WO2010090079A1 - Monoclonal antibody reacting specifically with stromelysin 1 - Google Patents
Monoclonal antibody reacting specifically with stromelysin 1 Download PDFInfo
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- WO2010090079A1 WO2010090079A1 PCT/JP2010/050710 JP2010050710W WO2010090079A1 WO 2010090079 A1 WO2010090079 A1 WO 2010090079A1 JP 2010050710 W JP2010050710 W JP 2010050710W WO 2010090079 A1 WO2010090079 A1 WO 2010090079A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96486—Metalloendopeptidases (3.4.24)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Definitions
- the present invention relates to a monoclonal antibody that specifically reacts with stromalysin 1, and an immunostaining method and an immunological measurement method using the same.
- MMPs matrix metalloproteases
- stromalysin 1, 2 and 3 belong to a subgroup of the stromalysin group.
- Stromlysin 1 is also called proteoglycanase or MMP-3, and is a substance produced by fibroblasts or tumor cells stimulated with various cytokines or various growth factors.
- Patent Document 1 In vivo, in addition to being produced in the joint area of patients with rheumatoid arthritis disease, it is present in blood or joint fluid (Patent Document 1). Therefore, quantification of stromycin 1 in patients has been used for diagnosis of rheumatoid arthritis.
- stromalisin 2 (MMP-10) has a substrate specificity similar to that of stromelysin 1 by cloning a gene by Muller et al. (Non-patent Document 1).
- Stromlysin 3 (MMP-11) has been cloned by Basset et al. And is thought to be related to cancer progression (Non-patent Document 2).
- Stromulysin 1, 2 and 3 have very similar amino acid sequences and substrate specificities, and when antibodies against strome lysin 1 are prepared, there is a high possibility of cross-reacting with strome lysin 2 and 3.
- Patent Document 1 a plurality of monoclonal antibodies against stromal lysin 1 are obtained, but these antibodies may have cross-reactivity with strome lysin 2 and 3.
- an object of the present invention is to quantify Stromlysin 1 with higher sensitivity and more accuracy.
- the present invention provides a monoclonal antibody that does not cross-react with strom lysin 2 and strom lysin 3, but specifically reacts with strom lysin 1. Since the monoclonal antibody of the present invention does not cross-react with strom lysin 2 and strom lysin 3, even if strom lysin 2 or strom lysin 3 is contained in the sample, it can specifically react only with strom lysin 1. .
- the above monoclonal antibody preferably has a cross-reactivity with respect to strotom lysin 2 and strotom lysin 3 of 5% or less of the reactivity with respect to strome lysin 1.
- Such monoclonal antibodies are more specific for stromalysin 1.
- the present invention also relates to a monoclonal antibody that specifically reacts with a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and that specifically reacts with stromalysin 1, wherein the amino acid sequence is SEQ ID NO: 1.
- Monoclonal antibodies comprising the sequence from 425 to 436, wherein the peptide is 12-20 amino acid residues in length are provided. It is preferable that the monoclonal antibody does not cross-react with stromalysin 2 and stromalysin 3.
- the amino acid sequence of SEQ ID NO: 1 represents the amino acid sequence of strome lysin 1, and the peptide consisting of the sequence from 425th to 436 of SEQ ID NO: 1 is considered to be involved in cross-reactivity.
- the present invention also provides a monoclonal antibody that specifically reacts with stromalysin 1, which loses its reactivity with stromalisin 1 by degrading stromalisin 1 with lysyl endopeptidase. It is preferable that the monoclonal antibody does not cross-react with stromalysin 2 and stromalysin 3. Monoclonal antibodies that lose reactivity due to degradation of lysyl endopeptidase may react by recognizing the higher-order structure of stromlysin 1 protein.
- the monoclonal antibody of the present invention can be used for immunostaining. According to the immunostaining method using a monoclonal antibody that does not cross-react with stromalysin 2 and stromalicin 3 of the present invention, even if stromalicin 2 or stromalicin 3 is contained in the sample, they are not stained, 1 can be specifically visualized.
- the monoclonal antibody of the present invention can be used for an immunological assay for stromalysin 1 to quantify strome lysin 1. According to the immunoassay using a monoclonal antibody that does not cross-react with stromalysin 2 and stromalysin 3 of the present invention, even if stromalysin 2 and stromalysin 3 are contained in the sample, they are not detected. Stromlysin 1 can be accurately quantified.
- the immunoassay is a competitive immunoassay using one type of monoclonal antibody, an immunoassay typified by a sandwich method using two types of monoclonal antibodies, or an immunoassay method using an aggregation method using two or more types of monoclonal antibodies.
- an immunoassay method using an aggregation method using two or more types of monoclonal antibodies it is preferable that at least one of the monoclonal antibodies used is the monoclonal antibody of the present invention.
- the specificity to stromalysin 1 becomes higher, and strome lysin 1 can be quantified more accurately.
- strome lysin 1 can be quantified with higher sensitivity and more accuracy.
- the monoclonal antibody used in the present invention may be a monoclonal antibody obtained by utilizing the cell fusion technique using myeloma cells disclosed by Kohler et al., Nature, 256, 495, 1975 and the like.
- the monoclonal antibody used in the present invention can be prepared by the following steps. 1. 1. Preparation of immunogenic antigen 2. Immunization of animals with immunogenic antigens. 3. Preparation of myeloma cells (myeloma cells) 4. Cell fusion of antibody-producing cells and myeloma cells 5. Selection and monocloning of hybridomas (fusion cells) 6. Production of monoclonal antibodies Selection of monoclonal antibodies that do not cross-react with stromalysin 2 and 3
- immunogenic antigen for example, from the supernatant of normal human skin fibroblast NB1RGB (RCB222), Obata et al., Clin. Chim. Stromlysin 1 obtained by purification according to the method of Acta, 211, 59-72, 1992 can be used. Stroemlysin 1 thus obtained may be further converted to an immunogenic conjugate or the like, but can be used as it is for immunization of animals by mixing it with an appropriate adjuvant.
- strome lysin 1 can be made into a hapten-protein immunogenic conjugate by binding a fragment thereof to various carrier proteins via an appropriate condensing agent.
- a DNA sequence or a modified DNA sequence is obtained using an enzyme or by chemical synthesis, and is obtained from a microorganism, animal, plant, Alternatively, it can be used as a recombinant antigen obtained by expression in insects.
- a peptide or a modified peptide containing an amino acid sequence specific to strome lysin 1 obtained by a peptide chemical synthesis method using such information can also be used.
- the carrier proteins When bound to carrier proteins, the carrier proteins can be activated first. For such activation, introduction of an activated linking group can be mentioned.
- the activated linking group include (1) activated ester or activated carboxy group, such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester group, etc.
- activated A dithio group such as a 2-pyridyldithio group
- carrier proteins include polypeptides such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, globulin, and polylysine, and bacterial cell components such as BCG.
- Immunization is performed using animals such as mice such as BALB / c.
- the dose of the antigen is, for example, about 1 to 400 ⁇ g / animal for mice, and is generally injected intraperitoneally or subcutaneously into the host animal, and thereafter intraperitoneally every 1 to 4 weeks, preferably every 1 to 2 weeks. Repeat booster immunization subcutaneously, intravenously or intramuscularly about 2 to 10 times.
- BA1 / c mice and other mice such as F1 mice can also be used as mice for immunization.
- an antibody titer measurement system can be prepared and the antibody titer can be measured to confirm the degree of animal immunity.
- the infinitely proliferative strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulins, such as P3-NS-1-Ag4-1 ( NS-1, Kohler et al., Eur. J. Immunol., 6, 511-519, 1976), SP2 / 0-Ag14 (SP2, Shulman et al., Nature, 276, 269-270, 1978), mouse myeloma MOPC-21 Cell line-derived P3-X63-Ag8-U1 (P3U1, Yelton et al., Current topics in Microbiol.
- P3-NS-1-Ag4-1 NS-1, Kohler et al., Eur. J. Immunol., 6, 511-519, 1976
- SP2 / 0-Ag14 SP2, Shulman et al., Nature, 276, 269-270, 1978
- 8-Azaguanine-resistant mouse myeloma cell lines are Dulbecco's modified Eagle medium (DMEM medium), RPMI-164 For example, antibiotics such as penicillin and amikacin, fetal calf serum (FCS), etc.
- DMEM medium Dulbecco's modified Eagle medium
- RPMI-164 Dulbecco's modified Eagle medium
- antibiotics such as penicillin and amikacin, fetal calf serum (FCS), etc.
- the required number of cell lines can be prepared by subculture in normal medium 2 to 5 days before cell fusion.
- the cell line used was prepared by thawing the cryopreserved strain completely at about 37 ° C., washing it with a normal medium such as RPMI-1640 medium three times or more, and then culturing in the normal medium to prepare the required number of cell lines. There may be.
- the spleen is removed 2 to 5 days after the final immunization to obtain a spleen cell suspension.
- lymph node cells in various parts of the body can be obtained and used for cell fusion.
- the spleen cell suspension thus obtained and 3.
- the myeloma cell line obtained according to the above step is placed in a cell medium such as minimum essential medium (MEM medium), DMEM medium, RPMI-1640 medium, etc., and a cell fusion agent such as polyethylene glycol is added.
- MEM medium minimum essential medium
- DMEM medium DMEM medium
- RPMI-1640 medium RPMI-1640 medium
- the cell fusion agent examples include inactivated Sendai virus (HVJ: Hemagglutinating virus of Japan).
- HVJ Hemagglutinating virus of Japan
- polyethylene glycol Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added.
- the molecular weight of such polyethylene glycol is preferably 1000 to 8000, and more preferably 1000 to 4000.
- concentration of polyethylene glycol in the fusion medium is preferably 30 to 60%. If necessary, for example, dimethyl sulfoxide can be added in a small amount to promote fusion.
- the ratio of spleen cells (lymphocytes): myeloma cell line used for the fusion is, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 10: 1.
- the fusion reaction is performed for 1 to 10 minutes, and then a cell culture medium such as RPMI-1640 medium is added.
- the fusion reaction treatment can be performed multiple times. After the fusion reaction treatment, the cells are separated by centrifugation or the like and then transferred to a selection medium.
- Hybridomas Fused Cells
- the selection medium include media such as FCS-containing MEM medium or RPMI-1640 medium containing hypoxanthine, aminopterin and thymidine, so-called HAT medium.
- the method of exchanging the selective medium can generally be performed by adding the volume dispensed to the culture plate and the same volume the next day, and then replacing the HAT medium half by half every 1 to 3 days. Can also be added. On the 8th to 16th day after the fusion, the medium can be changed every 1 to 4 days with a so-called HT medium excluding aminopterin.
- mouse thymocytes can be used as a feeder, which may be preferred.
- the culture supernatant of the culture well in which the hybridoma is proliferated is measured using a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or a fluorescence-induced cell separation device (FACS)
- a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or a fluorescence-induced cell separation device (FACS)
- RIA radioimmunoassay
- ELISA enzyme immunoassay
- FACS fluorescence immunoassay
- the target antibody is detected using stromalysin 1 or a fragment peptide thereof or a labeled anti-mouse antibody, and screened or separated.
- the hybridoma producing the target antibody is cloned. Cloning can be performed by picking colonies in an agar medium or by limiting dilution. It is more preferable
- the obtained hybridoma strain can be cultured in an appropriate growth medium such as FCS-containing MEM medium, RPMI-1640 medium, etc., and a desired monoclonal antibody can be obtained from the medium supernatant.
- an appropriate growth medium such as FCS-containing MEM medium, RPMI-1640 medium, etc.
- a desired monoclonal antibody can be obtained from the medium supernatant.
- ascites of the hybridoma can be mentioned.
- each hybridoma is transplanted into the abdominal cavity of a histocompatible animal syngeneic with the myeloma cell-derived animal and allowed to proliferate, or for example, each hybridoma is transplanted into a nude mouse and proliferated, and produced in the ascites of the animal.
- Monoclonal antibodies recovered can be obtained.
- mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered, and then the hybridoma is allowed to grow and ascites can be collected.
- Ascites fluid as it is or conventionally known methods, for example, salting out such as ammonium sulfate precipitation, gel filtration by Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, It can be purified and used as a monoclonal antibody by high performance liquid chromatography.
- the ascites containing the monoclonal antibody can be purified and separated by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column after ammonium sulfate fractionation.
- an anion exchange gel such as DEAE-Sepharose
- an affinity column such as a protein A column after ammonium sulfate fractionation.
- affinity chromatography in which an antigen or antigen fragment (for example, a site specifically recognized by an antibody such as a synthetic peptide, recombinant antigen protein or peptide) is immobilized, or affinity chromatography in which protein A is immobilized. .
- the antibodies that do not cross-react with stromalysin 2 and 3. are selected from the monoclonal antibodies against stromalysin 1 obtained in (1). Selection can be performed by immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and RIA, ELISA, etc. can be used, with or without BF separation. Preferably, ELISA by direct adsorption method or sandwich method is used.
- the assay may be direct or indirect. Indirect methods such as PAP method (peroxidase antiperoxidase method), ABC method (avidin biotin complex method), protein A method and the like can also be used.
- a fixed concentration of stromalisin 1 is immobilized as an antigen.
- a carrier shown later can be preferably used. After immobilization, it is preferable to adsorb and block proteins that are not involved in antigen-antibody reaction and enzyme reaction.
- an antigen-antibody reaction is carried out by bringing a monoclonal antibody against stromlysin 1 into contact with the solid phase.
- the monoclonal antibody may or may not be labeled with an enzyme. Excess antibody that has not reacted can be removed by washing the solid phase. If the monoclonal antibody is labeled with an enzyme, the enzyme substrate is added and the amount of the product of the enzyme reaction is measured.
- an enzyme-labeled antibody that reacts specifically with the monoclonal antibody is allowed to act on the monoclonal antibody.
- the secondary antibody not bound to the monoclonal antibody is removed, the enzyme substrate is added, and the amount of the product of the enzyme reaction is measured.
- the amount of enzyme reaction product is proportional to the reactivity to strome lysin 1.
- ELISA using the direct adsorption method described above is carried out using each of Stromlysin 2 and Stromlysin 3 as antigens. Measure the amount of product of the enzymatic reaction. Since the amount of the enzyme reaction product is proportional to the reactivity to each antigen, the amount of the enzyme reaction product when each of the stromal lysin 2 and 3 is used relative to the amount of the enzyme reaction product when the stromal lysin 1 is used. Are cross-reactive with each other. The cross-reactivity with respect to stromelysin 2 and 3 is measured, respectively, and a monoclonal antibody that does not cross-react with both is selected.
- the sandwich ELISA method can be used to produce a monoclonal antibody that specifically reacts with strom lysin 1 without cross-reacting with strom lysin 2 and strom lysin 3. Can be selected.
- two types of antibodies against stromolysin 1 are used. One of them, 6.
- the other monoclonal antibody known to react specifically with strome lysin As a monoclonal antibody that is known to specifically react with strome lysin, for example, a monoclonal antibody against strome lysin 1 described in Patent Document 1 can be used.
- One monoclonal antibody is immobilized.
- stromlysin 1 a certain concentration of stromlysin 1 and the other monoclonal antibody are added as antigens, and an antigen-antibody reaction is performed.
- the monoclonal antibody may be labeled with an enzyme or the like. Excess antibody that has not reacted can be removed by washing the solid phase. Enzyme substrate is added and the amount of enzyme reaction product is measured. The amount of enzyme reaction product is proportional to the reactivity to strome lysin 1.
- ELISA by the sandwich method described above is carried out using strom lysin 2 and strom lysin 3 as antigens. Measure the amount of product of the enzymatic reaction. Since the amount of the enzyme reaction product is proportional to the reactivity to each antigen, the amount of the enzyme reaction product when each of the stromal lysin 2 and 3 is used relative to the amount of the enzyme reaction product when the stromal lysin 1 is used. Are cross-reactive with each other. The cross-reactivity with respect to stromelysin 2 and 3 is measured, respectively, and a monoclonal antibody that does not cross-react with both is selected.
- “specifically react” or “show reactivity” with an antigen means that when an antigen-antibody reaction by ELISA of either the direct method or the sandwich method is performed, for example, at room temperature. Perform antigen-antibody reaction for 1 hour or longer or overnight at 4 ° C, wash the solid phase, perform color reaction with enzyme labeling using a color former such as tetramethylbenzidine (TMB), and then stop the color reaction with sulfuric acid A450 (difference when primary antibody is 0 ng / ml) is 0.05 or more under the measurement conditions.
- A450 is 0.08 or more, more preferably 0.1 or more.
- cross-reactivity with stromalysin 2 refers to the magnitude of reactivity with stromalisin 2 when the reactivity of the monoclonal antibody with respect to strome lysin 1 is taken as 100%.
- the magnitude of cross-reactivity may be measured by either the direct adsorption method or the sandwich ELISA.
- does not cross-react means that the cross-reactivity measured by ELISA of either the direct adsorption method or the sandwich method is 5% or less.
- the cross-reactivity measured by the sandwich method is 5% or less.
- the cross-reactivity of the monoclonal antibody of the present invention to stromalysin 2 and 3 is preferably 2.4% or less, more preferably 1.3% or less.
- the obtained peptide is fractionated by a conventionally known method such as chromatography, When the monoclonal antibody obtained in (1) is reacted with the peptides of each fraction, the highly reactive fraction contains the peptide that is the reaction site of stromlysin 1.
- the peptide contained in the highly reactive fraction can be further purified using a conventionally known method, and the amino acid sequence of the reaction site can be determined by a known amino acid sequence analysis method.
- the present invention specifically reacts with a peptide comprising a part of the amino acid sequence of SEQ ID NO: 1 and is specific to stromalysin 1
- a monoclonal antibody which reacts manually, wherein the amino acid sequence comprises the sequence from 425 to 436 of SEQ ID NO: 1, and the length of the peptide is 12 to 20 amino acid residues is provided.
- the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of strome lysin 1 protein.
- the peptide consisting of the sequence of Nos. 425 to 436 of SEQ ID No. 1 is considered to be involved in cross-reactivity.
- the monoclonal antibody described above is described in 6. It is possible to obtain a monoclonal antibody that reacts with the peptide comprising the sequence of Nos. 425 to 436 of SEQ ID No. 1 from the monoclonal antibodies against strome lysin 1 obtained in the above.
- the selection is 7.
- 7. In the ELISA based on the direct adsorption method shown in (1), a peptide consisting of the sequence of Nos. 425 to 436 of SEQ ID No. 1 may be used as an antigen in place of stromolysin 1. It is preferable that the monoclonal antibody does not cross-react with stromalysin 2 and stromalysin 3.
- Monoclonal antibodies that do not cross-react with stromalysin 2 and stromalysin 3 are: It is possible to select by the method shown in.
- the present invention also relates to a monoclonal antibody that specifically reacts with stromalisin 1, which is obtained by degrading stromalisin 1 with lysyl endopeptidase.
- Monoclonal antibodies are provided that lose reactivity to.
- Such a monoclonal antibody is a monoclonal antibody that recognizes a three-dimensional structure such as a secondary structure or a tertiary structure of stromlysin 1 protein.
- “Degrading Stromlysin 1 to eliminate the reactivity to Stromlysin 1” means that the reactivity after the degradation of Stromlysin 1 is lower than the reactivity before the degradation.
- Such monoclonal antibodies are described in 6. It can be obtained by selecting from the monoclonal antibodies against stromalisin 1 obtained in the following manner. Enzymatic degradation of stromolysin 1 into peptides. Compare the reactivity of the monoclonal antibody that specifically reacts with strome lysin 1 to strome lysin 1 before and after degradation, and select monoclonal antibodies whose reactivity after degradation of strome lysin 1 is lower than that before degradation. To do. The lower the reactivity after degradation, the more the monoclonal antibody reacts with strome lysin 1 by recognizing the three-dimensional structure of strome lysin 1 regardless of the primary structure of strome lysin 1.
- the reactivity after decomposition is 10% or less.
- Reactivity is 7.
- the reactivity after decomposition measured by ELISA of direct adsorption method is 10% or less of the reactivity before decomposition. . More preferably, it is 8.0% or less, More preferably, it is 7.3% or less.
- the monoclonal antibody thus obtained can be examined for the type of heavy chain and light chain of its antibody chain using a commercially available isotype-specific anti-mouse Ig antibody, such as an isotype-specific rabbit anti-mouse Ig antibody.
- these antibodies may be used as antibody fragments such as Fab, Fab ′, and F (ab ′) 2 obtained by treating with an enzyme such as trypsin, papain, pepsin, etc., and optionally reducing them.
- an IgG fraction for example, an IgG fraction obtained by treating an antibody-containing material with an ammonium sulfate fraction and then treating with an anion exchange gel such as DEAE-Sepharose, or the like, and further reducing after digestion with pepsin.
- the resulting specific binding part Fab ′ or the like can be used.
- labels in these cases include enzymes (peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, etc.), chemical substances, fluorescent substances, radioisotopes and the like as described below.
- the immunostaining method can be performed using a monoclonal antibody that does not cross-react with stromalysin 2 and 3 and specifically reacts with stromalysin 1 according to the present invention.
- the immunostaining method of the present invention comprises a step of reacting the monoclonal antibody of the present invention with an antigen in a sample to form an antigen-antibody complex, and a step of visualizing the antigen-antibody complex.
- the antigen-antibody complex can be visualized by previously labeling the antibody, changing the label after the antigen-antibody reaction, and detecting the change as a signal.
- the immunostaining method of the present invention may be a direct method of labeling an antibody (primary antibody) that reacts directly with an antigen, or an indirect method of labeling another antibody (secondary antibody) that recognizes an unlabeled primary antibody. Good. Indirect methods such as PAP method (peroxidase antiperoxidase method), ABC method (avidin biotin complex method), protein A method and the like can also be used. Alternatively, the sample can be electrophoresed, the electrophoresed sample can be transferred to a membrane, and the membrane can be immunostained.
- the immunostaining method using the monoclonal antibody of the present invention even when stromalisin 2 or strome lysin 3 is mixed in the sample, a signal is specifically detected only from strome lysin 1 and the presence of strome lysin 1 is present. And localization can be visualized. Moreover, immune cell staining or immune tissue staining can also be performed using the monoclonal antibody of the present invention.
- the monoclonal antibody of the present invention can be used in an immunological assay.
- the immunoassay method of the present invention comprises a step of reacting the monoclonal antibody of the present invention with an antigen in a sample to form an antigen-antibody complex, a step of detecting the formed antigen-antibody complex, And measuring the amount of the antigen in the sample from the amount of the antigen-antibody complex.
- the antigen-antibody complex can be detected as the turbidity of the antigen-antibody complex.
- a label can be added to the antibody in advance, the label can be changed after the antigen-antibody reaction, and the change can be detected as a signal. .
- the immunological assay can be performed by immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and RIA, ELISA, agglutination method, turbidimetric method, immunochromatography method, Western blot method, etc. can be used. It can be done with or without F separation.
- the immunological measurement method may be a direct method of labeling an antibody (primary antibody) that reacts directly with an antigen, or an indirect method of labeling another antibody (secondary antibody) that recognizes an unlabeled primary antibody. Indirect methods such as PAP method (peroxidase antiperoxidase method), ABC method (avidin biotin complex method), protein A method and the like can also be used.
- the monoclonal antibody of the present invention is used as a monoclonal antibody used in the competitive immunoassay by the direct method.
- the monoclonal antibody of the present invention is the monoclonal antibody of the present invention.
- the other is not particularly limited as long as it is an antibody that recognizes stromlysin 1. If the types are different, a monoclonal antibody that specifically reacts with strome lysin 1 without cross-reacting with strome lysin 2 and 3 of the present invention may be used.
- One of the two types of monoclonal antibodies may be a soluble antibody labeled with a label, and the other may be a solid-phased antibody.
- the monoclonal antibody of the present invention is the monoclonal antibody of the present invention. As long as the types are different, the monoclonal antibody of the present invention may be used. In addition, if at least one type is the monoclonal antibody of the present invention, three or more types of antibodies may be used.
- a concentration standard curve is prepared using a standard solution containing a known concentration of strome lysin 1, and the concentration of strome lysin 1 in the sample is measured from the concentration standard curve. Lysin 1 can be quantified. According to the immunological measurement method of the present invention, even if stromalisin 2 or strome lysin 3 is mixed in the sample, it is not detected and strome lysin 1 can be specifically quantified.
- a monoclonal antibody labeled with an enzyme or the like and an antibody bound to a carrier can be reacted sequentially or simultaneously.
- the monoclonal antibody of the present invention may be labeled with an enzyme or the like to form an immunostaining reagent or an immunological measurement reagent.
- an antigen and two or more kinds of unlabeled monoclonal antibodies can be reacted simultaneously or sequentially.
- the order in which the antibody and sample are added depends on the type of carrier system chosen.
- the carrier can be appropriately selected from those described below.
- the reaction can be carried out by adding beads such as.
- the reaction can be carried out in an appropriate buffer so as to maintain an optimum pH, for example, about pH 4-9.
- Particularly suitable buffers include, for example, acetate buffer, citrate buffer, phosphate buffer, Tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer, Tris-HCl A buffering agent etc. are mentioned.
- the buffering agents can be mixed and used at any ratio.
- the antigen-antibody reaction is preferably performed at a temperature between about 0 ° C and 60 ° C.
- the incubation treatment with the monoclonal antibody of the present invention, other antibodies, and antigens can be performed until equilibrium is reached, but the equilibrium of the antigen-antibody reaction has been achieved.
- the reaction can be stopped at a much earlier point, and the degree of turbidity and the presence of labels such as enzymes in either the liquid phase or the solid phase can be measured.
- the measurement operation can be performed using an automated measuring device. Using a colorimeter, a luminescence detector, a photo detector, etc., the turbidity and the signal generated when the substrate is converted by the action of the enzyme. Can also be measured.
- the antigen-antibody reaction it is possible to stabilize the reagent used, the substance to be measured, and the label such as an enzyme, or take appropriate measures to stabilize the antigen-antibody reaction itself.
- proteins, stabilizers, surfactants, chelating agents, etc. can be added to the incubation solution to eliminate non-specific reactions, reduce the inhibitory effect, or activate the measurement reaction. It can also be added.
- a blocking treatment for preventing a non-specific binding reaction that is commonly employed in the art or known to those skilled in the art may be performed, such as normal serum proteins such as mammals, albumin, skim milk, It can be treated with fermented milk material, collagen, gelatin and the like.
- these methods are not particularly limited and can be used.
- washing, stirring, shaking, filtration, or antigen pre-extraction are appropriately employed under specific circumstances.
- Other measurement conditions such as the concentration of a specific reagent, buffer, etc., temperature, or incubation time can be varied according to factors such as the concentration of antigen in the sample, the nature of the sample, and the like.
- a person skilled in the art can perform measurement by appropriately selecting optimum conditions effective for each measurement while using a normal experimental method.
- glass for example, activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloy and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride, polyfluoride, etc.
- Vinylidene polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, crosslinked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, etc., crosslinked albumin Collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate and other natural or modified cellulose, cross-linked dex Organic polymer materials such as polyamide such as orchid, nylon, polyurethane, polyepoxy resin, etc., and those obtained by emulsion polymerization of them, cells, erythrocytes, etc., if necessary, functional group with silane coupling agent etc. The thing which introduce
- transduced is mentioned.
- filter paper for example, test tubes, titer plates, titer wells, glass cells, synthetic resin cells such as synthetic resin cells, glass rods, synthetic material rods, thickened ends, etc.
- a solid substance such as a thinned rod, a rod having a round protrusion at the end or a flat protrusion, a thin plate, or the like may be used.
- An antibody can be bound to these carriers, and preferably a monoclonal antibody that specifically binds to strome lysin 1 obtained in the present invention can be bound.
- the binding between the carrier and those involved in the antigen-antibody reaction may be performed by a physical method such as adsorption, a chemical method using a condensing agent or the like, an activated one, and the like. It can be performed by a technique using a chemical bonding reaction.
- Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules, coenzymes, enzyme precursors, apoenzymes, fluorescent materials, dye materials, chemiluminescent compounds, luminescent materials, chromogenic materials, magnetic materials, metal particles such as gold Examples include colloids and radioactive materials.
- enzymes include oxidoreductases such as dehydrogenase, reductase, and oxidase, such as transferases that catalyze the transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, etc., such as ester bonds, Examples include hydrolase, lyase, isomerase, ligase and the like that hydrolyze glycoside bonds, ether bonds, peptide bonds and the like.
- the enzyme can be used for detection by using a plurality of enzymes in combination. For example, enzymatic cycling can be used.
- Typical enzyme labels include peroxidase such as horseradish peroxidase (HRP), galactosidase such as E. coli ⁇ -D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, acetylcholinesterase, catalase And alkaline phosphatase such as bovine small intestine alkaline phosphatase and Escherichia coli alkaline phosphatase.
- HRP horseradish peroxidase
- galactosidase such as E. coli ⁇ -D-galactosidase
- maleate dehydrogenase glucose-6-phosphate dehydrogenase
- glucose oxidase glucose oxidase
- glucoamylase acetylcholinesterase
- catalase And alkaline phosphatase such as bovine small intestine al
- alkaline phosphatase When alkaline phosphatase is used, substrates such as umbelliferone derivatives such as 4-methylumbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, dioxetane derivatives, etc. It can be measured by the fluorescence, luminescence, etc. that occur when used. A luciferin or luciferase system can also be used. When catalase is used, it reacts with hydrogen peroxide to produce oxygen, so that the oxygen can be detected by an electrode or the like.
- the electrode may be a glass electrode, an ion electrode using a hardly soluble salt film, a liquid film type electrode, a polymer film electrode, or the like.
- the enzyme label can be replaced with a biotin label and enzyme-labeled avidin (streptavidin).
- the label may use a plurality of different types of labels. In such cases, it may be possible to perform multiple measurements continuously or discontinuously and simultaneously or separately.
- fluorescent substances or chemiluminescent compounds include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate, tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, lumiferin, luciferase And luminol such as equolin, imidazole, oxalate ester, rare earth chelate compound, coumarin derivative and the like.
- fluorescein isothiocyanate for example, rhodamine derivatives such as rhodamine B isothiocyanate, tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, lumiferin, luciferase And luminol such as
- the labeling can be performed using a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc., and can be easily performed by a known method or a person skilled in the art. It is possible to apply the method appropriately selected from methods that can be used in addition to methods modified by them. Further, a condensing agent that can be used for preparing an immunogenic conjugate, a condensing agent that can be used for binding to a carrier, and the like can be used.
- condensing agent examples include glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N′-polymethylene bisiodoacetamide, N, N′-ethylene bismaleimide, ethylene glycol bissuccinimidyl succinate, bis Diazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate ( SMCC), N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimid 4- (1-maleimidophen
- Samples to be subjected to the immunological measurement method of the present invention include all forms of solutions, colloidal solutions, etc., preferably biological fluid samples such as blood, plasma, serum, joint fluid, cerebrospinal fluid. , Saliva, amniotic fluid, urine, other body fluids, cell culture fluid, tissue culture fluid, biopsy specimens such as cells, tissues, organs, tumor tissues and the like.
- the sample can be used for detection of rheumatoid arthritis in a patient.
- Particularly preferred are plasma, serum, joint fluid and the like.
- biopsy specimens such as cells, tissues, organs, and tumor tissues are preferably used.
- the monoclonal antibody of the present invention can be used for the above sample as a marker for rheumatoid arthritis, and an immune cell staining method or an immune tissue staining method can be performed.
- the sample can be immobilized as necessary before staining.
- immobilization those widely used in the art or those derived therefrom can be used.
- periodate-lysine-paraformaldehyde, glutaraldehyde, buane, formalin, paraformaldehyde, zambony, acrolein and the like can be used. It can also be fixed with paraffin or the like.
- the present invention also provides an immunological measurement reagent comprising the monoclonal antibody of the present invention.
- an immunological measurement reagent comprising the monoclonal antibody of the present invention.
- strome lysin 1 can be specifically detected easily and with high sensitivity.
- the reagent can also be used for detection of rheumatoid arthritis in patients.
- the present invention further relates to the field of medicine or clinical examination or analytical measurement, comprising one or more containers filled with one or more of the aforementioned substances used in the immunostaining method or immunoassay of the present invention. It also relates to acceptable packs and kits. Packs and kits, together with such container (s), are written in a form instructed by a government agency that regulates the manufacture, use or sale of pharmaceuticals, test drugs or biological products ( Document), which may be accompanied by a notice (package insert) indicating the agency's approval for the manufacture, use or sale of human-related products.
- Document a government agency that regulates the manufacture, use or sale of pharmaceuticals, test drugs or biological products
- Document may be accompanied by a notice (package insert) indicating the agency's approval for the manufacture, use or sale of human-related products.
- special conditions, operations and the like are not required to be set.
- a measurement system related to the measurement target substance of the present invention or a substance having substantially the same activity may be constructed by adding the usual technical considerations of
- the monoclonal antibody according to the present invention a method for producing the same, an immunostaining method using the monoclonal antibody, and a method for immunologically quantifying stromelysin 1 using the monoclonal antibody will be specifically described below. .
- the present invention is not limited to these examples.
- the cells were washed with 100 ml of RITC80-7 basal medium, cultured in 300 ml of serum-free RITC80-7 basal medium containing 2 g / l lactalbumin hydrolyzate and 10000 U / l interleukin 1 ⁇ , and the supernatant was collected.
- Sepharose 4B column has a monoclonal antibody 55-3G3 against Stromlysin 1 (International Deposit Number: FERM BP-3744, Deposit Date: June 12, 1991, Deposit Agency: National Institute of Advanced Industrial Science and Technology, Patent Biology)
- the deposit center, 1-1-1 Higashi Tsukuba, Tsukuba, Ibaraki Prefecture, center 6) is pre-bonded with CNBr-activated Sepharose (GE Healthcare) according to the procedure described in the manual.
- the column provided with the supernatant was washed with 30 mM Tris-HCl buffer (pH 8.0) containing 0.1 M NaCl, 5 mM CaCl 2 and 5 g / l CHAPS, and the protein bound to the column was eluted with 3 M KSCN.
- the above elution was performed on a Sephadex G-25 column (GE Healthcare) equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 0.4 M NaCl, 10 mM CaCl 2 and 0.5 g / l Brij 35. Liquid was provided.
- the void volume fraction was collected and applied to a Concanavalin A-Sepharose column (GE Healthcare) equilibrated with the same buffer as the Sephadex G-25 column equilibrated to collect proteins that did not bind to the column. . Finally, the protein fraction that did not bind to the concanavalin A column was applied to an Ultrogel AcA44 column (PALL) and chromatographed.
- GE Healthcare Concanavalin A-Sepharose column
- PALL Ultrogel AcA44
- the purity of the protein was confirmed by dodecyl sulfate polyacrylamide gel electrophoresis.
- the purified enzyme was homogeneous and showed a molecular weight of 57 kDa. This purified enzyme was used as an antigen and a standard.
- Stromlysin 2 was a standard product manufactured by R & D Systems
- Stromlysin 3 was a standard product manufactured by Abcam.
- Example 2 Preparation of monoclonal antibody
- a monoclonal antibody was prepared using stromalisin 1 purified in Example 1 as an antigen.
- RPMI 1640 medium RPMI 1640 (manufactured by JRH Biosciences) was added with 24 mM sodium bicarbonate, 1 mM sodium pyruvate, 50 U / ml penicillin G potassium, 50 ⁇ g / ml streptomycin sulfate and 100 ⁇ g / ml amikacin sulfate, and the pH was adjusted to 7. And sterilized by filtration through a 0.22 ⁇ m Millipore filter.
- NS-1 medium FBS (manufactured by JRH Biosciences) sterilized and filtered in the above RPMI-1640 medium is added to a concentration of 15% (v / v).
- PEG 4000 solution A 50% (w / w) serum-free solution of polyethylene glycol 4000 (PEG 4000, manufactured by Merck and CO., Inc.) was prepared in RPMI-1640 medium.
- HAT medium Hypoxanthine (100 ⁇ M), aminopterin (0.4 ⁇ M) and thymidine (16 ⁇ M) were further added to the NS-1 medium described in Example 2 (b).
- HT medium The same composition as the HAT medium except that aminopterin was removed.
- RPMI 1640 medium- (2) RPMI 1640 MEDIAUM (SIGMA R8758) 500 ml (1 bottle), FOETAL BOVINE SERUM (SIGMA F9423) 50 ml, PENICILLIN-STREPTOMYCIN SOLUTION (SIGMA P0781) 2.5 ml.
- Frozen cells were reconstituted in RPMI 1640 medium-(2), and using BriClone-containing RPMI 1640 medium, 5 wells per well, 36, 36 and 24 wells, 96 well microwells, 1 and 0.5, respectively. Hybridomas were added. On day 5, approximately 0.1 ml of RPMI 1640 medium- (2) was added to all wells. After the start of cloning, sufficient growth of the hybridoma was observed from the 11th day to the 14th day, and the ELISA described in the following (f) was performed. If all tested wells were not positive, the number of colonies in the antibody-positive well was confirmed, and 1 to 3 colonies (well with as few colonies as possible if not present) were selected and recloned. As a result, as shown in Table 1, 12 clones of monoclonal antibody-producing cells against stromelysin 1 were obtained.
- TMB One Component HRP Microwell Substrate BioFX, Product # TMBW-1000-01 was added at 100 ⁇ l / well and allowed to react at room temperature for about 5 to 10 minutes. The reaction was stopped by adding 100 ⁇ l / well of 1 M sulfuric acid, and the absorbance at a measurement wavelength of 450 nm was measured with a microplate reader (TECAN SPECTRA).
- Example 3 Identification of subclass of monoclonal antibody According to the ELISA described in Example 2 (d), the subclass of the monoclonal antibody was identified as follows. The operation up to blocking of 96-well microwells coated with stromelysin 1 as described in Example 2 (d) was performed.
- Example 4 Reactivity with Stromlysin 2 and 3 Using Culture Supernatant
- the culture supernatant of 12 types of monoclonal antibody-producing hybridomas obtained in Example 2 was subjected to ELISA according to the method of Example 2 (d), using stromalisin 2 or stromalicin 3 instead of stromalicin 1 as an antigen. Went.
- Table 2 shows the ratio of the absorbance values of strome lysin 2 and 3 when the absorbance value of strome lysin 1 is 100%.
- the antibodies of 3 clones of 296-29C1, 296-12H8 and 296-7B10 have a reactivity to strome lysin 2 of 1% when the reactivity of the culture supernatant to strome lysin 1 is 100%. It did not cross-react with stromalysin 2.
- the antibodies of other clones were 10% to 337% reactive with stromalysin 2, and cross-reactivity with strome lysin 2 was observed.
- the reactivity with respect to strome lysin 3 was 0% to 6%, and most of them did not cross-react.
- Example 5 Immunostaining Each antigen of Stromlysin 1 and 2 was subjected to electrophoresis with c-PAGE (Ato), and then transferred to Sequi-Blot PVDF Membrane for Protein Sequencing (thickness 0.2 ⁇ m) (Bio Rad). . In order to remove SDS, the transferred membrane was washed with 0.01 M phosphate buffer (pH 7.0) containing 0.1% Tween 20 and 0.1 M NaCl. Further, blocking was performed with 0.01 M phosphate buffer (pH 7.0) containing 1% BSA and 0.1 M NaCl. This membrane was allowed to stand at 4-8 ° C. overnight in the culture supernatant of the 12 types of monoclonal antibody-producing hybridomas obtained.
- Example 6 Purification of monoclonal antibody
- 296-21C4, 296-29C1, 296-12H8, 296-7B10, 296-33F3 and 296-23A8 were selected, and antibody purification was performed.
- cells were proliferated, the cells were administered into the peritoneal cavity of five Balb / c mice, and ascites was collected. The collected ascites was subjected to ammonium sulfate (ammonium sulfate) fractionation, and the antibody was purified by DEAE ion exchange chromatography.
- ammonium sulfate ammonium sulfate
- Example 7 Reactivity with Stromlysin 2 and 3 Using Purified Antibody
- the cross-reactivity of the antibody purified in Example 6 to stromalysin 2 and 3 was examined by ELISA. Absorbance at a measurement wavelength of 450 nm was measured using a purified antibody as a primary antibody at 100 ng / well. A450 (difference when the primary antibody is 0 ng / ml) is shown in Table 3.
- the cross-reactivity of the antibody to stromalysin 2 was strongly observed in the order of 296-33F3, 296-23A8, 296-21C4.
- Known monoclonal antibody 55-2A4 (International deposit number: FERM BP-3743, deposit date: June 12, 1991, deposit agency: independent administrative institution, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-Higashi, Tsukuba, Ibaraki 1-1 Tsukuba Center Central 6) has a high cross-reactivity with strome lysin 2 of 85.1%, and some of the known monoclonal antibodies against strome lysin 1 show cross-reactivity with strome lysin 2. Was confirmed. To stromalysin 3, the antibody of any clone was hardly reactive.
- Stromlysin 1 was quantified by immunoassay by sandwich ELISA.
- 100 ⁇ l / well of 0.1 M phosphate buffer (pH 7.0) containing 100 ⁇ g / ml of the purified antibody obtained in Example 6 was added to a 96-well microwell plate (Nunc) and allowed to stand at 4 ° C. for 16 hours.
- the antibody solution was removed, 300 ⁇ l / well of 0.01 M phosphate buffer (pH 7.0) containing 1% BSA, 0.1 M NaCl, and 10 mM EDTA was added, and blocking was performed overnight at 4 ° C. to form antibody-bound wells.
- the wells were washed with 0.01 M phosphate buffer (pH 7.0) containing 0.1% Tween 20 and 0.1 M NaCl (hereinafter referred to as “washing solution”).
- the monoclonal antibody 55-2A4 was labeled with HRP (Roche Diagnostics).
- the labeled antibody was diluted with an assay buffer to 1.3 ⁇ g / ml, and 100 ⁇ l was added to each well containing a standard solution or sample. 100 ⁇ l from the well was added to the antibody-bound well, and incubated at 4 ° C. for 16 hours (standing). After washing the antibody-bound wells with the washing solution three times, 100 ⁇ l / well of TMB One Component HRP Microwell Substrate (BioFX) was added, reacted at 25 ° C.
- a standard curve of the concentration of stromelysin 1 could be drawn with each selected purified antibody.
- the absorbance was lower than when other antibodies were used, and the binding site of these antibodies on one stromalysin molecule was the same as that used for the labeled antibody.
- the reactivity was thought to be low because it was the same as the binding site on one stromal lysin molecule of 2A4 or close to the binding site on one stromal lysin molecule of 55-2A4. All antibodies were less reactive to stromalysin 2 compared to reactivity to strome lysin 1 (Tables 4 and 5).
- the reactivity to stromalysin 2 was 0.5% to 26.9% of the reactivity to stromalysin 1, indicating the greatest cross-reactivity with the 296-21C4 antibody. If the cross-reactivity was allowed to be 5% or less, it was considered that the 296-21C4 and 296-33F3 antibodies could not be used for immunoassay of stromlysin 1 in sandwich ELISA. In the case of the 296-23A8 antibody, whose cross-reactivity to stromelysin 2 was 17.1% in the direct adsorption ELISA, the sandwich ELISA was in an acceptable range of 2.4%. It was thought that it could be used for immunoassay by ELISA.
- the antibodies of 296-29C1, 296-12H8, and 296-7B10 have low cross-reactivity to stromelysin 2, and were considered to be usable for immunoassay by ELISA using the sandwich method.
- the antibody to 296-21C4, 296-33F3, 296-7B10 did not react with serum.
- the antibody of 296-12H8 was less reactive to serum than the antibodies of 296-29C1 and 296-23A8.
- 296-29C1 International Deposit Number: FERM BP-11199, Deposit Date: December 19, 2008, Deposit Agency: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, Tsukuba City 1-1-1 Tsukuba, Ibaraki Prefecture Center Center No.
- Example 9 Analysis of reaction site on stromalicin 1.
- the reaction site on one stromlysin molecule was analyzed for the purified monoclonal antibody obtained in Example 6 as follows.
- the monoclonal antibody shown in Table 8 was diluted with an assay buffer so as to be 10 ⁇ g / ml, and 100 ⁇ l / well each was added to wells to which strome lysin 1 after degradation or strome lysin 1 before degradation was bound. Incubated for a period of time (standing). After washing the wells with a washing solution, 100 ⁇ l / well of goat anti-mouse Ig (G + M + A) immunoglobulin-POD (CAPPEL, Cat # 55556) diluted to 1/1000 with an assay buffer was added as a secondary antibody, and 40 ° C. at 25 ° C. Incubated for minutes (still).
- the column was washed with 6 ml of liquid A (1 ml / fraction), and then eluted with about 33.2 ml of a gradient solution of liquid A and liquid B (0.5 ml / fraction).
- the fraction was A1 after sample application, and the washed and eluted fractions were collected in succession.
- the fractions were numbered by 12 fractions such as A1 to A12 and B1 to B12.
- 296-29C1 and 296-7B10 were diluted with an assay buffer so as to be 10 ⁇ g / ml, 100 ⁇ l / well of each was added to the peptide-binding well, and incubated at 25 ° C. for 1 hour (stationary). After washing the wells with a washing solution, 100 ⁇ l / well of goat anti-mouse Ig (G + M + A) immunoglobulin-POD (CAPPEL, Cat # 55556) diluted to 1/3000 with assay buffer was added as a secondary antibody, and 40 ° C. was added at 25 ° C. Incubated for minutes (still). After washing, 100 ⁇ l / well of color former was added, reacted at 25 ° C.
- an assay buffer so as to be 10 ⁇ g / ml
- 100 ⁇ l / well of each was added to the peptide-binding well, and incubated at 25 ° C. for 1 hour (stationary). After washing the wells with a washing
- FIG. 296-29C1 and 296-7B10 showed reactivity with FPLC fractions C10 and D9 (hereinafter referred to as “Fr.C10” and “Fr.D9”, respectively). Since reaction peaks are observed in the two fractions, the two sequences may contain the same sequence due to partial degradation of lysyl endopeptidase.
- Fr. D9 is a fraction following the first and second peaks having a high absorbance at a measurement wavelength of 230 nm, and it was considered that the peptide was not sufficiently separated.
- C10 N-terminal amino acid sequence analysis was performed. N-terminal amino acid sequence analysis was outsourced to AproScience and performed by Edman degradation. Fr. Using 50 ⁇ l of C10 and measuring the N-terminal 5 residues, multiple amino acids were detected in each cycle, and the sequence was not finalized. Therefore, the peptide sequence was deduced from the detected amount and sequence from among the detected amino acids.
- a sequence that was QIAED was listed as a candidate.
- the detected amount of each amino acid was about 9 pmol.
- the candidate peptide sequence is verified from the entire amino acid sequence of strome lysin 1 (SEQ ID NO: 1), Fr. Peptides contained in C10 were estimated.
- the peptide was presumed to be QIAEDFPPGIDSK (sequence numbers 425 to 436).
- the amino acid sequence corresponding to the above peptide sequence among all the amino acid sequences of strome lysin 2 is LIADDDFPGVEPK (No. 424 to 435 of SEQ ID NO: 2), 7 of 12 amino acids (58% ) Amino acids matched.
- each monoclonal antibody shown in Table 9 was diluted to 6.3 ⁇ g / ml with an assay buffer, added to each well coated with the peptide at 100 ⁇ l / well, and incubated (standing) at 25 ° C. for 1 hour. Subsequent operations were performed in the same manner as described in Example 9 (b). Table 9 shows A450 (difference when the primary antibody was 0 ng / ml) when the peptide obtained in this example was used as an antigen.
- Table 9 shows that 296-29C1 showed reactivity with stromlysin 1 peptide QIAEDFPPGIDSK, but 296-7B10 had low reactivity.
- Fr. From the results of N-terminal amino acid sequence analysis of C10, Fr. The amino acid sequence of the peptide contained in C10 was estimated and synthesized. Numerous peptides were mixed in C10, indicating that the stromalysin 1 peptide QIAEDFPPGIDSK was not a peptide to which 296-7B10 reacted.
- Mouse-derived monoclonal anti-human stromal lysin antibody-producing hybridoma 55-3G3 International deposit number: FERM BP-3744, deposit date: June 12, 1991, deposit agency: National Institute of Advanced Industrial Science and Technology, Patent Organism) Center, Tsukuba Center Central, Tsukuba Center 1-1-1 Tsukuba City, Ibaraki Prefecture
- Mouse-derived monoclonal anti-human stromal lysin antibody-producing hybridoma 55-2A4 International deposit number: FERM BP-3743, deposit date: June 12, 1991, deposit agency: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit
- Tsukuba Center Central, Tsukuba Center 1-1-1 Tsukuba City, Ibaraki Prefecture Mouse-derived monoclonal anti-human stromal lysin 1 antibody-producing hybridoma 296-29C1 (International deposit number: FERM BP-11199, deposit date: December 19, 2008, depository organization: National Institute of Advanced Industrial Science and Technology, Patent
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Abstract
Description
1.免疫原性抗原の調製
2.免疫原性抗原による動物の免疫
3.ミエローマ細胞(骨髄腫細胞)の調製
4.抗体産生細胞とミエローマ細胞の細胞融合
5.ハイブリドーマ(融合細胞)の選択及びモノクローン化
6.モノクローナル抗体の製造
7.ストロムライシン2及び3と交差反応しないモノクローナル抗体の選抜 The monoclonal antibody used in the present invention may be a monoclonal antibody obtained by utilizing the cell fusion technique using myeloma cells disclosed by Kohler et al., Nature, 256, 495, 1975 and the like. The monoclonal antibody used in the present invention can be prepared by the following steps.
1. 1. Preparation of immunogenic antigen 2. Immunization of animals with immunogenic antigens. 3. Preparation of myeloma cells (myeloma cells) 4. Cell fusion of antibody-producing cells and myeloma cells 5. Selection and monocloning of hybridomas (fusion cells) 6. Production of monoclonal antibodies Selection of monoclonal antibodies that do not cross-react with stromalysin 2 and 3
抗原としては、例えば、正常ヒト皮膚線維芽細胞NB1RGB(RCB222)培養上清より、Obataら,Clin.Chim.Acta,211,59-72,1992の方法に従い精製されて得られたストロムライシン1を用いることができる。こうして得られたストロムライシン1は、さらに免疫原性コンジュゲート等にしてもよいが、そのまま適当なアジュバントと混合して動物を免疫するのに使用できる。 1. Preparation of immunogenic antigen As the antigen, for example, from the supernatant of normal human skin fibroblast NB1RGB (RCB222), Obata et al., Clin. Chim. Stromlysin 1 obtained by purification according to the method of Acta, 211, 59-72, 1992 can be used. Stroemlysin 1 thus obtained may be further converted to an immunogenic conjugate or the like, but can be used as it is for immunization of animals by mixing it with an appropriate adjuvant.
動物を免疫するには、例えば村松繁ら編,実験生物学講座14,免疫生物学,丸善株式会社,昭和60年、日本生化学会編,続生化学実験講座5,免疫生化学研究法,東京化学同人,1986年、日本生化学会編,新生化学実験講座12,分子免疫学III,抗原・抗体・補体,東京化学同人,1992年等に記載の方法に準じて行うことができる。抗原と共に用いられるアジュバントとしては、例えばフロイント完全アジュバント、リビ(Ribi)アジュバント、百日咳ワクチン、BCG、リピッドA、リポソーム、水酸化アルミニウム、シリカ等が挙げられる。免疫は、例えばBALB/c等のマウスをはじめとする動物を使用して行われる。抗原の投与量は、例えばマウスに対して約1~400μg/動物で、一般には宿主動物の腹腔内や皮下に注射し、以後1~4週間おきに、好ましくは1~2週間ごとに腹腔内、皮下、静脈内あるいは筋肉内に追加免疫を2~10回程度反復して行う。免疫用のマウスとしてはBALB/c系マウスの他、BALB/c系マウスと他系マウスとのF1マウス等を用いることもできる。必要に応じ、抗体価測定系を調製し、抗体価を測定して動物免疫の程度を確認できる。 2. Immunization of animals with immunogenic antigens To immunize animals, for example, Shigeru Muramatsu et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Japanese Biochemical Society, Second Life Chemistry Experiment Course 5 , Immunochemical Research, Tokyo Chemical Doujin, 1986, Japan Biochemical Society, New Chemistry Laboratory 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Chemical Doujin, 1992, etc. Can be done. Examples of the adjuvant used together with the antigen include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipid A, liposome, aluminum hydroxide, silica and the like. Immunization is performed using animals such as mice such as BALB / c. The dose of the antigen is, for example, about 1 to 400 μg / animal for mice, and is generally injected intraperitoneally or subcutaneously into the host animal, and thereafter intraperitoneally every 1 to 4 weeks, preferably every 1 to 2 weeks. Repeat booster immunization subcutaneously, intravenously or intramuscularly about 2 to 10 times. In addition to BALB / c mice, BA1 / c mice and other mice such as F1 mice can also be used as mice for immunization. If necessary, an antibody titer measurement system can be prepared and the antibody titer can be measured to confirm the degree of animal immunity.
細胞融合に使用される無限増殖可能株(腫瘍細胞株)としては免疫グロブリンを産生しない細胞株から選ぶことができ、例えばP3-NS-1-Ag4-1(NS-1、Kohlerら,Eur.J.Immunol.,6,511~519,1976)、SP2/0-Ag14(SP2、Shulmanら,Nature,276,269~270,1978)、マウスミエローマMOPC-21セルライン由来のP3-X63-Ag8-U1(P3U1、Yeltonら,Current topics in Microbiol.and Immunol.,81,1~7,1978)、P3-X63-Ag8(X63、Kohlerら,Nature,256,495~497,1975)、P3-X63-Ag8.653(653、Kearneyら,J.Immunol.,123,1548~1550,1979)等を用いることができる。8-アザグアニン耐性のマウスミエローマ細胞株はダルベッコ変法イーグル培地(DMEM培地)、RPMI-164
0培地等の細胞培地に、例えばペニシリン、アミカシン等の抗生物質、牛胎児血清(FCS)等を加え、さらに8-アザグアニン(例えば5~45μg/ml)を加えた培地で継代されるが、細胞融合の2~5日前に正常培地で継代して所要数の細胞株を用意することができる。また使用細胞株は、凍結保存株を約37℃で完全に解凍したのちRPMI-1640培地等の正常培地で3回以上洗浄後、正常培地で培養して所要数の細胞株を用意したものであってもよい。 3. Preparation of myeloma cells (myeloma cells) The infinitely proliferative strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulins, such as P3-NS-1-Ag4-1 ( NS-1, Kohler et al., Eur. J. Immunol., 6, 511-519, 1976), SP2 / 0-Ag14 (SP2, Shulman et al., Nature, 276, 269-270, 1978), mouse myeloma MOPC-21 Cell line-derived P3-X63-Ag8-U1 (P3U1, Yelton et al., Current topics in Microbiol. And Immunol., 81, 1-7, 1978), P3-X63-Ag8 (X63, Kohler et al., Nature, 256, 495-497, 1975), P3 X63-Ag8.653 (653, Kearney et al., J.Immunol., 123,1548 ~ 1550,1979) or the like can be used. 8-Azaguanine-resistant mouse myeloma cell lines are Dulbecco's modified Eagle medium (DMEM medium), RPMI-164
For example, antibiotics such as penicillin and amikacin, fetal calf serum (FCS), etc. are added to a cell culture medium such as 0 medium, and 8-azaguanine (for example, 5-45 μg / ml) is further subcultured. The required number of cell lines can be prepared by subculture in normal medium 2 to 5 days before cell fusion. The cell line used was prepared by thawing the cryopreserved strain completely at about 37 ° C., washing it with a normal medium such as RPMI-1640 medium three times or more, and then culturing in the normal medium to prepare the required number of cell lines. There may be.
上記2.の工程に従い免疫された動物、例えばマウスは最終免疫後、2~5日後にその脾臓が摘出され、脾細胞懸濁液を得る。脾細胞の他、生体各所のリンパ節細胞を得て、それを細胞融合に使用することもできる。こうして得られた脾細胞懸濁液と上記3.の工程に従い得られたミエローマ細胞株を、例えば最小必須培地(MEM培地)、DMEM培地、RPMI-1640培地等の細胞培地中に置き、細胞融合剤、例えばポリエチレングリコールを添加する。細胞融合剤としては、この他各種当該分野で知られたものを用いることができ、例えば不活性化したセンダイウイルス(HVJ:Hemagglutinating virus of Japan)等が挙げられる。好ましくは、例えば30~60%のポリエチレングリコールを0.5~2ml加えることができる。そのようなポリエチレングリコールの分子量は1000~8000であることが好ましく、さらに分子量が1000~4000であることが好ましい。融合培地中でのポリエチレングリコールの濃度は、30~60%となるようにすることが好ましい。必要に応じ、例えばジメチルスルホキシド等を少量加え、融合を促進することもできる。融合に使用する脾細胞(リンパ球):ミエローマ細胞株の割合は、例えば1:1~20:1とすることが挙げられるが、より好ましくは4:1~10:1とすることができる。融合反応を1~10分間行い、次にRPMI-1640培地等の細胞培地を加える。融合反応処理は複数回行うこともできる。融合反応処理後、遠心等により細胞を分離した後選択用培地に移す。 4). 1. Cell fusion of antibody-producing cells and myeloma cells In the animal immunized according to the above step, for example, a mouse, the spleen is removed 2 to 5 days after the final immunization to obtain a spleen cell suspension. In addition to spleen cells, lymph node cells in various parts of the body can be obtained and used for cell fusion. The spleen cell suspension thus obtained and 3. The myeloma cell line obtained according to the above step is placed in a cell medium such as minimum essential medium (MEM medium), DMEM medium, RPMI-1640 medium, etc., and a cell fusion agent such as polyethylene glycol is added. As the cell fusion agent, those known in various other fields can be used, and examples include inactivated Sendai virus (HVJ: Hemagglutinating virus of Japan). Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added. The molecular weight of such polyethylene glycol is preferably 1000 to 8000, and more preferably 1000 to 4000. The concentration of polyethylene glycol in the fusion medium is preferably 30 to 60%. If necessary, for example, dimethyl sulfoxide can be added in a small amount to promote fusion. The ratio of spleen cells (lymphocytes): myeloma cell line used for the fusion is, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 10: 1. The fusion reaction is performed for 1 to 10 minutes, and then a cell culture medium such as RPMI-1640 medium is added. The fusion reaction treatment can be performed multiple times. After the fusion reaction treatment, the cells are separated by centrifugation or the like and then transferred to a selection medium.
選択用培地としては、例えばヒポキサンチン、アミノプテリン及びチミジンを含む、FCS含有MEM培地又はRPMI-1640培地等の培地、いわゆるHAT培地が挙げられる。選択培地交換の方法は、一般的には培養プレートに分注した容量と当容量を翌日加え、その後1~3日ごとにHAT培地で半量ずつ交換して行うことができるが、適宜これに変更を加えて行うこともできる。また融合後8~16日目には、アミノプテリンを除いた、いわゆるHT培地で1~4日ごとに培地交換をすることができる。フィーダーとして、例えばマウス胸腺細胞を使用することができ、それが好ましい場合がある。ハイブリドーマの増殖のさかんな培養ウェルの培養上清を、例えば放射免疫分析(RIA)、酵素免疫分析(ELISA)、蛍光免疫分析(FIA)等の測定系、又は蛍光惹起細胞分離装置(FACS)等で、ストロムライシン1若しくはその断片ペプチド又は標識抗マウス抗体を用いて目的抗体を検出し、スクリーニングしたり分離したりする。次に、目的抗体を産生しているハイブリドーマをクローニングする。クローニングは、寒天培地中でコロニーをピックアップするか、あるいは限界希釈法により行うことができる。限界希釈法でクローニングすることがより好ましい。クローニングは複数回行うことが好ましい。 5). Selection and Monocloning of Hybridomas (Fused Cells) Examples of the selection medium include media such as FCS-containing MEM medium or RPMI-1640 medium containing hypoxanthine, aminopterin and thymidine, so-called HAT medium. The method of exchanging the selective medium can generally be performed by adding the volume dispensed to the culture plate and the same volume the next day, and then replacing the HAT medium half by half every 1 to 3 days. Can also be added. On the 8th to 16th day after the fusion, the medium can be changed every 1 to 4 days with a so-called HT medium excluding aminopterin. For example, mouse thymocytes can be used as a feeder, which may be preferred. The culture supernatant of the culture well in which the hybridoma is proliferated is measured using a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or a fluorescence-induced cell separation device (FACS) Then, the target antibody is detected using stromalysin 1 or a fragment peptide thereof or a labeled anti-mouse antibody, and screened or separated. Next, the hybridoma producing the target antibody is cloned. Cloning can be performed by picking colonies in an agar medium or by limiting dilution. It is more preferable to clone by the limiting dilution method. Cloning is preferably performed multiple times.
得られたハイブリドーマ株は、FCS含有MEM培地、RPMI-1640培地等の適当な増殖用培地中で培養し、その培地上清から所望のモノクローナル抗体を得ることが出来る。大量の抗体を得るためには、ハイブリドーマを腹水化することが挙げられる。この場合ミエローマ細胞由来の動物と同系の組織適合性動物の腹腔内に各ハイブリドーマを移植し、増殖させるか、例えばヌードマウス等に各ハイブリドーマを移植し、増殖させ、該動物の腹水中に産生されたモノクローナル抗体を回収して得ることが出来る。ハイブリドーマの移植に先立ち、プリスタン(2,6,10,14-テトラメチルペンタデカン)等の鉱物油を腹腔内投与した後、ハイブリドーマを増殖させ、腹水を採取すればよい。腹水液はそのまま、あるいは従来公知の方法、例えば硫酸アンモニウム沈殿法等の塩析、セファデックス等によるゲルろ過法、イオン交換クロマトグラフィー法、電気泳動法、透析、限外ろ過法、アフィニティークロマトグラフィー法、高速液体クロマトグラフィー法等により精製してモノクローナル抗体として用いることができる。好ましくは、モノクローナル抗体を含有する腹水は、硫安分画した後、DEAE-セファロースのような陰イオン交換ゲル及びプロテインAカラムのようなアフィニティーカラム等で処理し、精製分離処理できる。特に好ましくは抗原又は抗原断片(例えば合成ペプチド、組換え抗原タンパク質あるいはペプチド等、抗体が特異的に認識する部位)を固定化したアフィニティークロマトグラフィー、プロテインAを固定化したアフィニティークロマトグラフィー等が挙げられる。 6). Production of Monoclonal Antibody The obtained hybridoma strain can be cultured in an appropriate growth medium such as FCS-containing MEM medium, RPMI-1640 medium, etc., and a desired monoclonal antibody can be obtained from the medium supernatant. In order to obtain a large amount of antibody, ascites of the hybridoma can be mentioned. In this case, each hybridoma is transplanted into the abdominal cavity of a histocompatible animal syngeneic with the myeloma cell-derived animal and allowed to proliferate, or for example, each hybridoma is transplanted into a nude mouse and proliferated, and produced in the ascites of the animal. Monoclonal antibodies recovered can be obtained. Prior to hybridoma transplantation, mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered, and then the hybridoma is allowed to grow and ascites can be collected. Ascites fluid as it is or conventionally known methods, for example, salting out such as ammonium sulfate precipitation, gel filtration by Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, It can be purified and used as a monoclonal antibody by high performance liquid chromatography. Preferably, the ascites containing the monoclonal antibody can be purified and separated by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column after ammonium sulfate fractionation. Particularly preferred are affinity chromatography in which an antigen or antigen fragment (for example, a site specifically recognized by an antibody such as a synthetic peptide, recombinant antigen protein or peptide) is immobilized, or affinity chromatography in which protein A is immobilized. .
6.で得られたストロムライシン1に対するモノクローナル抗体の中から、ストロムライシン2及びストロムライシン3と交差反応しない抗体を選抜する。選抜は、イムノアッセイ、例えば競合型イムノアッセイ又は非競合型イムノアッセイで行うことができ、RIA、ELISA等を用いることができ、B-F分離を行ってもあるいは行わなくてもできる。好ましくは直接吸着法又はサンドイッチ法によるELISAが挙げられる。アッセイは直接法でも間接法でもよい。また間接法の変法、例えばPAP法(ペルオキシダーゼ アンチペルオキシダーゼ法)、ABC法(アビジン ビオチン コンプレックス法)、プロテインA法等を用いることもできる。 7). 5. Selection of antibodies that do not cross-react with stromalysin 2 and 3. The antibodies that do not cross-react with stromalysin 2 and stromalysin 3 are selected from the monoclonal antibodies against stromalysin 1 obtained in (1). Selection can be performed by immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and RIA, ELISA, etc. can be used, with or without BF separation. Preferably, ELISA by direct adsorption method or sandwich method is used. The assay may be direct or indirect. Indirect methods such as PAP method (peroxidase antiperoxidase method), ABC method (avidin biotin complex method), protein A method and the like can also be used.
上記のようにして得られた、ストロムライシン1と特異的に反応し、ストロムライシン2及びストロムライシン3と交差反応しないモノクローナル抗体は、ストロムライシン1の特定の反応部位と反応する。このような反応部位を同定し、この反応部位と反応するモノクローナル抗体を1.~6.で説明した方法で作製することにより、作製されたモノクローナル抗体の交差反応性を調節することが可能となる。反応部位は、以下のようにして同定することができる。酵素によってストロムライシン1をペプチドに分解する。得られたペプチドをクロマトグラフィー等従来公知の方法によって分画し、7.で得られたモノクローナル抗体と各画分のペプチドとを反応させれば、反応性の高い画分にストロムライシン1の反応部位であるペプチドが含まれている。該反応性の高い画分に含まれるペプチドは、従来公知の方法を用いてさらに精製することができ、公知のアミノ酸配列分析法により、反応部位のアミノ酸配列を決定することができる。 8). Identification of the reaction site of strome lysin 1 The monoclonal antibody obtained as described above, which specifically reacts with strome lysin 1 and does not cross-react with strome lysin 2 and strome lysin 3, React with. Such a reactive site is identified, and a monoclonal antibody that reacts with the reactive site is identified as follows. ~ 6. It is possible to regulate the cross-reactivity of the produced monoclonal antibody by the method described in the above. The reaction site can be identified as follows. Enzymatic degradation of stromolysin 1 into peptides. 6. The obtained peptide is fractionated by a conventionally known method such as chromatography, When the monoclonal antibody obtained in (1) is reacted with the peptides of each fraction, the highly reactive fraction contains the peptide that is the reaction site of stromlysin 1. The peptide contained in the highly reactive fraction can be further purified using a conventionally known method, and the amino acid sequence of the reaction site can be determined by a known amino acid sequence analysis method.
また、本発明は、配列番号1の一部のアミノ酸配列からなるペプチドと特異的に反応し、かつ、ストロムライシン1と特異的に反応するモノクローナル抗体であって、該アミノ酸配列は配列番号1の425番~436番の配列を含み、該ペプチドの長さが12~20アミノ酸残基である、モノクローナル抗体を提供する。配列番号1のアミノ酸配列はストロムライシン1タンパク質のアミノ酸配列である。配列番号1の425番~436番の配列からなるペプチドは交差反応性に関与すると考えられる。上記モノクローナル抗体は、6.で得られたストロムライシン1に対するモノクローナル抗体の中から、配列番号1の425番~436番の配列からなるペプチドと反応性を示すモノクローナル抗体を選抜することによって得ることができる。選抜は、7.で示したアッセイ法によってすることができ、例えば、7.に示した直接吸着法によるELISAにおいて、ストロムライシン1の代わりに配列番号1の425番~436番の配列からなるペプチドを抗原として用いればよい。上記モノクローナル抗体は、ストロムライシン2及びストロムライシン3と交差反応しないことが好ましい。ストロムライシン2及びストロムライシン3と交差反応しないモノクローナル抗体は、7.に示す方法により選抜することができる。 9. Selection of Monoclonal Antibody Reactive with Peptide Composed of Part of Stromlysin 1 In addition, the present invention specifically reacts with a peptide comprising a part of the amino acid sequence of SEQ ID NO: 1 and is specific to stromalysin 1 A monoclonal antibody which reacts manually, wherein the amino acid sequence comprises the sequence from 425 to 436 of SEQ ID NO: 1, and the length of the peptide is 12 to 20 amino acid residues is provided. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of strome lysin 1 protein. The peptide consisting of the sequence of Nos. 425 to 436 of SEQ ID No. 1 is considered to be involved in cross-reactivity. The monoclonal antibody described above is described in 6. It is possible to obtain a monoclonal antibody that reacts with the peptide comprising the sequence of Nos. 425 to 436 of SEQ ID No. 1 from the monoclonal antibodies against strome lysin 1 obtained in the above. The selection is 7. For example, 7. In the ELISA based on the direct adsorption method shown in (1), a peptide consisting of the sequence of Nos. 425 to 436 of SEQ ID No. 1 may be used as an antigen in place of stromolysin 1. It is preferable that the monoclonal antibody does not cross-react with stromalysin 2 and stromalysin 3. Monoclonal antibodies that do not cross-react with stromalysin 2 and stromalysin 3 are: It is possible to select by the method shown in.
また、本発明はストロムライシン1に特異的に反応するモノクローナル抗体であって、ストロムライシン1をリジルエンドペプチダーゼで分解することによりストロムライシン1に対する反応性を消失する、モノクローナル抗体を提供する。このようなモノクローナル抗体は、ストロムライシン1タンパク質の二次構造又は三次構造等の立体構造を認識するモノクローナル抗体である。「ストロムライシン1を分解することによりストロムライシン1に対する反応性を消失する」とは、ストロムライシン1分解後の反応性が分解前の反応性より低いことをいう。このようなモノクローナル抗体は、6.で得られたストロムライシン1に対するモノクローナル抗体の中から、以下のようにして選抜することによって得ることができる。酵素によってストロムライシン1をペプチドに分解する。ストロムライシン1と特異的に反応するモノクローナル抗体の、ストロムライシン1に対する反応性を、分解前と分解後で比較し、ストロムライシン1分解後の反応性が分解前の反応性より低いモノクローナル抗体を選抜する。分解後の反応性がより低いものほど、ストロムライシン1の一次構造によらず、ストロムライシン1の立体構造を認識してストロムライシン1と反応するモノクローナル抗体である。好ましくは、分解前のストロムライシン1に対する反応性を100%としたとき、分解後の反応性が10%以下である。反応性は7.に示す方法により直接吸着法又はサンドイッチ法のいずれかのELISAによって測定することができ、好ましくは、直接吸着法のELISAによって測定した分解後の反応性が分解前の反応性の10%以下である。より好ましくは、8.0%以下であり、さらに好ましくは7.3%以下である。 10. Selection of Monoclonal Antibody That Reacts with Degradation of Stromulysin 1 The present invention also relates to a monoclonal antibody that specifically reacts with stromalisin 1, which is obtained by degrading stromalisin 1 with lysyl endopeptidase. Monoclonal antibodies are provided that lose reactivity to. Such a monoclonal antibody is a monoclonal antibody that recognizes a three-dimensional structure such as a secondary structure or a tertiary structure of stromlysin 1 protein. “Degrading Stromlysin 1 to eliminate the reactivity to Stromlysin 1” means that the reactivity after the degradation of Stromlysin 1 is lower than the reactivity before the degradation. Such monoclonal antibodies are described in 6. It can be obtained by selecting from the monoclonal antibodies against stromalisin 1 obtained in the following manner. Enzymatic degradation of stromolysin 1 into peptides. Compare the reactivity of the monoclonal antibody that specifically reacts with strome lysin 1 to strome lysin 1 before and after degradation, and select monoclonal antibodies whose reactivity after degradation of strome lysin 1 is lower than that before degradation. To do. The lower the reactivity after degradation, the more the monoclonal antibody reacts with strome lysin 1 by recognizing the three-dimensional structure of strome lysin 1 regardless of the primary structure of strome lysin 1. Preferably, when the reactivity with respect to strome lysin 1 before decomposition is 100%, the reactivity after decomposition is 10% or less. Reactivity is 7. Can be measured by ELISA of either direct adsorption method or sandwich method, preferably, the reactivity after decomposition measured by ELISA of direct adsorption method is 10% or less of the reactivity before decomposition. . More preferably, it is 8.0% or less, More preferably, it is 7.3% or less.
以下のように、Obataら,Clin.Chim.Acta,211,59-72,1992に記載の方法に従って、正常ヒト皮膚線維芽細胞NB1RGB(RCB222)培養上清より、ストロムライシン1を精製した。NB1RGB細胞を、10%ウシ胎児血清、14mM HEPES、1.4g/l NaHCO3含有RITC80-7基礎培地(pH7.2)中、5%CO2存在下、37℃でコンフルエントまで培養した。細胞を100mlのRITC80-7基礎培地で洗浄後、2g/lラクトアルブミン水解物及び10000U/lインターロイキン1α含有無血清RITC80-7基礎培地300ml中で培養し、その上清を回収した。 (Example 1 Production of antigen and standard)
As described in Obata et al., Clin. Chim. Stromlysin 1 was purified from the culture supernatant of normal human dermal fibroblast NB1RGB (RCB222) according to the method described in Acta, 211, 59-72, 1992. NB1RGB cells were cultured to confluence at 37 ° C. in the presence of 5% CO 2 in RITC80-7 basal medium (pH 7.2) containing 10% fetal bovine serum, 14 mM HEPES, 1.4 g / l NaHCO 3. The cells were washed with 100 ml of RITC80-7 basal medium, cultured in 300 ml of serum-free RITC80-7 basal medium containing 2 g / l lactalbumin hydrolyzate and 10000 U / l interleukin 1α, and the supernatant was collected.
以下のように、Obataら,Clin.Chim.Acta,211,59-72,1992に記載の方法に従って、実施例1で精製したストロムライシン1を抗原としてモノクローナル抗体を調製した。 (Example 2 Preparation of monoclonal antibody)
As described in Obata et al., Clin. Chim. In accordance with the method described in Acta, 211, 59-72, 1992, a monoclonal antibody was prepared using stromalisin 1 purified in Example 1 as an antigen.
実施例1に記載の方法により調製したストロムライシン1を44.6μg、等重量のフロイント完全アジュバントと共に6週齢BALB/c雌マウス2匹に腹腔内投与し初回免疫とした。その後19日目に0.4MNaCl、10mM CaCl2及び0.5g/l Brij35含有50mMトリス塩酸緩衝液(pH7.5)に溶解した40.4μgのストロムライシン1で追加免疫した。最終免疫として55日目に43.2μgのストロムライシン1で免疫した。最終免疫から3日後にマウス脾臓を取り出し脾臓細胞を調製した。 (A) Immunization of animals with stromalysin 1 Stromulysin 1 prepared by the method described in Example 1 was intraperitoneally administered to 2 6-week-old BALB / c female mice together with 44.6 μg of an equal weight of Freund's complete adjuvant. First immunization was performed. On the 19th day, booster immunization was performed with 40.4 μg of stromalisin 1 dissolved in 50 mM Tris-HCl buffer (pH 7.5) containing 0.4 M NaCl, 10 mM CaCl 2 and 0.5 g / l Brij35. As the final immunization, immunization was performed on day 55 with 43.2 μg of stromalisin 1. Three days after the final immunization, the mouse spleen was removed and spleen cells were prepared.
以下の材料及び方法を用いた。
RPMI 1640培地:RPMI 1640(JRH Biosciences製)に24mM重炭酸ナトリウム、1mMピルビン酸ナトリウム、50U/mlペニシリンGカリウム、50μg/ml硫酸ストレプトマイシン及び100μg/ml硫酸アミカシンを加え、ドライアイスでpHを7.2にし、0.22μmミリポアフィルターで除菌ろ過した。
NS-1培地:上記RPMI-1640培地に除菌ろ過したFBS(JRHBiosciences製)を15%(v/v)の濃度に加える。
PEG4000溶液:RPMI-1640培地にポリエチレングリコール4000(PEG4000,Merck and CO.,Inc.製)50%(w/w)無血清溶液を調製した。 (B) Cell fusion of antibody-producing cells and myeloma cells The following materials and methods were used.
RPMI 1640 medium: RPMI 1640 (manufactured by JRH Biosciences) was added with 24 mM sodium bicarbonate, 1 mM sodium pyruvate, 50 U / ml penicillin G potassium, 50 μg / ml streptomycin sulfate and 100 μg / ml amikacin sulfate, and the pH was adjusted to 7. And sterilized by filtration through a 0.22 μm Millipore filter.
NS-1 medium: FBS (manufactured by JRH Biosciences) sterilized and filtered in the above RPMI-1640 medium is added to a concentration of 15% (v / v).
PEG 4000 solution: A 50% (w / w) serum-free solution of polyethylene glycol 4000 (PEG 4000, manufactured by Merck and CO., Inc.) was prepared in RPMI-1640 medium.
使用する培地は以下のとおりである。
HAT培地:前記実施例2(b)で述べたNS-1培地にさらにヒポキサンチン(100μM)、アミノプテリン(0.4μM)及びチミジン(16μM)を加えた。
HT培地:アミノプテリンを除去した以外は上記HAT培地と同一組成のものである。 (C) Selective growth of hybridoma using selective medium The following media are used.
HAT medium: Hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM) were further added to the NS-1 medium described in Example 2 (b).
HT medium: The same composition as the HAT medium except that aminopterin was removed.
以下のようにして、直接吸着法によるELISAを行った。96穴マイクロウェルプレート(Nunc社)に100ng/ウェルになるように0.1Mリン酸緩衝液(pH7.0)で希釈したストロムライシン1を抗原としてコートした。4℃で一晩以上放置後、コーティング液を吸引し、0.05%Tween20含有PBS(pH7.2)でマイクロウェルプレートを洗浄した。確認したいハイブリドーマの培養上清(一次抗体)を100μl/ウェル加えて、室温で約1時間インキュベート(静置)した。洗浄後、二次抗体としてヤギ抗マウスIg(G+M+A)immunoglobulin-ペルオキシダーゼ(POD)(CAPPEL社)をブロッキング液で1/10000に希釈したものを100μl/ウェル加え、室温で約1時間インキュベート(静置)した。洗浄後、基質である過酸化水素とテトラメチルベンジジン(TMB)を100μl/ウェル加え、室温で約20分反応させ、1M硫酸を100μl/ウェル加えて反応を停止させ、マイクロプレートリーダー(TOSOH、MPR A4)にて測定波長450nmでの吸光度を測定した。ストロムライシン1との反応が陽性を示した16ウェルについて、細胞の凍結及び復元を実施し、限界希釈法によるクローニングを実施した。 (D) Selection of hybridoma producing anti-strome lysin 1 antibody by ELISA immediately after cell fusion ELISA was performed by the direct adsorption method as follows. A 96-well microwell plate (Nunc) was coated with stromalisin 1 diluted with 0.1 M phosphate buffer (pH 7.0) as an antigen to 100 ng / well. After standing overnight at 4 ° C., the coating solution was aspirated and the microwell plate was washed with PBS containing 0.05% Tween 20 (pH 7.2). The culture supernatant (primary antibody) of the hybridoma to be confirmed was added at 100 μl / well and incubated at room temperature for about 1 hour (standing). After washing, 100 μl / well of goat anti-mouse Ig (G + M + A) immunoglobulin-peroxidase (POD) (CAPPEL) diluted to 1 / 10,000 as a secondary antibody with a blocking solution is added and incubated at room temperature for about 1 hour )did. After washing, hydrogen peroxide and tetramethylbenzidine (TMB) as substrates were added at 100 μl / well, reacted at room temperature for about 20 minutes, 1 M sulfuric acid was added at 100 μl / well to stop the reaction, and the microplate reader (TOSOH, MPR In A4), the absorbance at a measurement wavelength of 450 nm was measured. For 16 wells that showed a positive reaction with stromlysin 1, cells were frozen and reconstituted, and cloned by limiting dilution.
以下の材料及び方法を用いた。
RPMI 1640培地-(2):RPMI1640 MEDIUM(SIGMA R8758)500ml(1本)に、FOETAL BOVINE SERUM (SIGMA F9423)50ml、PENICILLIN-STREPTOMYCIN SOLUTION(SIGMA P0781)2.5mlを添加した。
BriClone含有RPMI 1640培地;RPMI 1640培地-(2)にBriClone(Hybridoma cloning Medium)(NICB社製)を5%になるように添加した。 (E) Cloning The following materials and methods were used.
RPMI 1640 medium- (2): RPMI 1640 MEDIAUM (SIGMA R8758) 500 ml (1 bottle), FOETAL BOVINE SERUM (SIGMA F9423) 50 ml, PENICILLIN-STREPTOMYCIN SOLUTION (SIGMA P0781) 2.5 ml.
BriClone-containing RPMI 1640 medium; RPMI 1640 medium—BriClone (Hybridoma cloning Medium) (manufactured by NICB) was added to 5%.
以下のようにして、直接吸着法によるELISAを行った。直接吸着法によるELISAでは、96穴マイクロウェルプレート(Nunc社)に50ng/ウェルになるように0.1Mリン酸緩衝液(pH7.0)で希釈したストロムライシン1を抗原としてコートした。4℃で一晩以上放置後、コーティング液を吸引し、200μl/ウェルのブロッキング液(1%BSA及び0.1M NaCl含有0.01Mリン酸緩衝液、pH7.0)を用い、室温で1時間以上ブロッキングを行った。0.1%Tween20及び0.1M NaCl含有0.01Mリン酸緩衝液(pH7.0)でマイクロウェルプレートを洗浄後、確認したいハイブリドーマの培養上清(一次抗体)を100μl/ウェル加えて、室温で約1時間インキュベート(静置)した。洗浄後、二次抗体としてヤギ抗マウスIg(G+M+A)immunoglobulin-ペルオキシダーゼ(POD)(CAPPEL社)をブロッキング液で1/3000に希釈したものを100μl/ウェル加え、室温で約1時間インキュベート(静置)した。洗浄後、TMB One Component HRP Microwell Substrate (BioFX社, Product# TMBW-1000-01)を100μl/ウェル加え、室温で約5~10分反応させた。1M硫酸を100μl/ウェル加えて反応を停止させ、マイクロプレートリーダー(TECAN SPECTRA)にて測定波長450nmでの吸光度を測定した。 (F) Selection of anti-strome lysin 1 antibody-producing hybridoma by ELISA during cloning ELISA by direct adsorption was performed as follows. In the ELISA by the direct adsorption method, stromal lysin 1 diluted with 0.1 M phosphate buffer (pH 7.0) was coated as an antigen to a 96-well microwell plate (Nunc) at 50 ng / well. After standing overnight at 4 ° C., the coating solution is aspirated and 200 μl / well blocking solution (0.01 M phosphate buffer containing 1% BSA and 0.1 M NaCl, pH 7.0) is used for 1 hour at room temperature. Blocking was performed as described above. After washing the microwell plate with 0.01 M phosphate buffer (pH 7.0) containing 0.1% Tween 20 and 0.1 M NaCl, 100 μl / well of the culture supernatant of the hybridoma to be confirmed (primary antibody) was added to room temperature. And incubated for about 1 hour. After washing, 100 μl / well of goat anti-mouse Ig (G + M + A) immunoglobulin-peroxidase (POD) (CAPPEL) diluted to 1/3000 with a blocking solution was added as a secondary antibody, and incubated at room temperature for about 1 hour. )did. After washing, TMB One Component HRP Microwell Substrate (BioFX, Product # TMBW-1000-01) was added at 100 μl / well and allowed to react at room temperature for about 5 to 10 minutes. The reaction was stopped by adding 100 μl / well of 1 M sulfuric acid, and the absorbance at a measurement wavelength of 450 nm was measured with a microplate reader (TECAN SPECTRA).
実施例2(d)記載のELISAに従って、以下のようにモノクローナル抗体のサブクラスを同定した。実施例2(d)に記載の、ストロムライシン1をコートした96穴マイクロウェルのブロッキングまでの操作を行った。0.1%Tween20及び0.1M NaCl含有0.01Mリン酸緩衝液(pH7.0)で洗浄後、各モノクローナル抗体産生ハイブリドーマの培養上清を100μl/ウェル加え、室温で約1時間インキュベート(静置)した。洗浄後、アイソタイプ特異的ウサギ抗マウスIg抗体(Mouse MonoAb-ID Kit、Zymed Laboratories社)を50μl/ウェル加え、室温で約1時間インキュベート(静置)した。更に洗浄後、西洋ワサビペルオキシダーゼ(HRP)標識ヤギ抗ウサギIgG(H+L)抗体をブロッキング液で1/50に希釈した溶液を50μl/ウェル加え、室温で約1時間インキュベート(静置)した。洗浄後、TMB One Component HRP Microwell Substrate (BioFX社、Product#TMBW-1000-01)を100μl/ウェル加えて発色させ、1M硫酸を100μl/ウェル加えて反応を停止させ、測定波長450nmでの吸光度を測定した。その結果、表1に示すように、得られたモノクローナル抗体は、γ1/κが4個、γ2b/κが3個、μ/κが4個、α/κが1個であった。 (Example 3) Identification of subclass of monoclonal antibody
According to the ELISA described in Example 2 (d), the subclass of the monoclonal antibody was identified as follows. The operation up to blocking of 96-well microwells coated with stromelysin 1 as described in Example 2 (d) was performed. After washing with 0.01 M phosphate buffer (pH 7.0) containing 0.1% Tween 20 and 0.1 M NaCl, 100 μl / well of the culture supernatant of each monoclonal antibody-producing hybridoma is added, and incubated at room temperature for about 1 hour (static ) After washing, an isotype-specific rabbit anti-mouse Ig antibody (Mouse MonoAb-ID Kit, Zymed Laboratories) was added at 50 μl / well and incubated at room temperature for about 1 hour. Further, after washing, a solution obtained by diluting horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG (H + L) antibody to 1/50 with a blocking solution was added and incubated at room temperature for about 1 hour. After washing, TMB One Component HRP Microwell Substrate (BioFX, Product # TMBW-1000-01) was added at 100 μl / well for color development, 1M sulfuric acid was added at 100 μl / well to stop the reaction, and the absorbance at a measurement wavelength of 450 nm was measured. It was measured. As a result, as shown in Table 1, the obtained monoclonal antibody had 4 γ1 / κ, 3 γ2b / κ, 4 μ / κ, and 1 α / κ.
実施例2で得られた12種類のモノクローナル抗体産生ハイブリドーマの培養上清に対し、抗原としてストロムライシン1の代わりにストロムライシン2又はストロムライシン3を用いて、実施例2(d)の方法に従ってELISAを行った。ストロムライシン1の吸光値を100%としたときの、ストロムライシン2及び3の吸光値の割合を表2に示す。296-29C1、296-12H8、296-7B10の3クローンの抗体は、同培養上清のストロムライシン1に対する反応性を100%とすると、ストロムライシン2に対しての反応性が1%であり、ストロムライシン2と交差反応しなかった。それ以外のクローンの抗体は、ストロムライシン2に対しての反応性が10%~337%であり、ストロムライシン2との交差反応性が認められた。また、ストロムライシン3に対する反応性は0%~6%であり、交差反応しないものがほとんどであった。 Example 4 Reactivity with Stromlysin 2 and 3 Using Culture Supernatant
The culture supernatant of 12 types of monoclonal antibody-producing hybridomas obtained in Example 2 was subjected to ELISA according to the method of Example 2 (d), using stromalisin 2 or stromalicin 3 instead of stromalicin 1 as an antigen. Went. Table 2 shows the ratio of the absorbance values of strome lysin 2 and 3 when the absorbance value of strome lysin 1 is 100%. The antibodies of 3 clones of 296-29C1, 296-12H8 and 296-7B10 have a reactivity to strome lysin 2 of 1% when the reactivity of the culture supernatant to strome lysin 1 is 100%. It did not cross-react with stromalysin 2. The antibodies of other clones were 10% to 337% reactive with stromalysin 2, and cross-reactivity with strome lysin 2 was observed. Moreover, the reactivity with respect to strome lysin 3 was 0% to 6%, and most of them did not cross-react.
ストロムライシン1及び2の各抗原をc-PAGEL(アトー社)でそれぞれ電気泳動に供した後、Sequi-Blot PVDF Membrane for Protein Sequencing(厚さ0.2μm)(Bio Rad社)にトランスファーを行った。SDSを除去するために0.1%Tween20及び0.1M NaCl含有0.01Mリン酸緩衝液(pH7.0)で、トランスファーした膜を洗浄した。さらに、1%BSA及び0.1M NaCl含有0.01Mリン酸緩衝液(pH7.0)でブロッキングを行った。この膜を、得られた12種類のモノクローナル抗体産生ハイブリドーマの培養上清中で、4~8℃で一晩静置した。膜を洗浄後、2次抗体反応として、ヤギ抗マウスIg(G+M+A)immunoglobulin-POD(CAPPEL社)をブロッキング液で1/3000に希釈した溶液中で、室温で約2時間振とうした。膜を洗浄後、基質である過酸化水素とジアミノベンジジン(DAB)を加えて発色を行った。結果を表2に示す。296-29C1、296-12H8、296-7B10の3クローンの抗体は、ストロムライシン1との反応が認められたが、ストロムライシン2との反応が認められなかった。296-21C4、296-21F3、296-21E4、296-33F3、296-33E4、296-23A8、296-24C8、296-30C12、296-31A4の9クローンの抗体は、ストロムライシン1との反応が認められたが、ストロムライシン2とも反応が認められた。 (Example 5) Immunostaining
Each antigen of Stromlysin 1 and 2 was subjected to electrophoresis with c-PAGE (Ato), and then transferred to Sequi-Blot PVDF Membrane for Protein Sequencing (thickness 0.2 μm) (Bio Rad). . In order to remove SDS, the transferred membrane was washed with 0.01 M phosphate buffer (pH 7.0) containing 0.1% Tween 20 and 0.1 M NaCl. Further, blocking was performed with 0.01 M phosphate buffer (pH 7.0) containing 1% BSA and 0.1 M NaCl. This membrane was allowed to stand at 4-8 ° C. overnight in the culture supernatant of the 12 types of monoclonal antibody-producing hybridomas obtained. After the membrane was washed, as a secondary antibody reaction, it was shaken at room temperature for about 2 hours in a solution obtained by diluting goat anti-mouse Ig (G + M + A) immunoglobulin-POD (CAPPEL) to 1/3000 with a blocking solution. After the membrane was washed, coloring was performed by adding hydrogen peroxide and diaminobenzidine (DAB) as substrates. The results are shown in Table 2. The antibodies of 3 clones of 296-29C1, 296-12H8, and 296-7B10 were found to react with stromalysin 1, but not with stromelysin 2. Nine clones of 296-21C4, 296-21F3, 296-21E4, 296-33F3, 296-33E4, 296-23A8, 296-24C8, 296-30C12, 296-31A4 were found to react with stromalysin 1 However, a reaction was also observed with stromelysin 2.
表1に示すクローンのうち、296-21C4、296-29C1、296-12H8、296-7B10、296-33F3及び296-23A8を選択し、抗体精製を行った。296-21C4、296-29C1、296-12H8、296-7B10の4種類については、細胞を増殖させ、その細胞をBalb/cマウス5匹の腹腔内に投与し、腹水を採取した。採取した腹水は、硫酸アンモニウム(硫安)分画を行い、DEAEイオン交換クロマトグラフィーにより抗体を精製した。296-33F3については、細胞を増殖させ、その細胞をBalb/cマウス5匹の腹腔内に投与し、腹水を採取した。採取した腹水を、硫安分画により抗体を精製した。296-23A8については、細胞を増殖させ、増殖後、培地を無血清培地(E-RDF培地)に交換し、約1lの培養上清を回収した。回収した培養上清より、硫安分画により抗体を精製した。 (Example 6 Purification of monoclonal antibody)
Of the clones shown in Table 1, 296-21C4, 296-29C1, 296-12H8, 296-7B10, 296-33F3 and 296-23A8 were selected, and antibody purification was performed. For the four types of 296-21C4, 296-29C1, 296-12H8, and 296-7B10, cells were proliferated, the cells were administered into the peritoneal cavity of five Balb / c mice, and ascites was collected. The collected ascites was subjected to ammonium sulfate (ammonium sulfate) fractionation, and the antibody was purified by DEAE ion exchange chromatography. For 296-33F3, cells were expanded, the cells were administered into the peritoneal cavity of five Balb / c mice, and ascites was collected. The antibody was purified from the collected ascites by ammonium sulfate fractionation. For 296-23A8, the cells were grown, and after growth, the medium was replaced with a serum-free medium (E-RDF medium), and about 1 l of the culture supernatant was collected. From the collected culture supernatant, the antibody was purified by ammonium sulfate fractionation.
実施例2(d)の方法に従って、ELISAにより、実施例6で精製した抗体のストロムライシン2及び3に対する交差反応性を調べた。一次抗体として精製抗体を100ng/ウェル用い、測定波長450nmでの吸光度を測定した。A450(一次抗体が0ng/mlのときとの差)を表3に示す。 Example 7 Reactivity with Stromlysin 2 and 3 Using Purified Antibody
According to the method of Example 2 (d), the cross-reactivity of the antibody purified in Example 6 to stromalysin 2 and 3 was examined by ELISA. Absorbance at a measurement wavelength of 450 nm was measured using a purified antibody as a primary antibody at 100 ng / well. A450 (difference when the primary antibody is 0 ng / ml) is shown in Table 3.
サンドイッチ法のELISAによる免疫学的測定により、ストロムライシン1の定量を行った。96穴マイクロウェルプレート(Nunc社)に、実施例6で得られた精製抗体100μg/ml含有0.1Mリン酸緩衝液(pH7.0)を100μl/ウェル加え、4℃で16時間静置した。抗体溶液を除き、1%BSA、0.1M NaCl及び10mM EDTA含有0.01M リン酸緩衝液(pH7.0)を300μl/ウェル加え、4℃で一晩以上ブロッキングを行い、抗体結合ウェルとした。使用時にウェルを0.1%Tween20及び0.1M NaCl含有0.01Mリン酸緩衝液(pH7.0)(以下、「洗浄液」という)で洗浄した。 Example 8 Immunological Measurement of Stromlysin 1
Stromlysin 1 was quantified by immunoassay by sandwich ELISA. 100 μl / well of 0.1 M phosphate buffer (pH 7.0) containing 100 μg / ml of the purified antibody obtained in Example 6 was added to a 96-well microwell plate (Nunc) and allowed to stand at 4 ° C. for 16 hours. . The antibody solution was removed, 300 μl / well of 0.01 M phosphate buffer (pH 7.0) containing 1% BSA, 0.1 M NaCl, and 10 mM EDTA was added, and blocking was performed overnight at 4 ° C. to form antibody-bound wells. . At the time of use, the wells were washed with 0.01 M phosphate buffer (pH 7.0) containing 0.1% Tween 20 and 0.1 M NaCl (hereinafter referred to as “washing solution”).
以下のようにして、実施例6で得られた精製モノクローナル抗体について、ストロムライシン1分子上の反応部位の解析を行った。 (Example 9 Analysis of reaction site on stromalicin 1)
The reaction site on one stromlysin molecule was analyzed for the purified monoclonal antibody obtained in Example 6 as follows.
エッペンドルフチューブに、0.1M Tris-HCl(pH8.5)34μl及び51μg(17μl)のストロムライシン1を加えた。さらにリジルエンドペプチダーゼ26.1mU(17μl)を加え、反応を開始した。反応は37℃、15分間行なった。反応後、反応液(分解液)をSDS-PAGEに供したところ、リジルエンドペプチダーゼのバンドは確認されたが、ストロムライシン1のバンドは認められず、充分分解されたことが示された。 (A) Degradation of Stromlysin 1 To an Eppendorf tube, 34 μl of 0.1 M Tris-HCl (pH 8.5) and 51 μg (17 μl) of stromelysin 1 were added. Furthermore, 26.1 mU (17 μl) of lysyl endopeptidase was added to start the reaction. The reaction was carried out at 37 ° C. for 15 minutes. After the reaction, when the reaction solution (decomposition solution) was subjected to SDS-PAGE, a lysyl endopeptidase band was confirmed, but a stromlysin 1 band was not observed, indicating that it was sufficiently decomposed.
リジルエンドペプチダーゼによる分解後のストロムライシン1に対する抗体の反応性を直接吸着法のELISAにより調べた。(a)で得られたストロムライシン1の分解液に932μlの0.1%トリフルオロ酢酸(TFA)を加えた液の一部を0.1Mリン酸緩衝液(pH7.0)で1/100(ストロムライシン1濃度に換算して510ng/ml)に希釈後、96穴マイクロウェルに100μlを加え、4℃で一晩静置してコーティングした。コントロールとして、51μg/mlの分解前のストロムライシン1を同様に510ng/mlになるように希釈してコーティングした。コーティング後、1%BSA及び0.1M NaCl含有0.01Mリン酸緩衝液(pH7.0)(アッセイbuffer)300μlを加え、一晩静置した。使用時にウェルを0.01%Tween20及び0.1M NaCl含有0.01Mリン酸緩衝液(pH7.0)(洗浄液)で洗浄した。 (B) Reactivity of Monoclonal Antibody to Stromlysin 1 after Degradation The reactivity of the antibody to stromlysin 1 after degradation by lysyl endopeptidase was examined by direct adsorption ELISA. A portion of a solution obtained by adding 932 μl of 0.1% trifluoroacetic acid (TFA) to the stromalicin 1 digestion solution obtained in (a) was 1/100 with 0.1 M phosphate buffer (pH 7.0). After dilution to (Stronglysin 1 concentration, 510 ng / ml), 100 μl was added to a 96-well microwell and allowed to stand overnight at 4 ° C. for coating. As a control, 51 μg / ml of stromelysin 1 before degradation was similarly diluted to 510 ng / ml and coated. After coating, 300 μl of 0.01 M phosphate buffer (pH 7.0) (assay buffer) containing 1% BSA and 0.1 M NaCl was added and allowed to stand overnight. At the time of use, the wells were washed with 0.01 M phosphate buffer (pH 7.0) (washing solution) containing 0.01% Tween20 and 0.1 M NaCl.
結果を表8に示す。 As a primary antibody, the monoclonal antibody shown in Table 8 was diluted with an assay buffer so as to be 10 μg / ml, and 100 μl / well each was added to wells to which strome lysin 1 after degradation or strome lysin 1 before degradation was bound. Incubated for a period of time (standing). After washing the wells with a washing solution, 100 μl / well of goat anti-mouse Ig (G + M + A) immunoglobulin-POD (CAPPEL, Cat # 55556) diluted to 1/1000 with an assay buffer was added as a secondary antibody, and 40 ° C. at 25 ° C. Incubated for minutes (still). After washing the well with a washing solution, 100 μl / well of color former was added, reacted at 25 ° C. for 15 minutes, the reaction was stopped by adding 100 μl / well of 1M sulfuric acid, and the measurement wavelength was 450 nm with a microplate reader (TECAN SPECTRA). Absorbance was measured. Table 8 shows A450 (difference when the primary antibody is 0 ng / ml) and reactivity (%) after degradation with respect to reactivity before degradation.
The results are shown in Table 8.
上記反応液に932μlの0.1%TFAを加え、950μlをFPLC(Fast protein liquid chromatography、AKTA explorer、GEヘルスケア社)に供するサンプルとした。カラムはC18Sephasil peptideを用い、溶出は0.1%TFA(A液)と、80%アセトニトリルを含む0.1%TFA(B液)とを用いて、20カラム容量でB液が100%となるようにした。サンプルアプライ後、6mlのA液でカラムを洗浄(1ml/画分)し、その後A液とB液のグラジエント溶液約33.2mlで溶出(0.5ml/画分)した。フラクションはサンプルアプライ後からA1とし、洗浄、溶出画分を連続して採取した。画分はA1~A12、B1~B12というように、12画分ずつ番号付けした。 (C) Fractionation of Stromlysin 1 Peptide by FPLC 932 μl of 0.1% TFA was added to the above reaction solution, and 950 μl was used as a sample to be subjected to FPLC (Fast protein liquid chromatography, AKTA explorer, GE Healthcare). The column uses C18 Sephasil peptide, and elution uses 0.1% TFA (liquid A) and 0.1% TFA (liquid B) containing 80% acetonitrile, and the liquid B becomes 100% in 20 column volumes. I did it. After sample application, the column was washed with 6 ml of liquid A (1 ml / fraction), and then eluted with about 33.2 ml of a gradient solution of liquid A and liquid B (0.5 ml / fraction). The fraction was A1 after sample application, and the washed and eluted fractions were collected in succession. The fractions were numbered by 12 fractions such as A1 to A12 and B1 to B12.
各FPLC画分に対するモノクローナル抗体の反応性を直接吸着法のELISAにより調べた。96穴マイクロウェルプレート(Nunc社)に各FPLC画分の10倍希釈溶液100μl(0.1Mリン酸緩衝液(pH7.0)で希釈)を加え、4℃で一晩静置した。各ウェルの液を除去し、アッセイbufferを300μl/ウェル加え、4℃で一晩ブロッキングを行いペプチド結合ウェルとした。使用時にウェルを洗浄液で洗浄した。次に、296-29C1及び296-7B10を10μg/mlになるようアッセイbufferで希釈し、ペプチド結合ウェルに各100μl/well加えて、25℃で1時間インキュベート(静置)した。洗浄液でウェルを洗浄後、二次抗体としてヤギ抗マウスIg(G+M+A)immunoglobulin-POD(CAPPEL社、Cat#55556)をアッセイbufferで1/3000に希釈したものを100μl/ウェル加え、25℃で40分間インキュベート(静置)した。洗浄後、発色剤を100μl/ウェル加え、25℃で15分間反応し、1M硫酸を100μl/ウェル加えて反応を停止させ、マイクロプレートリーダー(TECAN SPECTRA)にて測定波長450nmでの吸光度を測定した。各画分の吸光度のグラフを図1に示す。296-29C1及び296-7B10は、FPLC画分C10及びD9(以下それぞれ「Fr.C10」及び「Fr.D9」という)と反応性を示した。2つの画分に反応のピークが認められることから、リジルエンドペプチダーゼの部分分解により、2つの画分に同一の配列が含まれている可能性がある。 (D) Reactivity of monoclonal antibody to FPLC fraction The reactivity of the monoclonal antibody to each FPLC fraction was examined by direct adsorption ELISA. To a 96-well microwell plate (Nunc), 100 μl of a 10-fold diluted solution of each FPLC fraction (diluted with 0.1 M phosphate buffer (pH 7.0)) was added and allowed to stand at 4 ° C. overnight. The solution in each well was removed, 300 μl / well of assay buffer was added, and blocking was performed overnight at 4 ° C. to obtain peptide-binding wells. The wells were washed with a washing solution at the time of use. Next, 296-29C1 and 296-7B10 were diluted with an assay buffer so as to be 10 μg / ml, 100 μl / well of each was added to the peptide-binding well, and incubated at 25 ° C. for 1 hour (stationary). After washing the wells with a washing solution, 100 μl / well of goat anti-mouse Ig (G + M + A) immunoglobulin-POD (CAPPEL, Cat # 55556) diluted to 1/3000 with assay buffer was added as a secondary antibody, and 40 ° C. was added at 25 ° C. Incubated for minutes (still). After washing, 100 μl / well of color former was added, reacted at 25 ° C. for 15 minutes, the reaction was stopped by adding 100 μl / well of 1M sulfuric acid, and the absorbance at a measurement wavelength of 450 nm was measured with a microplate reader (TECAN SPECTRA). . A graph of the absorbance of each fraction is shown in FIG. 296-29C1 and 296-7B10 showed reactivity with FPLC fractions C10 and D9 (hereinafter referred to as “Fr.C10” and “Fr.D9”, respectively). Since reaction peaks are observed in the two fractions, the two sequences may contain the same sequence due to partial degradation of lysyl endopeptidase.
296-29C1及び296-7B10が反応したFPLC画分のうち、Fr.D9は測定波長230nmの吸光度が高い第1及び第2のピークの後に続く画分であり、ペプチドの分離が充分ではないと考えられたため、比較的シャープなピークであったFr.C10のN末端アミノ酸配列分析を行った。N末端アミノ酸配列分析は、アプロサイエンスへ外注し、Edman分解法により行なわれた。Fr.C10を50μl使用し、N末端5残基の測定をしたが、各サイクルで複数のアミノ酸が検出され、配列の確定には至らなかった。そこで、検出されたいくつかアミノ酸の中から、検出量および配列等からそのペプチド配列を推定した。その結果、QIAEDである配列が候補に挙げられた。各アミノ酸の検出量は約9pmolであった。ストロムライシン1の全アミノ酸配列(配列番号1)から候補ペプチド配列を照合し、Fr.C10に含まれるペプチドを推定した。ペプチドは、QIAEDFPGIDSK(配列番号1の425番~436番)であることが推定された。ストロムライシン2の全アミノ酸配列(配列番号2)の中で上記ペプチド配列に相応するアミノ酸配列は、LIADDFPGVEPK(配列番号2の424番~435番)であり、12個のアミノ酸中7個(58%)のアミノ酸が一致していた。 (E) Fr. Identification of N-terminal amino acid sequence of C10 Among FPLC fractions reacted with 296-29C1 and 296-7B10, Fr. D9 is a fraction following the first and second peaks having a high absorbance at a measurement wavelength of 230 nm, and it was considered that the peptide was not sufficiently separated. C10 N-terminal amino acid sequence analysis was performed. N-terminal amino acid sequence analysis was outsourced to AproScience and performed by Edman degradation. Fr. Using 50 μl of C10 and measuring the N-terminal 5 residues, multiple amino acids were detected in each cycle, and the sequence was not finalized. Therefore, the peptide sequence was deduced from the detected amount and sequence from among the detected amino acids. As a result, a sequence that was QIAED was listed as a candidate. The detected amount of each amino acid was about 9 pmol. The candidate peptide sequence is verified from the entire amino acid sequence of strome lysin 1 (SEQ ID NO: 1), Fr. Peptides contained in C10 were estimated. The peptide was presumed to be QIAEDFPPGIDSK (sequence numbers 425 to 436). The amino acid sequence corresponding to the above peptide sequence among all the amino acid sequences of strome lysin 2 (SEQ ID NO: 2) is LIADDDFPGVEPK (No. 424 to 435 of SEQ ID NO: 2), 7 of 12 amino acids (58% ) Amino acids matched.
ペプチドQIAEDFPGIDSKを合成し、モノクローナル抗体との反応性を調べた。ペプチドの合成はInvitrogen社に外注し、95.15%の純度で8mgのペプチドを得た。測定分子量は1318.40であり、理論分子量1319.45とほぼ一致していた。このペプチドを0.1Mリン酸緩衝液(pH7.0)で500ng/mlになるよう希釈後、96穴マイクロウェルに100μlを加え、4℃で一晩静置してコーティングした。洗浄液で洗浄後、表9に示す各モノクローナル抗体をアッセイbufferで6.3μg/mlに希釈し、ペプチドをコーティングしたウェルに100μl/ウェルずつ加えて、25℃で1時間インキュベート(静置)した。後の操作は実施例9(b)に記載の方法と同様に行った。本実施例で得られたペプチドを抗原としたときのA450(一次抗体が0ng/mlのときとの差)を表9に示す。 (F) Reactivity of Stromlysin 1 Peptide with Monoclonal Antibody Peptide QIAEDFPPGIDSK was synthesized and the reactivity with the monoclonal antibody was examined. The peptide synthesis was outsourced to Invitrogen to obtain 8 mg of peptide with a purity of 95.15%. The measured molecular weight was 1318.40, which almost coincided with the theoretical molecular weight of 1319.45. The peptide was diluted with 0.1 M phosphate buffer (pH 7.0) to 500 ng / ml, 100 μl was added to a 96-well microwell, and allowed to stand overnight at 4 ° C. for coating. After washing with the washing solution, each monoclonal antibody shown in Table 9 was diluted to 6.3 μg / ml with an assay buffer, added to each well coated with the peptide at 100 μl / well, and incubated (standing) at 25 ° C. for 1 hour. Subsequent operations were performed in the same manner as described in Example 9 (b). Table 9 shows A450 (difference when the primary antibody was 0 ng / ml) when the peptide obtained in this example was used as an antigen.
マウス由来単クローン性抗ヒトストロムライシン抗体産生ハイブリドーマ55-2A4(国際寄託番号:FERM BP-3743、寄託日:平成3年6月12日、寄託機関:独立行政法人 産業技術総合研究所 特許生物寄託センター、茨城県つくば市東1-1-1 つくばセンター中央第6)
マウス由来単クローン性抗ヒトストロムライシン1抗体産生ハイブリドーマ296-29C1(国際寄託番号:FERM BP-11199、寄託日:平成20年12月19日、寄託機関:独立行政法人 産業技術総合研究所 特許生物寄託センター、茨城県つくば市東1-1-1 つくばセンター中央第6)
マウス由来単クローン性抗ヒトストロムライシン1抗体産生ハイブリドーマ296-23A8(国際寄託番号:FERM BP-11198、寄託日:平成20年12月19日、寄託機関:独立行政法人 産業技術総合研究所 特許生物寄託センター、茨城県つくば市東1-1-1 つくばセンター中央第6) Mouse-derived monoclonal anti-human stromal lysin antibody-producing hybridoma 55-3G3 (International deposit number: FERM BP-3744, deposit date: June 12, 1991, deposit agency: National Institute of Advanced Industrial Science and Technology, Patent Organism) Center, Tsukuba Center Central, Tsukuba Center 1-1-1 Tsukuba City, Ibaraki Prefecture)
Mouse-derived monoclonal anti-human stromal lysin antibody-producing hybridoma 55-2A4 (International deposit number: FERM BP-3743, deposit date: June 12, 1991, deposit agency: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit) Center, Tsukuba Center Central, Tsukuba Center 1-1-1 Tsukuba City, Ibaraki Prefecture)
Mouse-derived monoclonal anti-human stromal lysin 1 antibody-producing hybridoma 296-29C1 (International deposit number: FERM BP-11199, deposit date: December 19, 2008, depository organization: National Institute of Advanced Industrial Science and Technology, Patent Biology) Deposit Center, 1-1-1 East Tsukuba City, Ibaraki Prefecture Tsukuba Center Central 6)
Mouse-derived monoclonal anti-human stromal lysin 1 antibody-producing hybridoma 296-23A8 (International deposit number: FERM BP-11198, deposit date: December 19, 2008, deposit agency: National Institute of Advanced Industrial Science and Technology, Patent Biology) Deposit Center, 1-1-1 East Tsukuba City, Ibaraki Prefecture Tsukuba Center Central 6)
Claims (9)
- ストロムライシン2及びストロムライシン3と交差反応せず、ストロムライシン1と特異的に反応する、モノクローナル抗体。 Monoclonal antibody that does not cross-react with stromalysin 2 and stromalysin 3, but specifically reacts with stromalysin 1.
- ストロムライシン2及びストロムライシン3に対する交差反応性がストロムライシン1に対する反応性の5%以下である、請求項1に記載のモノクローナル抗体。 2. The monoclonal antibody according to claim 1, wherein the cross-reactivity with respect to stromalysin 2 and stromalysin 3 is 5% or less of the reactivity with respect to stromalysin 1.
- 配列番号1の一部のアミノ酸配列からなるペプチドと特異的に反応し、かつ、ストロムライシン1と特異的に反応するモノクローナル抗体であって、該アミノ酸配列は配列番号1の425番~436番の配列を含み、該ペプチドの長さが12~20アミノ酸残基である、モノクローナル抗体。 A monoclonal antibody that specifically reacts with a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and that specifically reacts with stromalysin 1, wherein the amino acid sequence is from 425th to 436th of SEQ ID NO: 1. A monoclonal antibody comprising a sequence, wherein the peptide is 12-20 amino acid residues in length.
- 配列番号1の一部のアミノ酸配列からなるペプチドと特異的に反応し、該アミノ酸配列は配列番号1の425番~436番の配列を含み、該ペプチドの長さが12~20アミノ酸残基である、請求項1に記載のモノクローナル抗体。 It reacts specifically with a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence comprises the sequence of 425 to 436 of SEQ ID NO: 1, and the length of the peptide is 12 to 20 amino acid residues The monoclonal antibody according to claim 1, wherein
- ストロムライシン1に特異的に反応するモノクローナル抗体であって、ストロムライシン1をリジルエンドペプチダーゼで分解することによりストロムライシン1に対する反応性を消失する、モノクローナル抗体。 Monoclonal antibody that specifically reacts with strome lysin 1 and loses its reactivity to strome lysin 1 by degrading strome lysin 1 with lysyl endopeptidase.
- ストロムライシン1をリジルエンドペプチダーゼで分解することによりストロムライシン1に対する反応性を消失する、請求項1に記載のモノクローナル抗体。 The monoclonal antibody according to claim 1, wherein the reactivity with respect to strome lysin 1 is lost by degrading strome lysin 1 with lysyl endopeptidase.
- 請求項1~6のいずれか一項に記載のモノクローナル抗体を用いることを特徴とする、免疫染色法。 An immunostaining method using the monoclonal antibody according to any one of claims 1 to 6.
- 請求項1~6のいずれか一項に記載のモノクローナル抗体を用いて、ストロムライシン1を定量することを特徴とする、ストロムライシン1の免疫学的測定法。 A method for immunological measurement of strome lysin 1, characterized by quantifying strome lysin 1 using the monoclonal antibody according to any one of claims 1 to 6.
- ストロムライシン1に対する2種類以上のモノクローナル抗体を用いる免疫学的測定法であって、前記モノクローナル抗体のうちの少なくとも1種類が請求項1~6のいずれか一項に記載のモノクローナル抗体である、請求項8に記載の免疫学的測定法。 An immunological assay using two or more monoclonal antibodies against stromalisin 1, wherein at least one of the monoclonal antibodies is the monoclonal antibody according to any one of claims 1 to 6. Item 9. The immunological measurement method according to Item 8.
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WO1992013096A1 (en) * | 1991-01-21 | 1992-08-06 | Fuji Yakuhin Kogyo Kabushiki Kaisha | Antihuman stromelysin monoclonal antibody and diagnosis of rheumatoid arthritis by enzyme immunoassay |
JPH11318449A (en) * | 1998-03-10 | 1999-11-24 | Fuji Chem Ind Ltd | Antibody against stromelysin-2 (mmp-10) |
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