JP2008048738A - Specific antibody to active hepacite growth factor activator and method for using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody which specifically recognizes active type HGFA and does substantially not recognize inactive type HGFA, and to provide a method for measuring active type HGFA with the same antibody. <P>SOLUTION: A method for preparing a monoclonal antibody comprises selecting a hybridoma producing the monoclonal antibody exhibiting reactivity with active type HGFA and not exhibiting reactivity with inactive type HGFA with the recombinant inactive type HGFA prepared with HGFA cDNA encoding a HGFA precursor and the active HGFA obtained by treating the inactive type HGFA with a protease, and then preparing the objective monoclonal antibody from the culture supernatant of the obtained hybridoma. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to an antibody or monoclonal antibody for specific measurement against activated hepatocyte growth factor activator (HGFA), a method for using the same, and a measurement kit. In addition, patients with a disease state using active HGFA as an index, particularly detection methods for organ inflammation, glomerulonephritis, cancer, myocardial infarction, angina pectoris and thrombosis, and the optimal biological components and blood for the implementation of these methods It is related with the collection method.

Proteases (proteolytic enzymes) are proteins having a function of hydrolyzing peptide bonds in proteins and peptides, and play an important role in maintaining their homeostasis while being closely related to biological functions. For example, various protease groups existing in blood themselves form a dense cascade and control blood coagulation and fibrinolysis.

Hepatocyte Growth Factor Activator (hereinafter abbreviated as “HGFA”) is known as a kind of serine protease having a serine residue at its active center (Miyazawa et al., Non-Patent Document 1). HGFA differs from general blood proteases involved in the blood coagulation / fibrinolytic cascade, and is known as a hepatocyte growth factor (hereinafter referred to as “Hepatocyte Growth Factor”), which is known as a cytokine involved in hepatocyte proliferation and organ regeneration. (Abbreviated as “HGF”) (Naka et al., Non-Patent Document 2), and has a unique property of specifically decomposing and activating it (Shimomura et al., Non-Patent Document 3). However, the action of HGFA is known only for the activation of HGF by animal model experiments using rats and in vitro experiments (Miyazawa et al., Non-Patent Document 4), and the role, function and blood in human pathology are known. The relationship between medium concentration and disease state was completely unknown.

HGFA was subjected to limited degradation between the 372nd arginine and the 373rd valine counted from the translational starting amino acid, showing a molecular weight of about 96,000 to about 98,000 (hereinafter abbreviated as “98 kDa HGFA”) by SDS-PAGE. A C-terminal peptide having a molecular weight of about 34,000 to about 38,000 (hereinafter abbreviated as “36 kDa HGFA”) is known. Each molecular weight has a width based on the heterogeneity of the amount of sugar chain binding and the difference in molecular weight measurement values obtained by performing SDS-PAGE under reduction or non-reduction. It is. Furthermore, each HGFA usually exists in the blood as an inactive form, but is activated by undergoing limited degradation between the 407th arginine and the 408th isoleucine, counted from the translation initiation amino acid, HGFA is converted to a double-stranded HGFA heterodimerized by a disulfide bond. This activated HGFA is considered to specifically activate the substrate HGF (Shimomura et al., Non-Patent Document 5).

In order to analyze the relationship between the blood concentration of HGFA and the pathological condition, it is necessary to specifically measure active HGFA present in a biological component such as human tissue, body fluid, or blood. In order to specifically detect or measure active HGFA, an antibody having the property of recognizing the active HGFA very specifically and not substantially recognizing the inactive HGFA, particularly preferably a monoclonal antibody. Acquisition is essential. However, the existence of antibodies having such properties has not been known until now, and there is no measurement method or kit specific to active HGFA present in biological components such as human blood.
J. Biol. Chem., 268: 10024-10028 (1993) J. Biol. Chem., 267: 20114-20119 (1992) Cytotechnology, 8: 219-229 (1992) J. Biol. Chem., 271: 3615-3618 (1996) J. Biol. Chem., 268: 22927-22932 (1993)

The present invention specifically relates to an antibody that specifically recognizes active HGFA and does not substantially recognize inactive HGFA, a method for measuring active HGFA using the antibody, and a method for detecting a disease involving active HGFA And providing a kit.

The present inventors attempted to construct an HGFA quantification system in human blood in order to analyze the relationship between HGFA blood concentration in human pathology and various human diseases. First, 7E10, P1-4, A-1, A-6, A-23, A-32, A-51, A-75 (Miyazawa et al., J. Biol) known as existing mouse monoclonal antibodies against HGFA. Chem., 271: 3615-3618 (1996)), an enzyme-linked immunosorbent assay (hereinafter abbreviated as “ELISA measurement system”) using a two-antibody sandwich method was constructed. Furthermore, the reactivity of the HGFA monoclonal antibody was analyzed against blood (plasma or serum) of various diseases including healthy persons and organ diseases. However, the ELISA measurement system using these monoclonal antibodies shows uniformly strong reactivity to all human blood, including healthy individuals, and specific reactivity against human diseases, that is, HGFA concentration in blood No significant change was detected. Therefore, the present inventors analyzed the reactivity of HGFA possessed by an existing monoclonal antibody against HGFA and attempted to pursue this cause.

First, since HGFA has two forms, an active form and an inactive form, the reactivity of each monoclonal antibody against each HGFA was analyzed. Specifically, each of active HGFA and inactive HGFA was attached to a solid phase plate, and the reactivity of HGFA attached to the solid phase with each monoclonal antibody was analyzed. As a result, these monoclonal antibodies were found to be active HGFA. It has been found that it has the property of reacting with both inactive HGFA. These results show that inactive HGFA (Shimomura et al. J. Biol. Chem., 268: 22927-22932), which is considered to be present constantly and in large amounts in the blood by ELISA using existing monoclonal antibodies. (1993)), or a trace amount of both active HGFA and inactive HGFA may be measured. In the ELISA measurement system using these existing monoclonal antibodies, there is no relationship between HGFA and human diseases. It turns out that I don't understand.

Accordingly, the present inventors have focused on the fact that HGFA has two forms, an active form and an inactive form, and specifically detected the activation state of HGFA present in the human body, and various human diseases and blood thereof. Investigation was made on the relationship with the amount of active HGFA present therein. As a result of extensive research, the inventors succeeded for the first time in producing polyclonal and monoclonal antibodies that recognize active HGFA and do not substantially recognize inactive HGFA. Furthermore, by using an immunological assay using these antibodies, it reacts with active HGFA present in human biological components and does not substantially react with inactive HGFA, that is, active HGFA is specifically selected. To complete the method and the kit.

Furthermore, when the active HGFA specific measurement method and kit thereof according to the present invention are used, the amount of active HGFA present in the blood of patients with organ disorders such as glomerulonephritis and cancer patients, compared to healthy individuals Was found for the first time to increase significantly. In addition, it has been found for the first time that thrombosis such as angina pectoris, myocardial infarction, cerebral infarction, etc. can be predicted with high sensitivity using the increase in the amount of active HGFA in blood as an index. .

On the other hand, when measuring the amount of active HGFA present in a biological component such as human blood, a method for collecting active HGFA present therein in a stable and reproducible manner is required. As a result of examining the addition of various protease inhibitors and the like regarding the method, the present inventors can measure active HGFA extremely stably and reproducibly by adding argatroban which is a selective thrombin inhibitor. I found out. In addition, as a result of repeated studies, it is effective to add argatroban even when collecting human blood such as whole blood, serum, and plasma, and it is extremely good to use citrated plasma in the blood. I found that the result was obtained.

This invention is made | formed from the said knowledge, The summary is as follows.
(1) An antibody that recognizes an active hepatocyte growth factor activator (HGFA) and does not substantially recognize an inactive HGFA.
(2) The antibody according to (1), wherein the dissociation constant for active HGFA is 1 × 10 ⁻⁸ M or less.
(3) The antibody according to (1) or (2), which is a monoclonal antibody.
(4) The antibody according to (3), which recognizes active HGFA showing about 34,000 to about 98,000 by SDS-PAGE method and does not substantially recognize inactive HGFA.
(5) The antibody according to (4), which recognizes active HGFA showing about 34,000 to about 38,000 by SDS-PAGE.
(6) The monoclonal antibody according to (4) produced by a hybridoma having the accession number FERM BP-7779.
(7) Recognizing active HGFA activated by undergoing limited degradation between 407th arginine and 408th isoleucine, counted from the translation initiation amino acid of inactive HGFA, which is a precursor of active HGFA, A monoclonal antibody that does not substantially recognize inactive HGFA.
(8) A monoclonal antibody that recognizes active HGFA and does not substantially recognize inactive HGFA and a complex of active HGFA and a protease inhibitor.
(9) A hybridoma cell line that produces the monoclonal antibody according to any one of (3) to (8).
(10) Measurement of active HGFA, wherein active HGFA is specifically measured by an immunological method using one or more of the antibodies according to any one of (1) to (8) Law.
(11) The method according to (10), wherein the specimen for measuring active HGFA is a biological component collected from a subject or animal suspected of having a disease.
(12) The method according to (11), wherein the disease is organ inflammation, glomerulonephritis, cancer, myocardial infarction, angina pectoris, cerebral infarction or thrombosis.
(13) A disease detection method comprising detecting or measuring active HGFA in a biological component collected from a subject suspected of having a disease.
(14) The method according to (13), wherein the disease is organ inflammation, glomerulonephritis, cancer, myocardial infarction, angina pectoris, cerebral infarction or thrombosis.
(15) The method according to (13) or (14), wherein the biological component is blood or a fraction or processed product thereof.
(16) The method according to (15), wherein the biological component is plasma.
(17) The method according to (16), wherein the plasma is citrated plasma.
(18) The method according to any one of (13) to (17), wherein argatroban is added to the biological component.
(19) A kit for detecting or measuring active HGFA, comprising one or more of the antibodies according to any one of (1) to (8).
(20) The kit according to (19) used for diagnosis of organ inflammation, glomerulonephritis, cancer, myocardial infarction, angina pectoris, cerebral infarction or thrombosis.
(21) The kit according to (19) or (20), wherein active HGFA is measured in a biological component collected from a patient suspected of having a disease.
(22) The kit according to any one of (19) to (21), wherein active HGFA is detected or measured by immunostaining.
(23) A blood collection tube for obtaining serum or plasma or whole blood, to which argatroban is added.
(24) The blood collection tube according to (23) used for collecting serum or plasma or whole blood used for measurement of active HGFA.

In the present specification, a monoclonal antibody that recognizes active HGFA and does not substantially recognize inactive HGFA is referred to as “active HGFA-specific monoclonal antibody”, which recognizes active HGFA and substantially recognizes inactive HGFA. Polyclonal antibodies that do not are sometimes referred to as “active HGFA-specific polyclonal antibodies”. Furthermore, these may be collectively referred to as “active HGFA-specific antibodies”. “Recognizing active HGFA” refers to binding to active HGFA by an antigen-antibody reaction, preferably a dissociation constant for active HGFA of 1 × 10 ⁻⁸ M or less, more preferably 1 × It means 10 ^-9 M or less. “Substantially does not recognize inactive HGFA” means that it does not substantially bind to inactive HGFA by an antigen-antibody reaction. More specifically, “substantially does not recognize inactive HGFA” means that inactive HGFA cannot be detected by ordinary immunoassay, and preferably, It means that the dissociation constant for active HGFA is 1 × 10 ⁻⁵ M or more.

The present invention provides monoclonal and polyclonal antibodies that specifically bind to active HGFA and do not bind to inactive HGFA. The antibody of the present invention can be used for specific measurement and detection of active HGFA.
The method for measuring active HGFA of the present invention can be used for diagnosis of various diseases reflected in the blood concentration of active HGFA.

The present invention is described in detail below.
<1> Immune antigen and screening antigen for production of specific antibody against active HGFA “Active HGFA” used in the present invention refers to its inactive form acting on its substrate hepatocyte growth factor (HGF). The single-chain HGF (Naka et al. J. Biol. Chem., 267: 20114-20119 (1992)) having the ability to convert to the active double-chain HGF. Here, the single chain type refers to a product obtained by removing the signal peptide from a single precursor protein generated from the HGF gene, and the double chain type refers to the 494th counting from the translation start amino acid of HGF. This refers to a product obtained by subjecting the resulting two chains to heterodimerization by disulfide bonds after limited degradation between arginine and the 495th valine. The activity of HGFA indicates the activity of converting inactive single-stranded HGF into active double-stranded HGF.

Specifically, active HGFA is activated by undergoing limited degradation between 407th arginine and 408th isoleucine, counted from the translation initiation amino acid of the HGFA precursor protein described in, for example, JP-A No. 6-15946 HGFA is included. This active HGFA has a molecular weight of about 34,000 to 98,000, usually by SDS-PAGE, and active HGFA showing various molecular weights with limited N-terminal degradation is known (Mitsubishi Kasei R & D Review, 8 (1), 26-35 (1994)). For example, a molecular weight of about 98,000 in SDS-PAGE under reduction, or a peptide on the C-terminal side subjected to limited degradation between the 88th arginine and the 89th alanine counted from the translation initiation amino acid. There are those showing about 80,000, and those showing a molecular weight of about 36,000, which is a peptide on the C-terminal side subjected to limited degradation between the 372rd arginine and the 373rd valine counted from the translation initiation amino acid. Those showing the molecular weight of about 98,000 by the heterogeneity of the amount of sugar chain binding, the molecular weight measurement method, or the purification method may show a range of about 96,000 to 98,000, and those showing the molecular weight of about 36,000 , May exhibit a width of about 34,000-38,000. Therefore, the active HGFA used here is not limited to its molecular weight, and it only needs to have the ability to activate its substrate, HGF.
Active HGFA or inactive HGFA used for screening of immune antigens or monoclonal antibodies is purified from blood such as humans according to the method of Shimomura et al. (J. Biol. Chem., 268: 22927-22932 (1993). In addition, a set using microorganisms such as E. coli, insect cells, yeast, animal cells and animals using the HGFA cDNA described in JP-A-6-153966 and JP-A-6-153946. Those using HGFA, which is a replacement protein, can also be used.

In particular, in order to obtain an active HGFA-specific antibody, high-purity active HGFA that is not contaminated with inactive HGFA, and high that is not contaminated with active HGFA as antigens for immunization and screening. It is necessary to prepare pure inactive HGFA. For these purposes, the active HGFA and inactive HGFA used here are preferably recombinant proteins using HGFA cDNA. For example, using HGFA cDNA encoding the HGFA precursor described in JP-A-6-153946, the full length or a part thereof is inserted into an appropriate expression vector, and this is inserted into microorganisms such as E. coli, insect cells, yeast, It is possible to obtain HGFA, which is a recombinant protein, by introducing it into animal cells or animals and further purifying it from the culture supernatant or intracellular, tissue, or body fluid of these gene-transfected cells that express HGFA It is. It is also possible to produce the target HGFA using a transcription / translation system in a test tube using Rapid Translation System RTS500 (Roshe Diagnostics) without using a cell system.

As a specific example, HGFA cDNA encoding the full length of the inactive HGFA precursor described in JP-A-6-1553946 and having a signal sequence of 655 amino acids was inserted downstream of the promoter of the animal cell expression vector. Animal cells in which expression vectors are used, or, as described in JP-A-6-153966, an appropriate signal sequence and cDNA encoding the C-terminal side of the 373rd valine after the translation initiation amino acid of the HGFA precursor are bound Examples thereof include a method of using an expression vector or an animal cell expression vector in which an appropriate signal sequence and a region cDNA having an ability to exert HGFA activity are bound. After introducing these expression vectors into animal cells, cells expressing the HGFA cDNA are selected and purified from the culture supernatant to obtain a recombinant protein HGFA.

HGFA, which is a recombinant protein obtained by such a method, is generally inactive. Therefore, referring to the method of Shimomura et al. (J. Biol. Chem., 268: 22927-22932 (1993)), adding appropriate amounts of thrombin and dextran sulfate or kallikrein and thrombin to this inactive HGFA This inactive HGFA can be activated. The HGFA activated by this treatment can be purified by purification by gel filtration using HPLC or affinity chromatography.

Highly purified active HGFA and inactive HGFA are not only important as immunogens, but also when selecting active HGFA-specific polyclonal antibodies by affinity purification or screening active HGFA-specific monoclonal antibodies It becomes a very important material.

As a result of detailed studies, the present inventors have found that purified inactive HGFA is extremely unstable and is activated by the incorporation of slight impurities. Using such inactive HGFA mixed with active HGFA, or conversely using active HGFA mixed with inactive HGFA, an active HGFA-specific polyclonal antibody or an active HGFA-specific monoclonal is used. Obtaining antibodies is difficult. Therefore, the present inventors examined various protease inhibitors as inhibitors that prevent artificial activation when preparing inactive HGFA, and as a result, Argatroban (Mitsubishi Tokyo Pharmaceutical Co., Ltd.), a selective thrombin inhibitor, was investigated. It was found useful to add)) to HGFA. That is, when argatroban is added to inactive HGFA during purification of inactive HGFA or during monoclonal antibody screening, it is possible to prevent artificial activation and prevent contamination with active HGFA. The active HGFA prepared here is suitable as an antigen for immunization for obtaining an active HGFA-specific antibody, and further selects an antibody that recognizes active HGFA and does not substantially recognize inactive HGFA, It can also be used as a material to be purified.

On the other hand, inactive HGFA is suitable as an antigen for immunization for obtaining an antibody that recognizes inactive HGFA and does not substantially recognize active HGFA, and recognizes inactive HGFA and recognizes inactive HGFA. It can be used as a material for selecting, absorbing or removing antibodies other than antibodies that are not substantially recognized.

As an immunization antigen or screening antigen necessary for obtaining an active HGFA-specific antibody, it is located at a site of difference in primary structure sequence (amino acid sequence) between active HGFA and inactive HGFA, or a site of difference in steric structure It is possible to utilize peptides that For example, a peptide that is exposed only on the protein surface of active HGFA and not exposed on the protein surface of inactive HGFA can be used. Such protein three-dimensional structure prediction is generally available to those skilled in the art, and a three-dimensional conformation structure is schematically represented from the amino acid sequences of active HGFA and inactive HGFA by a computer program. It is available. By comparing the two, it is possible to find a region that becomes a binding site (antigen site) of an antibody that exhibits a specific reaction to active HGFA, and to use a peptide obtained by synthesizing the partial amino acid sequence as an immunizing antigen. .

In addition, using protein structure analysis software such as GENETYX (SOFTWARE DEVELOPMENT CO., LTD), from the information on the amino acid sequence of HGFA, the hydrophilic / hydrophobic differences between active HGFA and inactive HGFA, Cho- A region that is a different site of the secondary structure analysis result by Fasman method or Robson method, or a region that is a different site of antigenicity may be found, and a peptide synthesized based on the amino acid sequence may be used as an immune antigen or a screening antigen. For example, active HGFA has an amino acid sequence subjected to limited degradation between the 407th arginine and the 408th isoleucine, counting from the translation initiation amino acid of the HGFA precursor protein. On the other hand, inactive HGFA has an amino acid sequence in which the 407th arginine and the 408th isoleucine are continuous. Therefore, as an amino acid sequence that does not exist in inactive HGFA but exists only in active HGFA, a peptide containing 407th arginine as the C-terminus and several amino acid sequences present in the N-terminal direction is available. . For example, the following can be selected. The amino acid sequences of sequences 2 to 12 are sequences that are deleted by one amino acid residue from the N-terminal side of the amino acid sequence of sequence 1 (SEQ ID NO: 1).

Sequence 1: GRRHKKRTFLRPR Sequence 2: RRKKKRFLRPR Sequence 3: RHKKRTFLRPR Sequence 4: HKKRTFLRPR Sequence 5: KKRTFLRPR Sequence 6: KRTFLRPR Sequence 7: RTFLRPR Sequence 8: TFLRPR Sequence 9: PRLOPR Sequence 9: FLOPR Sequence 9: FLOPR Sequence 9: FLOPR

Further, as an amino acid sequence that is not present in inactive HGFA but is present only in active HGFA, the following is selected as a peptide containing several amino acid sequences present at the N-terminus of 408th isoleucine and in the C-terminal direction. It is possible. The amino acid sequences of Sequences 14 to 24 are sequences that are deleted by one amino acid residue from the C-terminal side of the amino acid sequence of Sequence 13 (SEQ ID NO: 2).

Sequence 13: IIGGSSSSLPGSH Sequence 14: IIGGSSSSLPGSH Sequence 1
5: IIGGSSSLPGS sequence 16: IIGGSSSSLPG sequence 17: IIGGSSSSLP sequence 18: IIGGSSSL sequence 19: IIGGSSS sequence 20: IIGGSS sequence 21: IIGGS sequence 22: IIGG sequence 23: IIG sequence 24: II

In addition, as an amino acid sequence that is not present in active HGFA but only in inactive HGFA, a position between 407th arginine and 408th isoleucine counted from the translation initiation amino acid of the HGFA precursor protein (positions 13 and 14 of sequence 25). For example, a peptide such as SEQ ID NO: 25 (SEQ ID NO: 3) can be selected that comprises a contiguous amino acid sequence.

Sequence 25: GRRHKKRTFLRPRIIGGSSSPGPSHP

When these peptides are used as antigens for immunization, they are generally bound to proteins or macromolecules such as KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), OVA (ovalbumin) as carriers. It is desirable to use those as antigens for immunization. For example, the peptide described in SEQ ID NO: 1 to SEQ ID NO: 12 with an additional cysteine added to the N-terminus, the peptide described in SEQ ID NO: 13 to SEQ ID NO: 24, further added with a cysteine, described in SEQ ID NO: 25 A peptide in which cysteine is added to the N-terminus or C-terminus of the peptide and synthesized with PIMACE Imprint Maleimide Activated Carrier Proteins can be used as an antigen for immunization.

Also obtained by binding cDNA corresponding to sequence 1 to sequence 25 and cDNA of a protein serving as a carrier, expressing a fusion protein in which the peptides encoded by both are expressed in various cells by genetic engineering techniques, and purifying them. It can be used. Those containing any one of Sequence 1 to Sequence 24 are suitable as immunizing antigens for obtaining active HGFA-specific antibodies, and can also be used as materials for selecting and purifying active HGFA-specific antibodies. I can do it. On the other hand, a peptide such as sequence 25 is suitable as an antigen for immunization for obtaining an antibody that recognizes inactive HGFA and does not substantially recognize active HGFA, and an antibody other than an active HGFA-specific antibody. It can be used as a material to be absorbed or removed.

<2> Preparation of monoclonal antibody that recognizes active HGFA and does not substantially recognize inactive HGFA To obtain an active HGFA-specific monoclonal antibody, the above-mentioned active HGFA is usually used as an immunizing antigen. The performed immunological method may be performed. For example, active 98 kDa HGFA or active 36 kDa HGFA or a mixture of both can be used.

Alternatively, a peptide comprising any one of Sequence 1 to Sequence 24 may be prepared by fusing with a carrier by the method described above, and one or more of these may be mixed and used as an immunizing antigen. Furthermore, a peptide containing an antigen site specific for active HGFA may be used.

The animal used for immunization is not particularly limited, but any of rabbits, goats, sheep, mice, rats, guinea pigs, chickens and the like can be used. Inoculation of animals with an immunizing antigen is performed by thoroughly mixing Freund's adjuvant or incomplete Freund's adjuvant with an immunizing antigen subcutaneously, intramuscularly or intraperitoneally. The inoculation is carried out every 2 to 5 weeks and continued until the antibody titer of the immunized animal against the inoculated antigen is sufficiently increased. Thereafter, the immunized animal is intravenously injected with the antigen alone, and 3 days later, the spleen or lymph node considered to contain antibody-producing cells is collected, and the spleen cells or lymphocytes are fused with tumor cells. Thereafter, antibody-producing cells (hybridomas) that have become immortalized by cell fusion are isolated. The tumor cells used here are preferably of the same species as the spleen cells or lymphocytes generally prepared from the immunized animal, but can also be between different animals.

Examples of tumor cells include myeloma cells such as p3 (p3 / x63-Ag8), P3U1, NS-1, MPC-11, SP2 / 0, FO, x63.6.5.3, S194, R210. Cell fusion may be performed according to a commonly used method, for example, “Monoclonal Antibody Experiment Manual” (Kodansha Scientific 1987). Cell fusion can be performed by adding a cell fusion promoter to a fusion medium in which cells to be fused are suspended. Examples of cell fusion promoters include Sendai virus and polyethylene glycol having an average molecular weight of 1000-6000. At this time, in order to further increase the fusion efficiency, an auxiliary agent such as dimethyl sulfoxide and a cytokine such as IL-6 can be added to the fusion medium. The mixing ratio of tumor cells to immunized spleen cells or lymphocytes may be, for example, about 1 to 10 times that of spleen cells or lymphocytes relative to tumor cells.

Various normal media such as ERDF media, RPMI-1640 media, and MEM media can be used as the above-mentioned fusion media. During fusion, serum such as fetal bovine serum (FBS) is usually removed from the media. Is good. For fusion, a predetermined amount of spleen cells or lymphocytes and tumor cells subjected to the above immunization are mixed well in the above medium, and a polyethylene glycol solution that has been heated to about 37 ° C. in advance is 20% to 50%. %, Preferably by reacting at 30 to 37 ° C. for 1 to 10 minutes. Thereafter, the operation of adding an appropriate medium sequentially, centrifuging, and removing the supernatant is repeated.

The target hybridoma is cultured in a normal selective medium such as HAT medium (medium containing hypoxanthine, aminopterin and thymidine). The culture in the HAT medium may be performed for a time sufficient for the cells other than the target hybridoma (unfused cells etc.) to die, usually several days to several weeks. When obtaining an active HGFA-specific monoclonal antibody, the most technically important point is its screening. Hybridomas producing active HGFA-specific monoclonal antibodies can be screened by using various materials such as the above-mentioned active HGFA and inactive HGFA and analyzing them by various immunochemical methods. For example, active HGFA or inactive HGFA is used as a screening antigen, and the binding between these screening antigen and a monoclonal antibody secreted into the hybridoma culture supernatant is determined by enzyme immunoassay such as ELISA or Western blotting. The target hybridoma can be selected by analysis using a method or the like.

Specifically, a hybridoma secreting an antibody recognizing active HGFA by adding the culture supernatant of the hybridoma to an adherent active HGFA attached to a screening plate or the like and blocking with BSA or the like Sort out. Further, the selected hybridoma is attached to an inactive HGFA on a screening plate or the like and blocked with BSA or the like, and a hybridoma secreting an antibody that does not recognize this inactive HGFA is selected. For example, the culture supernatant of the selected hybridoma is added to and reacted with an ELISA plate to which active HGFA or inactive HGFA is attached, and after a sufficient washing operation, a labeled anti-mouse IgG polyclonal antibody is further added. React. After the washing operation, the label is detected, and a hybridoma having a culture supernatant that is reactive to the plate to which active HGFA is attached and that has no reactivity to the plate to which inactive HGFA is attached is selected. As the label, various enzymes, fluorescent substances, chemiluminescent substances, radioisotopes, biotin, avidin, etc. described later are used.

By the above screening, a hybridoma that produces a monoclonal antibody that recognizes active HGFA and does not substantially recognize inactive HGFA is obtained. On the other hand, when screening such a hybridoma, it may be used as an indicator that it reacts with any of the peptides including the sequence 1 to the sequence 24 shown above and does not react with the peptide such as the sequence 25. Regarding the antibody produced by the hybridoma selected by such a method, it may be confirmed that it reacts with active HGFA and does not react with inactive HGFA. The reactivity of the antibody can be confirmed by measuring a dissociation constant for active HGFA or inactive HGFA. The dissociation constant of the antibody of the present invention for active HGFA is preferably 1 × 10 ⁻⁸ M or less, more preferably 1 × 10 ⁻⁹ M or less. Moreover, the dissociation constant for inactive HGFA is preferably 1 × 10 ⁻⁵ M or more.

The obtained hybridoma can be cloned by a limiting dilution method to obtain a hybridoma clone that produces a single monoclonal antibody. This hybridoma clone is cultured using a medium or serum-free medium supplemented with about 1 to 5% of FBS excluding bovine antibody (IgG) contained in FBS in advance, and the obtained culture supernatant is used as a target culture supernatant. A raw material for purifying monoclonal antibodies. On the other hand, the obtained hybridoma clone was transplanted into the abdominal cavity of a Balb / C mouse or Balb / c (nu / nu) mouse to which pristene had been administered in advance, and ascites containing a high concentration of monoclonal antibody was collected 10 to 14 days later. The target monoclonal antibody may be used as a raw material for purification. As a method for purifying a monoclonal antibody, an ordinary immunoglobulin purification method may be used, for example, ammonium sulfate fractionation method, polyethylene fractionation method, ethanol fractionation method, anion exchange chromatography, protein A or protein G bound thereto. It can be carried out by affinity chromatography or the like.

<3> Preparation of polyclonal antibody that recognizes active HGFA and does not substantially recognize inactive HGFA Active HGFA-specific polyclonal antibodies were obtained using active HGFA or inactive HGFA as an immunizing antigen. The polyclonal antibody derived from the immunized animal can be obtained by purifying an antibody that recognizes active HGFA and does not substantially recognize inactive HGFA.

In addition, as a immunizing antigen for obtaining a polyclonal antibody, the aforementioned peptide-carrier fusion containing an antigen site specific for active HGFA can also be used. For example, one or more of the peptides containing any of the aforementioned sequences 1 to 25 and a carrier protein or polymer fused together may be used as an immunizing antigen. The animal used for immunization is not particularly limited, but any of rabbits, goats, sheep, mice, rats, guinea pigs, chickens and the like can be used. Inoculation of animals with antigens for immunization is performed by mixing thoroughly with Freund's adjuvant or incomplete Freund's adjuvant subcutaneously, intramuscularly or intraperitoneally. Inoculation is carried out every 2 to 5 weeks and continued until the antibody titer of the immunized animal against the inoculated antigen is sufficiently increased. Thereafter, the immunized animal is intravenously injected with the antigen alone, and antiserum is obtained 3 to 5 days later.

The method for purifying the polyclonal antibody from the obtained antiserum may be a normal immunoglobulin purification method, for example, ammonium sulfate fractionation method, polyethylene fractionation method, ethanol fractionation method, anion exchange chromatography, protein A or It can be carried out by affinity chromatography to which protein G is bound.

The purification operation for obtaining an active HGFA-specific polyclonal antibody may be any method that can fractionate or purify a polyclonal antibody that recognizes active HGFA and does not substantially recognize inactive HGFA, Examples thereof include ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, reverse phase chromatography, hydroxyapatite chromatography, affinity chromatography, gel electromigration method, and immunoelectromigration method. One specific method is affinity column chromatography using a resin on which active HGFA or inactive HGFA is immobilized. For example, affinity chromatography is performed using the polyclonal antibody obtained by the above-described method as a material and a resin on which inactive HGFA is immobilized. By this method, the polyclonal antibody present in the non-adsorbed fraction that does not bind to inactive HGFA is recovered. Next, using this non-adsorbed fraction as a material, affinity chromatography using a resin on which active HGFA is immobilized is carried out to recover the polyclonal antibody present in the adsorbed fraction bound to active HGFA.

By these methods, an active HGFA specific polyclonal antibody can be obtained. As the affinity chromatography method, a peptide having an amino acid sequence that is considered to be an active HGFA-specific antigenic site can be used. For example, affinity chromatography using a resin in which at least one of Sequence 1 to Sequence 25 described above is immobilized can be used. For example, a resin on which a peptide comprising sequence 1 to sequence 24 is immobilized can be used as an immobilization carrier for affinity chromatography for purifying an active HGFA specific polyclonal antibody. On the other hand, a resin on which a peptide such as sequence 25 is immobilized can be used as an immobilization carrier for affinity chromatography that absorbs or removes an antibody other than an active HGFA-specific antibody.

<4> Method for specifically measuring active HGFA The method for specifically measuring active HGFA means a method comprising an operation of reacting an active HGFA-specific antibody with active HGFA. Therefore, this method is characterized in that active HGFA is measured and inactive HGFA is not substantially measured. This method can be used in various diagnostic methods, measurement methods, and assay methods for qualitatively or quantitatively determining active HGFA in a biological sample using the active HGFA-specific antibody of the present invention. The method is not particularly limited as long as it is for the purpose of detecting active HGFA. For example, a tissue staining method or immunoprecipitation method that specifically detects active HGFA, a competitive binding assay method that specifically measures active HGFA, or a direct or indirect sandwich assay method, a two-antibody sandwich assay method Etc. Examples of the detection method include enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, immunoblotting method, immunochromatography method, latex agglutination method and the like.

Examples of immunoblotting applications include those in which an active HGFA-specific antibody is added to or immobilized on a microarray or chip. It is also possible to fluorescently label an active HGFA specific antibody and detect an interaction with active HGFA using a fluorescence deflection elimination method or a fluorescence correlation dispersion method. It is also possible to measure the interaction between an active HGFA-specific antibody and active HGFA using a surface plasmon resonance apparatus. For example, after binding this antibody on a sensor chip, a biological sample containing active HGFA is flowed to a surface plasmon resonance apparatus to which this chip is attached, and the time change of the response signal is traced, whereby the activity in the biological component is monitored. It is possible to quantify type HGFA.

An antibody used in a method for specifically measuring active HGFA, for example, an active HGFA-specific antibody, may be used as it is, or Fab obtained by papain treatment or F (ab obtained by pepsin treatment, which is a standard method. ') An antibody having the form ₂ or F (ab') may be used. Antibody fragments that have the property of recognizing active HGFA and not substantially recognizing inactive HGFA are also included in the present invention. For example, a fragment containing a complementarity determining region (CDR) or a hypervariable region in both the heavy chain and light chain variable domains of an active HGFA specific antibody is included.

The two-antibody sandwich assay for quantifying active HGFA in biological components is, for example, a method for specifically measuring active HGFA, which includes (1) one or more active HGFA-specific antibodies A step of reacting a reagent consisting of active HGFA in a specimen to generate an immune reaction product, (2) separating the immune reaction product, and then reacting with a labeled antibody that recognizes HGFA in the immune reaction product, And (3) measuring the labeled antibody bound to the immunoreactant, or

A method for specifically measuring active HGFA, comprising: (1) a reagent comprising active HGFA and one or more of active HGFA specific antibodies as a primary antibody, and active HGFA in a sample; A step of reacting to generate an immune reaction product, (2) a step of separating the immune reaction product and then reacting with a secondary antibody that recognizes HGFA in the immune reaction product to produce an immune reaction product, (3) And a method comprising a step of reacting a labeled antibody that recognizes a secondary antibody in the immune reaction after separating the immune reaction, and (4) a step of measuring the labeled antibody bound to the immune reaction. .

Specifically, an active HGFA-specific polyclonal antibody or an active HGFA-specific monoclonal antibody is immobilized on a solid phase such as a microtiter well or micromagnetic beads as a primary antibody by a conventional method. Next, this excessive protein binding site on the solid surface is blocked with bovine serum albumin, skim milk, gelatin or the like. Thereafter, a biological component containing active HGFA is added to form an immune reaction product on the solid phase, followed by washing. Next, a labeled polyclonal antibody or labeled monoclonal antibody that recognizes HGFA is added and reacted as a secondary antibody. In this case, when a monoclonal antibody is used as the primary antibody, an active HGFA-specific monoclonal antibody having an epitope different from that of the primary antibody may be used as the secondary antibody. Further, the amount of active HGFA in the biological component can be measured by measuring the amount of labeled antibody after washing.

The label of the polyclonal antibody or monoclonal antibody used here may be an enzyme such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, glucose oxidase, or a fluorescent substance such as a fluorescein derivative or a rhodamine derivative. Further, it may be a chemiluminescent substance such as acridinium ester or a radioisotope such as ¹²⁵ I, ³ H, ¹⁴ C, or ³² P. That is, the present invention includes quantifying the amount of active HGFA in a biological component using methods for measuring color development, fluorescence, chemiluminescence, electrochemiluminescence, and radioactivity. In addition, secondary antibodies are labeled with biotinylated, and complexed with avidin such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, glucose oxidase, fluorescein derivatives, rhodamine derivatives, acridinium esters, etc. The detection of radioisotopes such as chemiluminescent materials, ¹²⁵ I, ³ H, ¹⁴ C, ³² P is also included in the present invention.

<5> Kit that specifically measures or stains active HGFA Active HGFA-specific measurement kit or active HGFA-specific staining kit is a diagnosis of a disease characterized by measuring or detecting active HGFA It is a kit. The specific mentioned here means that inactive HGFA does not affect the measured or detected value of active HGFA when measuring or detecting active HGFA. By measuring active HGFA, patients in disease state, for example, glomerulonephritis, nephritis, hepatitis, pancreatitis, pneumonia, enteritis, gastritis and other organ disorder patients, cancer patients, angina pectoris, myocardial infarction It has been found for the first time by the present invention that it is possible to diagnose and predict patients with thrombosis including cerebral infarction. Therefore, various diseases as described above can be detected by measuring or detecting active HGFA. In the present invention, as long as it is an active HGFA-specific measurement kit for the purpose of diagnosing a disease, it is not limited to materials and methods constituting the kit.

Specifically, for example, a kit for diagnosing a disease by measuring or detecting active HGFA using an electrokinetic method, HPLC method, various column chromatography methods, various arrays, chips, surface plasmon resonance devices, etc. Is mentioned. More specifically, a kit for measuring or detecting active HGFA by an immunological method using an antibody can be mentioned. As the antibody, at least one antibody that recognizes the active HGFA and does not substantially recognize the inactive HGFA is used.

For example, when the kit of the present invention is based on a two-antibody sandwich assay, the kit specifically measures active HGFA, and (1) one of active HGFA and active HGFA-specific antibody or A step of reacting a reagent containing plural substances with active HGFA in a specimen to generate an immunoreactant; (2) after separating the immunoreactant, reacting with a labeled antibody that recognizes HGFA in the immunoreactant A kit comprising the steps of: and (3) measuring the labeled antibody bound to the immunoreactant, or

A kit for specifically measuring active HGFA, wherein (1) a reagent comprising one or more active HGFA-specific antibodies as a primary antibody is reacted with active HGFA in a sample to immunize A step of generating a reaction product, (2) a step of separating an immune reaction product and then reacting with a secondary antibody that recognizes HGFA in the immune reaction product to generate an immune reaction product, and (3) an immune reaction product Examples of the kit include a step of reacting a labeled antibody that recognizes a secondary antibody in the immune reaction after separation, and a step of (4) measuring the labeled antibody bound to the immune reaction.

This kit includes at least an active HGFA-specific monoclonal or active HGFA-specific polyclonal antibody, and may further include components necessary for the operation of detecting or measuring active HGFA. Examples of the constituent elements include active HGFA, inactive HGFA, enzymes, substrates and the like as standard proteins. The monoclonal antibody or the polyclonal antibody included in the kit may be in a state in which a labeling substance such as an enzyme is bound thereto, or may include a labeled antibody that recognizes these antibodies. Various appropriate buffers, antigen diluents, reaction diluents, substrate solutions, reaction stop solutions, and the like may also be included. The kit may include a container that is labeled and encloses the materials required for active HGFA detection and quantification. Examples of suitable containers include containers made of various plastics such as glass, polypropylene, polystyrene, polycarbonate, nylon, and Teflon (registered trademark). The kit preferably includes an instruction describing a method for detecting or measuring active HGFA together with materials and containers necessary for detecting or measuring active HGFA as described above.

<6> Active HGFA-specific antibodies relating to human diseases and methods of use thereof Activity in biological components collected from patients in disease states by using methods and kits using active HGFA-specific antibodies according to the present invention Type HGFA can be detected and quantified. The biomaterial for detecting active HGFA is not particularly limited, and any biomaterial such as tissue, serum, plasma, urine, serum, spinal fluid, tissue extract, etc. can be applied by performing appropriate pretreatment. . By detecting or quantifying active HGFA present in the biomaterial of a patient in a disease state, it is possible to determine the diagnosis, prediction and course of the disease. Examples of diseases include glomerulonephritis, nephritis, hepatitis, pancreatitis, pneumonia, enteritis, gastritis and other organ inflammation, cancer, myocardial infarction, angina pectoris, thrombosis including cerebral infarction, etc. It is done.

Examples of nephritis include mesangial proliferative nephropathy, IgA nephropathy, membranoproliferative nephritis, membranous nephropathy, focal glomerulosclerosis, acute renal failure, acute glomerulonephritis after streptococcal infection, chronic / acute Examples include interstitial nephritis and nephrotic syndrome. Examples of angina pectoris and myocardial infarction are stable angina pectoris, unstable angina pectoris, acute myocardial infarction, old myocardial infarction, stable angina pectoris, patients undergoing coronary intervention, and transesophageal It is also possible to know the disease status and prognosis of patients undergoing echocardiography, patients undergoing lower limb arterial bypass surgery, patients undergoing aortic balloon pumping, and patients with acute aortic dissection.

Active HGFA-specific antibodies are expected to be used as therapeutic agents for diseases caused by active HGFA because they can be expected to specifically suppress the activity of active HGFA. For example, active HGFA has the property of acting on and inactivating inactive HGF. Therefore, an antibody that suppresses the activity of active HGFA suppresses an increase in the amount of active HGF that appears in the living body, and is a disease that is assumed to be caused by active HGF (WO96 / 38557: Genentic Incorporated) , Molecular Medicine of HGF (published in 1998) Medical Review, Inc.), for example, cancers such as gastric cancer, lung cancer, colon cancer, spleen cancer, liver cancer and their metastasis, therapeutic drugs for various nephritis including glomerulonephritis, prevention It can be used as a medicine. The presence of a mouse monoclonal antibody that suppresses the activity of active HGFA (monoclonal antibody P1-4) is described in Miyazawa et al. (J. Biol. Chem., 271: 3615-3618 (1996)). It has been revealed by the present inventors that the antibody 4 reacts with both active HGFA and inactive HGFA. Since a large amount of inactive HGFA exists in the living body, it is considered that an extremely large amount of P1-4 antibody is necessary for suppressing the activity of active HGFA. On the other hand, since the active specific antibody of the present invention is presumed to be an antibody that suppresses the activity of active HGFA, it seems to exhibit extremely excellent effects as a therapeutic agent for the above-mentioned diseases in a small amount. The antibody used for this purpose is preferably humanized using genetic engineering techniques. Humanization of antibodies can be performed by methods well known to those skilled in the art described in, for example, JP-A-11-506327.

<7> A blood collection method for measuring active HGFA and a biological component for detecting or measuring blood collection active HGFA are not particularly limited, but are usually blood or a fraction or processed product thereof, preferably Biological components that can be collected from blood vessels such as blood, serum, citrate plasma, heparin plasma, and EDTA plasma, or fractions or processed products thereof, and urine that can be easily collected are desirable. In particular, at the time of collecting and storing the biological component, an operation for preventing the inactive HGFA mixed in the biological component from being artificially converted into the active HGFA is necessary. For example, it is desirable that collected serum or plasma, urine, etc. be immediately placed at a low temperature such as ice-cold after collection.

It is also desirable to use a blood collection tube in order to quickly collect blood, serum, citrate plasma, heparin plasma, and EDTA plasma from the blood vessel. In particular, blood for detecting or measuring active HGFA is preferably in the state of plasma, particularly citrated plasma. Further, a method of adding various protease inhibitors to a biological component for detecting or measuring active HGFA may be used. For example, in order to prevent the inactive HGFA contained in the biological component from being artificially converted to the active HGFA at the time of collection and storage of the biological component, for example, a biological sample obtained by collecting argatroban which is a selective thrombin inhibitor Good results are obtained when added to. In particular, blood, serum, citrate plasma, heparin plasma, and EDTA plasma collection tubes to which argatroban has been added in advance are preferably used.

<8> Screening method of protease inhibitor against active HGFA Use of an antibody that recognizes active HGFA, which does not substantially recognize inactive HGFA and does not recognize a complex of active HGFA and protease inhibitor By doing so, it is possible to screen for protease inhibitors that act on active HGFA.

An antibody having the above properties can be obtained by selecting an antibody that does not recognize a complex of active HGFA and a protease inhibitor from active HGFA-specific antibodies.

Specifically, the screening method includes (1) a step of mixing and reacting a compound that is a candidate for active HGFA and its protease inhibitor, and (2) a mixture of a compound that is a candidate for active HGFA and a protease inhibitor. A step of adding a monoclonal antibody which recognizes active HGFA, which does not substantially recognize inactive HGFA and does not recognize a complex of active HGFA and protease inhibitor, (3) immune reaction After separating the product, a step of measuring a decrease in the amount of an immunoreactive product composed of a complex composed of a compound that is a candidate for active HGFA and a protease inhibitor and the antibody is mentioned. Microarrays that are application examples of enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, immunoblotting, immunochromatography, latex agglutination, and immunoblotting A method using a method, a fluorescence deflection elimination method, a fluorescence correlation dispersion method, or a surface plasmon resonance apparatus can be used. For example, an antibody that recognizes active HGFA, which does not substantially recognize inactive HGFA and does not recognize a complex of active HGFA and a protease inhibitor is bound on a sensor chip, and then this chip is used. It is possible to screen a protease inhibitor by flowing a biological sample containing active HGFA through an attached surface plasmon resonance apparatus and tracking the time change of the response signal.

The antibody used here may be used as it is, or an antibody having the form of F (ab ′) ₂ or F (ab ′) obtained by Fab or pepsin treatment obtained by papain treatment, which is a standard method. It may be used. In addition, antibody fragments having the properties of the antibodies used here can also be used.

[Example]
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these.

Preparation of inactive HGFA and active HGFA Inactive HGFA was prepared using HGFA cDNA encoding the HGFA precursor described in JP-A-6-153946, and the method described in JP-A-6-153966 The recombinant inactive HGFA was prepared according to the above. That is, HGFA cDNA encoding 655 amino acids, which is the full length of the inactive HGFA precursor, was inserted downstream of the CMV promoter of pcDNA3.1 (Invitrogen), which is an animal cell expression vector, by a conventional method.

The obtained expression vector was introduced into animal cell lines and CHO cells derived from Chinese hamster ovary cells using BIOSEPRA TRANSFECTAM according to the attached method. Thereafter, CHO cells expressing HGFA cDNA were selected using the property of the neomycin resistance gene present on the introduced expression vector. That is, cells capable of growing at 37 ° C. under 5% CO _{2 in} ERDF (Kyokuto Pharmaceutical Co., Ltd.) medium containing 400 μg / ml neomycin and 5% fetal calf serum were selected. Further, CHO cells expressing the selected HGFA cDNA were cultured for about 10 days in an ERDF (manufactured by Kyokuto Pharmaceutical Co., Ltd.) medium containing 100 μM nafamostat mesylate, 400 μg / ml neomycin and 5% fetal calf serum. A culture supernatant was obtained.

After filtering the obtained culture supernatant, a protease inhibitor containing 10 mM EDTA, 10 mM benzamidine, 100 μM nafamostat mesylate, soybean trypsin inhibitor, 1000 KIU / ml aprotinin was added, and 0.5 M acetate buffer (pH 4.5) Was added to adjust the pH of the culture supernatant to about 5.8. The culture supernatant was added to a sulfated cellulose fine column (Seikagaku Corporation) equilibrated with 50 mM acetate buffer (pH 5.5) containing 100 mM NaCl, and 50 mM acetate buffer (pH 5) containing 100 mM NaCl. Washing was performed in step 5). Then, elution was performed with 50 mM sodium phosphate buffer (pH 7.5) containing 500 mM NaCl, 0.05% CHAPS (3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid) to obtain an inactive HGFA fraction. It was.

The obtained fraction was dialyzed against 50 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl, and then A6 monoclonal antibody affinity column (Shimomura et al., J. Biol. Chem., 268: 22927-22932 ( 1993)) and elution was carried out with 50 mM glycine-HCl (pH 3.0) buffer. The obtained inactive HGFA fraction was replaced with 10 mM sodium phosphate buffer (pH 7.3) containing 100 mM NaCl and 0.05% CHAPS. A portion of this was added with protease inhibitor argatroban to a final concentration of 40 μM and stored at −40 ° C. as inactive HGFA. On the other hand, active 36 kDa HGFA was obtained by adding 1/100 amount of plasma kallikrein and thrombin to the inactive HGFA to which argatroban was not added and sufficiently reacting at 37 ° C.

Further, to inactive HGFA to which no argatroban was added, 1/100 of thrombin and 10 μg / ml dextran sulfate were added at a weight ratio to HGFA, and reacted at 37 ° C. for 20 minutes, whereby active 98 kDa was reacted. Got HGFA. Each active HGFA fraction was again purified with an A6 monoclonal antibody affinity column and then equilibrated with 10 mM sodium phosphate buffer (pH 7.3) containing 100 mM NaCl and 0.05% CHAPS (Showa GS) After the HPLC operation using Denki Kogyo Co., Ltd., it was stored as active 36 kDa HGFA and active 98 kDa HGFA at −40 ° C.

Preparation of ELISA plate for screening of active HGFA-specific monoclonal antibody Physiological hydrogen phosphate to a final concentration of 1 μg / ml using active 36 kDa HGFA or active 98 KDa HGF prepared in Example 1 as antigen for hybridoma screening After dilution in a buffer solution (PBS (−)), 100 μl was added to each well of a 96-well plate. Thereafter, it was stored at 4 ° C. for 24 hours, and each antigen was adsorbed to a 96-well plate. Remove the solution from the antigen-attached plate and add 250 μl of PBS (-) containing 5% bovine serum albumin (hereinafter abbreviated as “BSA”) to the wells at 4 ° C. overnight (about 12 hours) or 37 ° C. Was kept for 2 hours or longer at 4 ° C. as an ELISA plate for active HGFA screening.

On the other hand, the inactive HGFA prepared in Example 1 was diluted with PBS (−) to a final concentration of 1 μg / ml as an antigen for hybridoma screening. Thereafter, 100 μl was added to each well of a 96-well plate and stored at 4 ° C. for 24 hours to adsorb each antigen to the 96-well plate. Remove the solution from this antigen-attached plate, add PBS (-) containing 5% BSA to the wells in a volume of 250 μl, and block at 4 ° C overnight (about 12 hours) or at 37 ° C for 2 hours or longer. And stored at 4 ° C. as an ELISA plate for inactive HGFA screening. These ELISA plates were used with the blocking solution in the plate removed immediately before use.

Production of active HGFA-specific monoclonal antibody A solution containing 100 µg of active 36 kDa HGFA or 100 µg of 98 KDa HGFA prepared in Example 1 together with the same volume of Freund's complete adjuvant was subcutaneously and intraperitoneally injected into Balb / c mice at 2 week intervals. Administered once. After confirming that the antibody was produced in the mouse serum, a solution containing 100 μg of HGFA was administered into the tail vein. Three days later, the spleen was removed, and spleen cells were fused with myeloma cells P3U1 using polyethylene glycol 1500 according to the “Monoclonal Antibody Experiment Manual” (published by Kodansha Scientific 1987) and injected into a 96-well plate. Thereafter, HAT medium was added and cultured for 14 days.

Thereafter, a hybridoma producing a monoclonal antibody specific for each active HGFA in the medium was selected. That is, the culture supernatant of the selected hybridoma was added to the 36 kDa HGFA or active 98 KDa HGFA screening ELISA plate prepared in Example 2, and the reactivity of the monoclonal antibody present in the culture supernatant was analyzed. The culture supernatant of the hybridoma to be selected was added at 100 μl / well to each active screening ELISA plate, followed by reaction at 4 ° C. for 2 hours or more.

Thereafter, the plate was thoroughly washed with a PBS (−) solution containing 0.05% Tween 20 (hereinafter abbreviated as “PBST solution”), and an HRP (horseradish peroxidase) -labeled sheep anti-mouse IgG / Fc polyclonal antibody (DAKO) 100 μl of PBS (−) containing 1 μg / ml and 1% BSA was added to each well and allowed to react at room temperature for 1 hour. After thoroughly washing with PBST solution, citrate-phosphate buffer (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015-0.03% hydrogen peroxide solution. ) Was added and reacted at room temperature to develop color. Thereafter, 1N H ₂ SO ₄ solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm.

Next, the culture supernatant of the monoclonal antibody-producing hybridoma having reactivity with the active 36 kDa HGFA or active 98 KDa HGFA obtained here was used for the inactive HGFA screening ELISA plate prepared in Example 2. Similarly, screening was performed to select hybridomas that did not show any reactivity to inactive HGFA. By this screening, a monoclonal antibody recognizing active 36 kDa HGFA, which does not substantially recognize inactive HGFA, or a monoclonal antibody recognizing active 98 KDa HGFA, which recognizes inactive HGFA Hybridomas (AHGA-A, AHGA-B, AHGA-C) that produce monoclonal antibodies that do not. Each of the obtained hybridomas was subjected to cloning operations four times by the limiting dilution method, and the culture product was collected and purified for monoclonal antibodies by affinity chromatography (Amersham Pharmacia Biotech) to which protein A was bound. The hybridoma clone AHGA-A was launched on October 19, 2001 from the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba City, Ibaraki, 305-8566, Japan) It is deposited under the deposit number of FERM BP-7779.

Analysis of reaction specificity of active HGFA-specific monoclonal antibody Among active HGFA-specific monoclonal antibodies prepared and purified in Example 3, monoclonal antibodies derived from hybridoma clones AHGA-A, AHGA-B and AHGA-C The reactivity was analyzed. Therefore, the hybridoma clones AHGA-A, AHGA-B and AHGA-C were derived from the active 36 kDa HGFA or active 98 KDa HGFA screening ELISA plate or inactive HGFA screening ELISA plate prepared in Example 2. About 100 μl of PBS (−) containing about 1 μg / ml of monoclonal antibody and 1% BSA was added to each well and reacted at 4 ° C. for 2 hours or more.
Thereafter, the plate was thoroughly washed with a PBS (−) solution containing 0.05% Tween 20 (hereinafter abbreviated as “PBST solution”), and 1 μg / ml of HRP-labeled sheep anti-mouse IgG polyclonal antibody (DAKO) and 1%.
100 μl of PBS (−) containing BSA was added to each well, and further reacted at room temperature for 1 hour. After thoroughly washing with PBST solution, citrate-phosphate buffer (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015-0.03% hydrogen peroxide solution. ) Was added and reacted at room temperature to develop color.

Thereafter, the reaction was stopped by adding a 1N H ₂ SO ₄ solution, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. The measurement results are shown in FIG. Monoclonal antibodies derived from hybridoma clones AHGA-A and AHGA-B reacted with active 36 kDa HGFA and did not react with inactive HGFA. Hybridoma clone AHGA-C reacted with active 98 kDa HGFA and did not react with inactive HGFA (FIG. 1). On the other hand, regarding existing monoclonal antibodies against HGFA (7E10, P1-4, A-1, A-6, A-23, A-32, A-51, A-75), active 36 kDa HGFA and inactive HGFA As a result of analyzing the reactivity with respect to the active HGF, the active HGF showed the same reactivity with both the active and inactive HGFA, like the monoclonal antibody obtained in the present invention.
It did not have specific reactivity with A.

Measurement of dissociation constant of active HGFA-specific monoclonal antibody Among the active HGFA-specific monoclonal antibodies prepared and purified in Example 3, the dissociation constant of the monoclonal antibody derived from hybridoma clone AHGA-A was measured. Activated HGFA was immobilized and added to an activated HGFA immobilized plate that had been blocked with BSA at different antibody concentrations and allowed to react well until equilibrium was reached (over 2 hours). Thereafter, the plate was thoroughly washed with a PBS (−) solution containing 0.05% Tween 20 (hereinafter abbreviated as “PBST solution”), and an HRP (horseradish peroxidase) -labeled goat anti-mouse IgG / Fc polyclonal antibody (ICN) 100 μl of PBS (−) containing 1 μg / ml and 1% BSA was added to each well and allowed to react at room temperature for 1 hour. After thoroughly washing with PBST solution, citrate-phosphate buffer (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015-0.03% hydrogen peroxide solution. ) Was added and reacted at room temperature to develop color. Thereafter, the reaction was stopped by adding a 1N H ₂ SO ₄ solution, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. Scattered plots were performed from the measurement results to determine dissociation constants. The result was 4.05 × 10 ⁻¹⁰ M. This result shows that the affinity of the antibody of the present invention is high.

Production of monoclonal antibody that specifically recognizes active HGFA and does not recognize complex of active HGFA and protease inhibitor. Recognizes specifically active HGFA selected in Example 3 and recognizes inactive HGFA The following screening was further carried out on hybridomas producing non-reactive monoclonal antibodies. After removing the blocking solution of the active 36 kDa HGFA or active 98 KDa HGFA screening ELISA plate prepared in Example 2, 20 μl of PBS (-) containing 200 μM of the protease inhibitor nafamostat mesylate was added to each well. The reaction was carried out at room temperature for 1 hour to form a complex of active HGFA and nafamostat mesylate. Thereafter, the culture supernatant of the selected hybridoma was added at 100 μl / well and reacted at 4 ° C. for 2 hours.

Thereafter, the plate was thoroughly washed with a PBS (−) solution containing 0.05% Tween 20 (hereinafter abbreviated as PBST solution), and PBS containing 1 μg / ml of HRP-labeled sheep anti-mouse IgG polyclonal antibody (DAKO) and 1% BSA was used. 100 μl of (−) was added to each well and allowed to react at room temperature for 1 hour. After thoroughly washing with PBST solution, citrate-phosphate buffer (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015-0.03% hydrogen peroxide solution. ) Was added and reacted at room temperature to develop color.

Thereafter, 1N H ₂ SO ₄ solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. By this screening, a monoclonal antibody that did not recognize the complex of active 36 kDa HGFA and nafamostat mesylate was selected. The hybridoma clone producing the selected monoclonal antibody was designated as AHGA-D. Each of the obtained hybridomas was cloned four times by the limiting dilution method, and then the culture product was recovered and purified for monoclonal antibodies by affinity chromatography to which Protein A was bound.

Analysis of reaction specificity of monoclonal antibody that specifically recognizes active HGFA and does not recognize complex of active HGFA and protease inhibitor Reactivity of monoclonal antibody derived from hybridoma clone AHGA-D selected in Example 6 Analyzed. 20 μl of PBS (−) containing nafamostat mesylate at concentrations of 0 μM, 0.820 μM, 7.40 μM, 22.20 μM, and 2000 μM was added to the wells of the active 36 kDa HGFA screening ELISA plate prepared in Example 2. The mixture was reacted at room temperature for 1 hour to form a complex of active HGFA and nafamostat mesylate. This was followed by PBS (-) containing about 1 μg / ml of monoclonal antibody derived from hybridoma clone AHGA-D, 1% BSA and various concentrations of nafamostat mesylate (0 μM, 0.820 μM, 7.40 μM, 22.20 μM, 2000 μM). 100 μl was added to each well and reacted at 4 ° C. for 2 hours or longer.

Thereafter, the plate was thoroughly washed with a PBS (−) solution containing 0.05% Tween 20 (hereinafter abbreviated as “PBST solution”), and HRP-labeled sheep anti-mouse IgG polyclonal antibody (DAKO) 1 μg / ml and 1% BSA PBS (-) containing 100 μl was added to each well, and further reacted at room temperature for 1 hour. After thoroughly washing with PBST solution, citrate-phosphate buffer (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015-0.03% hydrogen peroxide solution. ) Was added and reacted at room temperature to develop color.

Thereafter, 1N H ₂ SO ₄ solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. The measurement results are shown in FIG. The monoclonal antibody derived from the hybridoma clone AHGA-D was significantly less reactive with HGFA bound to the protease inhibitor nafamostat mesylate.

Preparation of polyclonal antibody against HGFA and its labeled form Polyclonal antibodies against HGFA were prepared by mixing 100 μg each of active 36 kDa HGFA and active 98 kDa HGFA and inactive HGFA prepared in Example 1 into the rabbit subcutaneously. Seven doses were given at weekly intervals. After confirming that antibodies were produced in the serum, an additional 10 μg of each antigen was administered into the pulse and antiserum was obtained 5 days later. Further, after an ammonium sulfate precipitation operation, an anti-HGFA polyclonal antibody was obtained by a purification operation using a protein A column. Thereafter, the obtained HGFA polyclonal antibody was labeled with biotin to prepare a biotin-labeled anti-HGFA polyclonal antibody.

Construction of active HGFA-specific measurement system The monoclonal antibody derived from hybridoma clone AHGA-A used in Example 4 was used as the primary antibody. This monoclonal antibody is dissolved in 0.05 M carbonate-bicarbonate buffer (pH 9.6) to a concentration of 30 μg / ml, added to a 96-well plate at 100 μl / well, and then overnight at 4 ° C. for 12 hours. More than about) After removing the primary antibody solution from the primary antibody-attached plate, PBS (-) containing 1% BSA was added in an amount of 250 to 300 μl / well, and the whole was added at 4 ° C. overnight (about 12 hours) or at 37 ° C. for 2 hours. That's it. The blocking solution was removed from this plate and various concentrations dissolved in 0.15M NaCl, 0.1% CHAPS, 0.05% Tween 20, 0.05% Az (sodium azide), 0.1% BSA, 20 mM sodium phosphate buffer pH 7.5 ( 100 μl each of active 36 kDa HGFA or inactive 98 kDa HGFA (0, 0.69, 2.1, 6.2, 18.5, 55.6, 167, 500 ng / ml) was added and reacted at 4 ° C. for about 2 hours or more.

Thereafter, the plate was sufficiently washed using a washing solution having a composition of 500 mM NaCl, 0.05% Tween 20, 20 mM Tris-HCl pH 7.5, and then 1 μg / ml of the biotin-labeled anti-HGFA polyclonal antibody prepared in Example 8 PBS (-) containing 1% BSA and 100 μl each were added to the wells and allowed to react in a 37 ° C. incubator for 2 hours.

Next, thorough washing was performed using the above-mentioned washing solution, and PBS (-) containing 1% BSA diluted with HRP-labeled streptavidin (Amersham Pharmacia Biotech, code RPN1231) 2000 times was added to each well, and 100 μl was further added. The reaction was allowed to proceed for 1 hour at room temperature. After thoroughly washing with washing solution, 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0
A citrate-phosphate buffer solution (pH 5.0) containing a .015 to 0.03% hydrogen peroxide solution was added in an amount of 100 μl / well and reacted at room temperature to develop color.

Thereafter, 100 μl / well of 1N H ₂ SO ₄ solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. The result is shown in FIG. FIG. 3A shows the concentration of HGFA in the range of 0 to 500 ng / ml. FIG. 3B shows an enlarged range of 0 to 55.6 ng / ml among the HGFA concentrations shown in FIG. 3A.

Measurement of the amount of active HGFA in the blood of healthy persons Using the active HGFA-specific measurement system prepared in Example 9, the serum of 144 healthy persons was measured. Healthy human serum was frozen and stored after collection, and was thawed and used immediately before this experiment. First, with respect to the 96-well plate subjected to the blocking operation after attaching the primary antibody prepared in Example 9, 0.15 M NaCl, 0.1% CHAPS, 0.05% Tween 20, 0.05% Az, 0.1% BSA, 20 mM sodium phosphate buffer, pH 7 in advance. .5 was added to each well by 50 μl, and 50 μl each of healthy human serum or standard 36 kDa active HGFA (0, 0.69, 2.1, 6.2, 18.5, 55.6, 167, 500 ng / ml) was added. After this, the reaction is performed at 4 ° C. for about 2 hours or more, and 500 mM NaCl, 0.05% Tween 20, 20 mM.
After thorough washing using a washing solution composed of Tris-HCl pH 7.5, 100 μl of PBS (−) containing 1 μg / ml of biotin-labeled anti-HGFA polyclonal antibody prepared in Example 8 and 1% BSA was used. It added to the well one by one, and it was made to react for 2 hours in a 37 degreeC incubator. Next, thorough washing was performed using the above-mentioned washing solution, and PBS (-) containing 1% BSA diluted with HRP-labeled streptavidin (Amersham Pharmacia Biotech, code RPN1231) 2000 times was added to each well, and 100 μl was further added. The reaction was allowed to proceed for 1 hour at room temperature.

After thoroughly washing with the washing solution, a citrate-phosphate buffer (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015-0.03% hydrogen peroxide solution. ) Was added at a rate of 100 μl / well and reacted at room temperature to develop color. Thereafter, 100 μl / well of 1N H ₂ SO ₄ solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. Thereafter, a calibration curve was prepared based on the standard 36 kDa active HGFA concentration and the amount of color development, and the concentration in the serum of a healthy person was calculated. The result is shown in FIG. In healthy human serum, the range was from 0 ng / ml to 58.5 ng / ml, and the average was 19 ng / ml.

Measurement of the amount of active HGFA in the blood of various human disease patients The serum of various human disease patients was measured according to the method of Example 10 using the active HGFA-specific measurement system prepared in Example 9. The number of patients was 5 for each disease. Each serum was stored frozen after collection, and was thawed and used immediately before this experiment. The results are shown in Table 1. In the blood of patients with glomerulonephritis, pancreatitis, cancer, myocardial infarction, angina pectoris, cerebral infarction, the concentration of active HGFA is higher than that of healthy individuals (9 samples average value 19 ng / ml) I understood.

Blood collection method for active HGFA measurement and examination of stability Collect serum, citrate plasma, heparin plasma or EDTA plasma, or serum containing 40 μM final concentration of argatroban, citrate plasma, heparin plasma or EDTA plasma from healthy individuals, Each was prepared and stored at 4 ° C. or 37 ° C. for 12 hours, and then the amount of active HGFA was measured by the method described in Example 10. The result is shown in FIG. It was found that the increase in the amount of active HGFA due to storage of serum or plasma was less in plasma than in serum, and this increase was well suppressed by addition of argatroban.

The figure which shows the reactivity of the monoclonal antibody with respect to active type HGFA and inactive type HGFA. The figure which shows the reactivity of the composite_body | complex of active type HGFA and a protease inhibitor, and a monoclonal antibody. It is the figure which showed the reactivity of active type HGFA and inactive type HGFA, and a monoclonal antibody in an active type HGFA measuring system. FIG. 3A shows the concentration of HGFA in the range of 0 to 500 ng / ml, and FIG. 3B shows the enlarged range of 0-55.6 ng / ml of the concentration of HGFA shown in FIG. 3A. It is the figure which showed the histogram of the amount of active HGFA in a healthy subject serum. It is the figure which showed the effect of the storage stability of active type HGFA value in a healthy subject's serum, citrate plasma, heparin plasma, and EDTA plasma, and the addition of argatroban.

Claims (5)

A method for producing a monoclonal antibody, comprising the following steps.
(1) Mice are treated with activated HGFA activated as a result of limited degradation between 407th arginine and 408th isoleucine, counted from the translation initiation amino acid of inactive hepatocyte growth factor activator (HGFA). Immunize.
(2) A hybridoma is prepared by fusing antibody-producing cells obtained from mouse spleen and myeloma cells.
(3) Inactive HGFA is prepared by adding argatroban.
(4) Using the active HGFA and the inactive HGFA prepared in (3) above among the hybridomas obtained in (2) above, the dissociation constant for the active HGFA is 1 × 10 ⁻⁸ A hybridoma that produces an antibody that is M or less and has a dissociation constant for inactive HGFA of 1 × 10 ⁻⁵ M or more is selected.
(5) The monoclonal antibody is prepared from the culture supernatant of the obtained hybridoma. The dissociation constant for active HGFA activated by undergoing limited degradation between 407th arginine and 408th isoleucine, counted from the translation initiation amino acid of inactive HGFA, produced by the method according to claim 1 Antibody having a dissociation constant of not more than 1 × 10 ⁻⁸ M and a dissociation constant for inactive HGFA of 1 × 10 ⁻⁵ M or more. The monoclonal antibody according to claim 2, which is produced by a hybridoma having the accession number FERM BP-7779. A hybridoma cell line producing the monoclonal antibody according to claim 2 or 3. A method for measuring active HGFA, wherein the active HGFA is specifically measured by an immunological method using one or more of the monoclonal antibodies according to claim 2 or 3.

Images