein emulsions using Freund's incomplete adjuvant were prepared in the same manner, and booster immunizations were repeated 4 times every 2 weeks at 1 mg / feather. Blood was collected from the ear vein before immunization and one week after each immunization after the third immunization, and the increase in serum antibody titer was confirmed by ELISA using an immunoplate coated with the protein. One week after final immunization, carotid blood was collected from 3 rabbits under anesthesia, approximately 70 ml of antiserum was obtained, and antiseptic (0.05% sodium azide) was added. Saved in.

Of these, 6 ral of the rabbit antiserum with the highest antibody titer was diluted 2-fold with MAPS II binding buffer (Nippon Bio-Rad Co., Ltd.), and the centrifuged supernatant was pre-equilibrated with the same buffer. Adsorbed to A Sepharose FF (16 mm ID X 50 mm, 50 μιη, 10 ml) (Amersham Biosciences), washed with protein A MAPS II binding buffer, then protein A MAPSII elution buffer ( The IgG fraction was eluted with Nippon Bio-Rad Co., Ltd. The eluted fraction was neutralized with 1 M Tris-HCl (pH 8.0), and dialyzed overnight at 4 ° C with PBS.

The antigen column prepared by immobilizing the recombinant MEPE protein 3 rag prepared in Example 3 on HiTrap NHS-Activated HP (Amersham Biosciences) was equilibrated with PBS, and the above antibody dialysis was then performed. Liquid was provided. After washing the column with PBS, the non-adsorbed fraction was added with 0.1 M Tris-HCl (pH 8.0) containing 0.5 M NaCl, and 0.1 M containing 0.5 M NaCl. After removing low-affinity antibodies with acetate buffer (pH 4.5), elute high-titer antibodies with 0.5 M NaCl-containing 0.1 M Glycine-HC1 (pH 2.7). The eluted fraction was immediately neutralized with 1 M Tris-HCl (pH 8.0). This was dialyzed against PBS to obtain 16 mg of a purified anti-MEPE antibody preparation. Example 5 Secretory expression of recombinant MEPE-FLAG in insect cells

Phosphor phosphatonin mounting serial in Example 6 of WO International patent application yamada T. (TO No. 01/98485) (MEPE) - FLAG adduct animal cell expression vector _(P T- PH0SF- 11, FIG. 4) A vector produced by ligating the MEPE (phosphatonin) -FLAG structural gene excised from BsiM I and Ssl I from the pFastBacl vector treated with the same restriction enzyme into a JM109 competent cell was transformed. Donor plasmid DNA (pFastBac-PH0SF) was prepared from the obtained transformant (JM109 / pFastBac-PHOSF), and the nucleotide sequence was confirmed. This donor plasmid DNA was added to a competent cell containing Bacmid and helper (MAX Efficiency DHlOBac ™) (Invitrogen ™ life tec hologies), and a transformant was obtained by antibiotic selection. Furthermore, genetically modified Bacraid DNA (Bacraid-PH0SF) was prepared from the transformant, introduced into an insect cell line (SF +) in the presence of CelFECTIN reagent, and a baculovirus for expression of recombinant MEPE-FLAG. A stock solution was prepared. This was infected with an insect cell line (SF +), and the recombinant MEPE-FLAG protein with a molecular weight of 65 kDa was secreted and expressed in the culture supernatant. In addition, a Western plot using an HRP0 labeled anti-rabbit IgG goat antibody was performed. Example 6 Preparation of insect cell expression recombinant MEPE-FLAG

Insect cell lines (SF +) were cultured on a 6 L scale, and the bacterial mouth virus stock solution obtained in Example 5 was added to induce secretion of recombinant MEPE-FLAG. Over time, the amount of recombinant MEPE-FLAG protein in the culture supernatant was monitored by Western blot, and the culture was stopped when the expression level reached the maximum. Insect cell expression recombinant MEPE-FLAG was purified according to the method described in Example 7 of Yamada T.'s international patent application (WO01 / 98485). 4.5 ml of 1 L culture supernatant after infection of insect cells with baculovirus in anti-MEPE rabbit antibody ram (5 ml) equilibrated with 50 mM Tris-HCl (pH 8.0) Adsorbed at a flow rate of / min. After washing this column with Tris-HC1 (pH 8) containing 0.5 M NaCl, recombinant MEP E bound to the column with 0.1 M glycine-HC1 (pH 3.0) containing 0.5 M NaCl -The FLAG protein was eluted and neutralized with 1M Tris-HCl (pH 8.0). The eluate was concentrated and replaced with PBS using an ultrafiltration membrane (Vivapine 20: molecular weight cut off 10 kDa) (Zalto'Rius Co., Ltd.), and this was used as the final sample. A series of operations was repeated 6 times, and 18 mg of recombinant MEPE-FLAG preparation was obtained from 6 L of culture supernatant of insect cells infected with baculovirus. In order to measure the purity of the insect expression MEPE-FLAG sample obtained in this way, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The sample buffer was added with 100 mM DTT. After mixing with [Laemml i, Nature, 227 680 (1979)] and heating at 95 ° C for 1 minute, it was subjected to electrophoresis in Multigel 10/20 (first chemical). After the electrophoresis, the gel was stained with Coomassie 'priliant' blue, and as a result, a nearly single band protein was observed at about 70 kDa (Fig. 5, lane 2).

The N-terminal amino acid sequence of this MEPE-FLAG purified sample was determined using a gas phase protein sequencer (Applied Biosystem Model 4 ⁹² ). As a result, the N-terminal amino acid sequence of this product was consistent with the sequence (APTFQPQTE) obtained by removing the signal peptide sequence from the N-terminal amino acid sequence of MEPE deduced from the base sequence. Example 7 Production of anti-MEPE mouse monoclonal antibody

Emulsion prepared by mixing equal volumes of recombinant MEPE-FLAG sample (1 rag / ml PBS solution) and Freund's complete adjuvant prepared in Example 6 was used in BALB mice (8 weeks old, Female; Japan Claire) Immunized with 40 _μ§ / animal subcutaneously and intradermally in 8 animals. From the second time on, the MEPE-FLAG preparation and Freund's incomplete adjuvant The same prepared emulsion was boosted every 2 weeks.

Prior to the start of immunization and one week after the third immunization, all mice were anesthetized with ether and blood was collected from the fundus to obtain antisera. In other words, mouse antiserum diluted serially with PBS containing 0.2% ushi serum albumin (BSA) was coated with recombinant MEPE-FLAG and blocked with 2% BSA-containing PBS. Nunc) was added at 100 μΐ / well and allowed to react at room temperature for 2 hours. The plate was washed 4 times with PBS containing 0.05% Tween20, and HRP0-labeled anti-mouse IgG goat antibody (Chemicon) diluted 3,000 times with PBS containing 0.2% BSA was added at 100 μΐ / well. The reaction was further allowed to proceed for 2 hours at room temperature. After washing the plate 6 times with PBS containing 0.05% Tweeny, add 3, 3 ', 5, 5, -tetramethyl benzidine (TMB) solution (TMB Peroxidase EIA Substrate Kit; Bio-Rad Laboratories; 100 μΐ / wenore). Addition [J and color at room temperature for 5 minutes, 1 N sulfuric acid (Wako Pure Chemical Industries) was added at 100 μΐ / well to stop the enzymatic reaction. Absorbance (450 nra) of each well was measured using a plate reader Labsystems) was used to create a graph with the serum dilution ratio on the horizontal axis and the absorbance on the vertical axis, and the serum antibody titers were compared.

Recombinant MEPE-FLAG was injected at 10 g / mouse into the tail vein of the top 2 mice that were confirmed to have a sufficient increase in serum antibody titer as described above, and exsanguinated 3 days later to remove the spleen. did. Mouse spleen cells prepared by loosening these spleens and haus myeloma cells (P3X63Ag8U. 1) previously conditioned and cultured in serum-free medium (S'Clon SF-B; Sanko Junyaku) The mixture was mixed at a ratio of 1 and fused using polyethylene glycol (PE G) 1,500 (Rocheda Ignostics). Cell fusion was performed according to the manual attached to the reagent. The fused cells are seeded in a 96-well culture plate at 1 x 10 ⁵ splenocytes / 100 μΐ / well in serum-free medium for cloning (S-Clone CM-B; Sanko Junyaku), and C0 ₂ incubator (5% C0 _2, 37 ° C) were cultured for 1 day in, 0. 1 mM hypoxanthine -0 4 μΜ Aminoputerin -. 0. 016 mM thymidine emissions (HAT) added es ■ Kron CM- B medium 100 μΐ / Ueru And continue culturing in the C0 ₂ incubator, adding 3/4 of the culture supernatant twice every 3 days. The medium was changed.

Culture day 7 to day 14. The culture supernatant of the wells in which the growth of the hybridoma colony was observed over the period of time was subjected to the above-mentioned ELISA for measuring the anti-MEPE mouse antibody to obtain a number of positive wells. From these, highly reactive IgG antibody-producing hybridomas were selected and cloned by limiting dilution, and 8 types of anti-MEPE mouse monoclonal antibody-producing hybridomas [ME-2D11. 9

_s ME-9D2.15 (Ig G _r / K), ME-9H6.8 (IgG ^ K) ME-10E4.2 (IgG K), ME-12C9.2 (IgG _2a / i), ME-12E 2 1 (IgG K) ME-16F4.3 (IgG κ)] was established and stored frozen. The subclass of monoclonal antibodies produced and produced by each hybridoma was determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

Cultivate 500 ml of the above 8 hybridomas in serum-free medium (S. Clon SF-B), and centrifuge (1,200 rpraX for 5 minutes). Further, filter through a 0.2 μηι filter. A culture supernatant containing the monoclonal antibody was prepared. Protein A Sepharose FF (16 mm ID X 25 mm, 50 μηι, 5 ml) mixed with protein A MAPS II binding buffer and equilibrated with the same buffer (Amersham Bioscience) Adsorbed to (Co., Ltd.). The column was thoroughly washed with protein A MAPS II binding buffer, and then the IgG fraction was eluted with protein A MAPSII elution buffer. The obtained fraction was neutralized with 1 M Tris-HC1 (pH 8.0), dialyzed overnight at 4 ° C against PBS, and purified with 8 anti-MEPE mouse monoclonal antibodies. Got. The MEPE binding activity of these antibodies was confirmed by the ELISA described above (FIG. 6). Example 8 Plasma Enzyme Immunoassay for Measurement of MEPE-derived Antigen Concentration (ELISA)

Of the 8 types of anti-MEPE mouse monoclonal antibodies prepared in Example 7, MAB-ME-9D2.15 was used as the solid phase antibody, and the HRP0 labeled antibody of the anti-MEPE Usagipolik oral antibody prepared in Example 4 was liquid. As a phase antibody, a sample designed to measure MEPE concentration in human plasma Doi's ELISA was established.

HRP0-labeled anti-MEPE Usagi Polyclonal antibody is an EZ-link Plus Activated Peroxidase (Pierce Biotechnology, Inc .; # 31487) 1 tng compared to the anti-MEPE Usagi Polyclonal Antibody prepared in Example 4 Was prepared by reacting. For the details of the preparation method, the method described in the document attached to the kit was followed.

The optimized sandwich ELISA procedure is described below. MAB- ME-9D2. 15 is diluted to a concentration of 5 g / ml with 50 mM sodium carbonate / bicarbonate buffer ( _PH 9.6) containing 0.05% sodium azide. (Nunk) was added at 100 μΐ / well and allowed to react at room temperature for 5 hours. After removing the reaction solution, 200 μΐ / ul of a 4-fold diluted solution of Plock Ace (Dainippon Pharmaceutical Co., Ltd.) was added and reacted at 4 ° C overnight to prepare an ELISA plate. Insect cell expression recombinant ME PE-FLAG prepared in Example 6 was concentration determined by amino acid composition analysis, it was dispensed subdivision content those adjusted to 10 μ _§ / ηι1 10% proc Ace containing PBS, _80 The one stored at ° C was used as the standard stock solution. The MEPE standard solution was prepared by thawing this standard stock solution and diluting it further with PBS containing 10% Plocace. Human plasma was collected from ethylenediaminetetraacetate (EDTA) from normal volunteers and rheumatoid arthritis patients with informed consent and dispensed at -80 ° C. The ELISA plate prepared above was washed twice with PBS containing 0.05% Tween20, and 100 μΐ / well of human plasma sample diluted with PBS containing MEPE standard and PBS containing 10% Plockace was added at room temperature. The reaction was performed for 2 hours. After washing the plate 4 times with PBS containing 0.05% Tween20, the above HRP0-labeled anti-MEPE Usagi polyclonal antibody solution diluted to 1 g / ral with PBS containing 10% Block Ace was added at 100 μΐ / well. The reaction was allowed to proceed at room temperature for 2 hours. Furthermore, the plate was washed 6 times with PBS containing 0.05% Tween20, TMB solution (TMB Peroxidase EIA Substrate Kit; Bio-Rad Laboratories) was added at 100 μΐ / well, color was developed for 30 minutes at room temperature, and 2Ν sulfuric acid was added. (Wako Pure Chemical Industries) was added at 100 μ 100 / well to stop the enzyme reaction. The absorbance (450 nm) of each well is measured using a plate reader (Multiskan BICHROMATC; Labsysteras), and the MEPE standard is measured. The MEPE concentration in human plasma was quantified from a calibration curve (Fig. 7) prepared with the quasi-solution. The daily fluctuation coefficient and daily fluctuation coefficient of this ELISA are 2.3-6. 9% and 3. 1-3. 9 ° /, respectively. (Table 1). Quantitative values of human plasma samples by this ELISA are not affected by plasma components if diluted more than 2-fold with PBS containing 10% Block Ace, and human plasma samples are allowed to stand at room temperature for 1 day and at -80 ° C It was confirmed that the quantitative values were not affected by 1 month storage (Figs. 8 and 9). Table 1 Accuracy of MEPE measurement by EUSA

Atsushi Nadauchi Days Atssay

Sample MEPE (ng / ml) ^ CV (%) ^b) MEPE ml) ^ CV (%) *

A 0.576 ± 0.013 2.3 0.589 ± 0.020 3.4

B 0.282 ± 0.009 3.2 0.290 ± 0.009 3.1

C 0.102 ± 0.007 6.9 0.103 ± 0.004 3.9

a) Values represent the mean ± S.D. of 5 experiments.

W CV = 100 x S.D_ / average

Example 9 Correlation between plasma MEPE-derived antigen concentration, femur / tibia bone density, and bone resorption markers in patients with rheumatoid arthritis

The plasma concentration of MEPE-derived antigen in 44 women with rheumatoid arthritis who did not take bisphosphonate and was taking vitamin D (average 63.2 ± 9. years, disease duration 19.3 ± 7.7 years) The measurement was performed by ELISA described in Example 8, and the relationship between the measured value, bone density, and bone metabolism marker was examined. As a result, the MEPE-derived antigen concentration measured by ELISA described in Example 8 was significantly negatively correlated with the bone density t-score and the distal tibia density of the proximal femur (correlation coefficient: 0 363, -0. 414; p-value: 0.032, 0.009, respectively, and a significant positive correlation with urinary deoxypyridinoline (DPD), a bone resorption marker (phase) Number of relations: 0.355; p-value: 0.024) (Fig. 10). Example 1 0 Antigenic determinant analysis of anti-MEPE mouse monoclonal antibody

In order to investigate the antigenic determinant of the anti-MEPE mouse monoclonal antibody (MAB-ME-9D2.15) used in the ELISA of Example 8 and Example 9, the C-terminal sequence was deleted. FfiPE fragments of species [ΜΕΡΕ (Δ 507-525), ΜΕΡΕ (Δ 331-525) and ΜΕΡΕ (Α 24 1-525); each number is the corresponding amino acid number of the amino acid sequence shown in SEQ ID NO: 2 3 types of Escherichia coli expression vectors to which FLA G peptide is added at the C-terminal of each of the C-terminals of the specified range], and these are used as enterococci competent cells BL ² Transformants were obtained by transforming to 1 (DE3).

The obtained transformant was cultured in a small scale in the same manner as described in Example 2 to obtain a MEPE mutein-expressing E. coli strain culture solution. A portion of these were subjected to SDS ^ PAGE, and it was confirmed that the target MEPE mucin was expressed by Western blotting using protein-stained anti-FLAG antibody with Commassie brilliant pull.

A culture solution of mature recombinant MEPE-FLAG and the above three MEPE mutein-expressing E. coli strains (2.5 μ 混合) is mixed with an equal volume of sample buffer to which 100 mM DTT has been added, and heated at 95 ° C for 5 minutes. SDS-PAGE was performed on Multigel 10/20 (Daiichi Kagaku). The electrophoretic protein was transferred to PVDF membrane from the gel after electrophoresis, and anti-MEPE Usagi polyclonal antibody prepared in MAB-ME-9D2.15 and Example 4, and HRP0-labeled anti-mouse. IgG goat antibody (Chemicon) Western plots were performed using HRP0-labeled anti-rabbit IgG goat antibody (Chemicon) (Figure 11). Multiple bands were detected for each mutin, but the uppermost main band matched with the molecular size predicted from the amino acid sequence and stained with anti-FLAG antibody was the target MEPE mutin and the lower molecular band It was judged as the decomposition product.

Anti-MEPE Usagi polyclonal antibody bound to mature recombinant MEPE-FLAG and any of the above three MEPE mutins. In addition, since MAB-ME-9D2.15 used as a solid phase antibody in the ELISA of Example 8 also bound to mature recombinant MEPE-FLAG and any of the above three MEPE mutins, Was found to recognize an epitope in the N-terminal region of MEPE (Ala ¹⁷ _Gly ²⁴ ). . Industrial applicability

The two anti-MEPE antibodies of the present invention can specifically detect MEPE-derived molecules in biological samples that can serve as diagnostic markers for secondary osteoporosis in rheumatoid arthritis. Useful for the diagnosis of osteoporosis. While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims. This application is based on Japanese Patent Application No. 2005-269071 filed in Japan, and the contents disclosed therein are incorporated in full herein. In addition, the contents of all publications including the patents and patent application specifications mentioned here are incorporated herein by reference to the same extent as if all of them were explicitly stated. Is. '

Claims

The scope of the claims

1. The following antibody (a) or (b).

(a) Mouse monoclonal antibody MAB- ME-9D2.15 (FERM BP-10327)

2. The antibody according to claim 1, wherein the antigenic determinant is present in the amino acid sequence represented by amino acid numbers 17 to 240 in the amino acid sequence represented by SEQ ID NO: 2. '

3. A method for diagnosing secondary osteoporosis in rheumatoid arthritis, comprising using the antibody according to claim 1 and a polyclonal antibody against MEPE. .

4. The method according to claim 3, characterized in that the child uses an immunoassay by the sandwich method.

5. The method according to claim 3, wherein blood, serum or plasma is used as the test sample.

6. A diagnostic kit for secondary osteoporosis in rheumatoid arthritis, comprising the antibody according to claim 1 and a polyclonal antibody against MEPE. '