JP5840274B2 - Monoclonal antibody that specifically reacts with strome lysin 1 - Google Patents

Description

  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.

  A group of enzymes collectively referred to as matrix metalloproteinases (MMPs) are involved in the degradation of the constituent proteins of blood vessels, lymphatic vessels and articular cartilage, and the so-called extracellular matrix. There is a subgroup in MMPs, and 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. 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.

  Stroemlysin 2 (MMP-10) is cloned by Muller et al. And has the same substrate specificity as stromlysin 1 (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).

Japanese Patent Publication No. 8-5920

Muller et al., Biochem. J. et al. , 253, 187-192, 1988 Basset et al., Nature, 348, 699-704, 1990.

  Stromlysin 1, 2 and 3 have very similar amino acid sequences and substrate specificities, and when an antibody against stromalysin 1 is prepared, there is a high possibility of cross-reacting with stromalysin 2 and 3. In 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. In order to more accurately diagnose rheumatoid arthritis, it is necessary to quantify stromlysin 1 with higher accuracy.

  Therefore, an object of the present invention is to quantitate stromal lysin 1 with higher sensitivity and more accurately.

The present invention relates to a monoclonal antibody produced by a mouse-derived monoclonal anti-human stromal lysin 1 antibody-producing hybridoma 296-29C1 (international deposit number: FERM BP-11199) or 296-23A8 (international deposit number: FERM BP-11198). I will provide a. Monoclonal antibodies produced by the above two types of hybridomas have high reactivity with stromalysin 1 and serum. Therefore, the monoclonal antibody can be used for immunological measurement of strome lysin 1 in serum.

The monoclonal antibody of the present invention can be used for immunostaining. According to the immunostaining method using the monoclonal antibody of the present invention, stromalysin 1 can be specifically visualized.

The monoclonal antibody of the present invention can be used in an immunological assay for stromalysin 1 to quantify strome lysin 1. According to the immunoassay using the monoclonal antibody of the present invention, stromalisin 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. When two or more types of monoclonal antibodies are used, it is preferable that at least one of the monoclonal antibodies used is the monoclonal antibody of the present invention. By using two or more kinds of monoclonal antibodies, the specificity to stromalysin 1 becomes higher, and strome lysin 1 can be quantified more accurately.

  According to the present invention, stromal lysin 1 can be quantified with higher sensitivity and more accuracy.

It is a graph which shows the reactivity of the peptide of each fraction obtained by fractionating strom lysin 1 peptide, and a monoclonal antibody.

In one embodiment, a monoclonal antibody is provided that does not cross-react with stromalisin 2 and strome lysin 3 but specifically reacts with strome 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 monoclonal antibody preferably has a cross-reactivity with stromlysin 2 and strom- lysin 3 of 5% or less of the reactivity with strom- lysin 1. Such monoclonal antibodies are more specific for stromalysin 1.

In one embodiment, the present invention also relates to a monoclonal antibody that specifically reacts with a peptide consisting of a partial 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 A monoclonal antibody comprising the sequence of Nos. 425 to 436 in which the peptide has a length of 12 to 20 amino acid residues. 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 425 to 436 of SEQ ID NO: 1 is considered to be involved in cross-reactivity.

In one embodiment, there is provided a monoclonal antibody that specifically reacts with strome lysin 1 and that loses reactivity with strome lysin 1 by degrading strome lysin 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 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 can be used in an immunological assay for strome lysin 1 for quantifying 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. When two or more types of monoclonal antibodies are used, it is preferable that at least one of the monoclonal antibodies used is the monoclonal antibody of the present invention. By using two or more kinds of monoclonal antibodies, the specificity to stromalysin 1 becomes higher, and strome lysin 1 can be quantified more accurately.

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

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.

  In addition, 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. For example, from a DNA sequence obtained by molecular cloning from a natural cell or a known genomic sequence, 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. Moreover, 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. When bound to carrier proteins, the carrier proteins can be activated first. For such activation, introduction of an activated linking group can be mentioned. Examples of 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. (2) activated A dithio group, such as a 2-pyridyldithio group, can be mentioned. Examples of 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.

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.

3. Preparation of myeloma cells (myeloma cells) The infinitely proliferative strain (tumor cell strain) 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, an antibiotic such as penicillin or amikacin, fetal calf serum (FCS) or the like is 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 2-5 days prior to 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 cultivating it in the normal medium to prepare the required number of cell lines. There may be.

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 a 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 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.

5. Selection and Monocloning of Hybridoma (Fused Cell) 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 selective medium replacement method can 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.

6). Production of Monoclonal Antibody The obtained hybridoma strain can be cultured in an appropriate growth medium such as FCS-containing MEM medium or RPMI-1640 medium, 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. .

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.

  For example, in an ELISA using a direct adsorption method, a fixed concentration of strome lysin 1 is immobilized as an antigen. As the solid phase, 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. Next, 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. When the enzyme is not labeled on the monoclonal antibody, an enzyme-labeled antibody (secondary 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.

  Next, ELISA using the direct adsorption method is performed using stromal lysin 2 and strome 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.

  In addition to the direct adsorption ELISA assay described above, for example, 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. In the sandwich method, two types of antibodies against stromolysin 1 are used. One of them, 6. And 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. After immobilization, it is preferable to adsorb and block proteins that are not involved in antigen-antibody reaction and enzyme reaction. Next, 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.

  Next, ELISA using the sandwich method is performed using stromal lysin 2 and strome 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.

  As used herein, “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. Preferably, A450 is 0.08 or more, more preferably 0.1 or more. The term “cross-reactivity with stromalysin 2” as used herein 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 same applies to “cross-reactivity with stromlysin 3”. The magnitude of cross-reactivity may be measured by either the direct adsorption method or the sandwich ELISA. As used herein, “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. Preferably, 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.

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.

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 of Nos. 425 to 436 of SEQ ID NO: 1, and the length of the peptide is 12 to 20 amino acid residues. 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 consisting of the sequence of Nos. 425 to 436 of SEQ ID No. 1 from the monoclonal antibodies against stromelysin 1 obtained in the above. The selection is 7. For example, 7. In the ELISA based on the direct adsorption method shown in (2), a peptide consisting of the sequence from 425th to 436th of SEQ ID NO: 1 may be used as an antigen instead of stromelysin 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.

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.

  The thus obtained monoclonal antibody can be examined for the type of heavy chain and light chain of the antibody using a commercially available isotype-specific anti-mouse Ig antibody, such as an isotype-specific rabbit anti-mouse Ig antibody.

It is also possible to determine the base sequence encoding the obtained monoclonal antibody and prepare the antibody by gene recombination technology. Furthermore, 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. As an antibody to be labeled, an IgG fraction, for example, an IgG fraction obtained by treatment with an anion exchange gel such as DEAE-Sepharose after fractionating the antibody-containing material with ammonium sulfate, and further reduced after digestion with pepsin. The resulting specific binding part Fab ′ or the like can be used. Examples of labels in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, radioisotopes and the like as described below.

  The immunostaining method can be performed using the monoclonal antibody that does not cross-react with stromalysin 2 and 3 and specifically reacts with stromalicin 1 of 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. According to the immunostaining method using the monoclonal antibody of the present invention, even if stromlysin 2 or stromal lysin 3 is mixed in the sample, a signal is specifically detected only from strome lysin 1, and the presence of strome lysin 1 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. In addition, 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 immunoassay can be performed by immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and RIA, ELISA, agglutination method, turbidimetric method, immunochromatography, Western blotting, 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 the monoclonal antibody used in the competitive immunoassay by the direct method. When two types of monoclonal antibodies are used in the sandwich method, at least one of them 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. Of the two or more types of monoclonal antibodies used in the immunoassay such as the agglutination method, at least one type 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.

  In the above immunological measurement method, for example, 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.

  In the immunostaining method and immunological measurement method of the present invention, a monoclonal antibody labeled with an enzyme or the like and an antibody bound to a carrier can be reacted sequentially or simultaneously. In addition, 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. In the aggregation method, 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. For example, the carrier can be appropriately selected from those described below. When using beads such as sensitized plastic, put the monoclonal antibody labeled with an enzyme together with a specimen sample containing the substance to be measured together in an appropriate test tube, and then sensitized plastic The reaction can be carried out by adding beads such as. The reaction can be performed 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.

  In the immunostaining method and immunological assay of the present invention, 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. In 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. In addition, 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. As long as the purpose is to prevent non-specific binding reaction, these methods are not particularly limited and can be used. For example, 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.

  Many carriers capable of immobilizing an antigen or antibody are known, and in the present invention, they can be appropriately selected and used. Particularly preferably used are, for example, glass, such as 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. 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 substances such as polyamide such as orchid, nylon, polyurethane, polyepoxy resin, and those obtained by emulsion polymerization of these, cells, erythrocytes, etc., functional groups with silane coupling agents, etc. as necessary The thing which introduce | transduced is mentioned.

  Furthermore, filter paper, beads, inner walls of test containers, 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. Alternatively, the surface of a solid substance (object) 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. Examples of 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 β-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. When alkaline phosphatase is used, 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.

  4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine, etc., horseradish peroxidase, umbelliferyl galactoside, nitrophenyl galactoside, etc., β-D-galactosidase, glucose Combinations of enzyme reagents such as -6-phosphate dehydrogenase can also be used, and quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone, thiol compounds such as lipoic acid and glutathione, phenol derivatives and ferrocene derivatives are formed by the action of enzymes, etc. Possible compounds can be used.

  Examples of the fluorescent substance or chemiluminescent compound include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate and 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. 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.

  Examples of the condensing agent 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-succinimidyl -(1-maleimidophenyl) butyrate, N- (ε-maleimidocaproyloxy) succinimide (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4′-dithiopyridyl) propionimi Date, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate, N-succinimidyl-S-acetylmercaptoacetate.

  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. In the immunostaining method using the monoclonal antibody of the present invention, 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. For immobilization, those widely used in the art or those derived therefrom can be used. For example, 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 immunoassay reagent comprising the monoclonal antibody of the present invention. According to the immunological measurement reagent, 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. In applying the measurement reagent or kit to the immunostaining method or immunological measurement method of the present invention, 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 those skilled in the art to the normal conditions and operation methods in each method. .

  Examples Hereinafter, 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. . However, the present invention is not limited to these examples.

(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 skin fibroblasts 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.

The supernatant was applied to a Sepharose 4B column. This 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 1-1-1 Tsukuba City, Ibaraki Prefecture, Tsukuba Center Central 6) is preliminarily bound to CNBr-activated Sepharose (GE Healthcare) according to the operation method 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. Next, 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.

  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 manufactured by R & D Systems was used as a standard product, and Stromlysin 3 manufactured by Abcam was used as a standard product.

(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.

(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.

(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 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.

Fusion of spleen cells with 8-azaguanine resistant myeloma cells SP2 (SP2 / 0-Ag14) is described in Oi et al., Selected Methods in Cellular Immunology, 351-371, W. et al. H. Freeman & Co. , 1980. The spleen cells prepared in (a) (live cell rate 100%) and myeloma cells (live cell rate 100%) were fused at a ratio of 5: 1. Spleen cells and myeloma cells were each washed with the RPMI-1640 medium. Next, 3.2 × 10 ⁸ spleen cells suspended in the same medium and 6.4 × 10 ⁷ myeloma cells were mixed. Next, 1000 r. p. m. The cells were precipitated by centrifugation at 10 minutes and the supernatant was completely aspirated off. To the precipitated cells, 2.1 ml of a PEG 4000 solution heated to 37 ° C. was added dropwise over 1 minute while stirring gently, and the cells were resuspended and dispersed by stirring for 1 minute. Next, 4.2 ml of RPMI-1640 medium heated to 37 ° C. was added dropwise over 2 minutes. 14.7 ml of the same medium was added dropwise for 2-3 minutes with constant stirring to disperse the cells. This is 1000 r. p. m. The supernatant was completely removed by aspiration. Next, 21 ml of NS-1 medium heated to 37 ° C. was quickly added to the precipitated cells, and a large cell mass was carefully dispersed by pipetting. Further, 42 ml of the same medium was added for dilution, and 6 × 10 ⁵ cells / 0.1 ml of cells were added to a 96-well microwell made of polystyrene (manufactured by Iwaki Glass). The microwells were cultured in 7% CO ₂ /93% air at a temperature of 37 ° C. and a humidity of 100%.

(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.

  On the next day (first day) after the start of the culture in (b), 2 drops (about 0.1 ml) of HAT medium were added to the cells by pipette. On days 2, 3, 5 and 8, half of the medium (approximately 0.1 ml) was replaced with fresh HAT medium. On all the wells where sufficient growth of the hybridoma was observed on the 11th day, positive wells were examined by ELISA described in (d) below.

(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.

(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%.

  Frozen cells were reconstituted in RPMI 1640 medium- (2) and using BriClone-containing RPMI 1640 medium, 36, 36 and 24 wells of 96-well microwells were each 5, 1 and 0.5 per well, 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. When all the tested wells were not positive, the number of colonies in the antibody-positive well was confirmed, and 1 to 3 colonies (well having as few colonies as possible) were selected in the wells and recloned. As a result, as shown in Table 1, 12 clones of monoclonal antibody-producing cells against stromelysin 1 were obtained.

(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).

(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 (standing). 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, 100 μl / well of TMB One Component HRP Microwell Substrate (BioFX, Product # TMBW-1000-01) was added to cause color development, 100 μl / well of 1M sulfuric acid was added 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.

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%. 296-29C1,296-12H8, and 296-7B10 have three clone antibodies, the reactivity of the same culture supernatant with stromlysin 1 is 100%, and the reactivity with stromlysin 2 is 1%, 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 stromalysin 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 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 overnight at 4-8 ° C. 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 stromalysin 2. Nine clones of 296-21C4, 296-21F3, 296-21E4, 296-33F3, 296-33E4, 296-23A8, 296-24C8, 296-30C12, and 296-31A4 were found to react with stromalysin 1. However, a reaction was also observed with stromelysin 2.

(Example 6 Purification of monoclonal antibody)
Among 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. As for four types of 296-21C4, 296-29C1, 296-12H8, and 296-7B10, the 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 grown, the cells were administered intraperitoneally to 5 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. The antibody was purified from the collected culture supernatant by ammonium sulfate fractionation.

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.

  The three clone antibodies, 296-29C1,296-12H8, 296-7B10, did not cross-react with stromelysin 2. The cross-reactivity of the antibody to stromalysin 2 was strongly observed in the order of 296-33F3, 296-23A8, and 296-21C4. Known monoclonal antibody 55-2A4 (International deposit number: FERM BP-3743, deposit date: June 12, 1991, deposit agency: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Higashi 1- Tsukuba, Ibaraki 1-1 Tsukuba Center No. 6) has a high cross-reactivity to strome lysin 2 of 85.1%, and some 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.

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”).

  Standard Stromlysin 1 to 1000, 250, 62.5, 15.6 ng / ml, Standard Stromlysin 2 and Stromlysin 3 to 20000, 5000, 1250, 313, 78 ng / ml, 1% The solution was diluted with 0.01 M phosphate buffer (pH 7.0) containing BSA and 0.1 M NaCl (hereinafter referred to as “assay buffer”) to prepare a standard solution, and 20 μl was taken on a vinyl plate. As a sample, 20 μl of human serum was similarly taken on a vinyl plate.

  On the other hand, Obata et al., Clin. Chim. According to the method described in Acta, 211, 59-72, 1992, the monoclonal antibody 55-2A4 was labeled with HRP (Roche Diagnostics). The labeled antibody was diluted with an assay buffer to 1.3 μg / ml, and 100 μl was added to each well containing a standard solution or sample. 100 μl from the well was added to the antibody-bound well, and incubated at 4 ° C. for 16 hours (standing). After washing the antibody-bound well 3 times with a washing solution, 100 μl / well of TMB One Component HRP Microwell Substrate (BioFX) was added, reacted at 25 ° C. for 20 minutes, and 100 μl / well of 1M sulfuric acid was added to stop the reaction. Absorbance at a measurement wavelength of 450 nm was measured with a reader (TECAN SPECTRA). A450 (difference when the labeled antibody is 0 ng / ml) when each antibody is used is shown in Table 4 for stromelysin 1, Table 5 for stromelysin 2, and Table 6 for serum.

  With each of the selected purified antibodies, it was possible to draw a standard curve of the concentration of stromlysin 1. However, when the antibodies 296-21C4 and 296-33F3 were used, 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 absorbance when using strome lysin 2 was plotted on a log-log graph for each concentration, and the absorbance at 1000 ng / ml was calculated and compared with the absorbance of strome lysin 1 at 1000 ng / ml (Table 7).

  At 1000 ng / ml, 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 the immunoassay of stromlysin 1 in the sandwich ELISA. In the case of the 296-23A8 antibody, whose cross-reactivity to stromelysin 2 was 17.1% in the ELISA by the direct adsorption method, it was in the acceptable range of 2.4% in the ELISA by the sandwich method. 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 are considered to be usable for immunoassay by ELISA by sandwich method. On the other hand, when serum was used as a sample, the 296-21C4, 296-33F3, and 296-7B10 antibodies did not react with serum. In addition, the 296-12H8 antibody was less reactive to serum than the 296-29C1 and 296-23A8 antibodies. 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, 1-1-1 Higashi Tsukuba, Ibaraki Center Center No. 6) and 296-23A8 (International Deposit Number: FERM BP-11198, Deposit Date: December 19, 2008, Deposit Agency: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, East of Tsukuba City, Ibaraki Prefecture 1-1-1 The antibody at Tsukuba Center Central No. 6) has good reactivity with serum and is considered to be usable for immunological measurement of stromalisin 1 in serum.

(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.

(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, the lysyl endopeptidase band was confirmed, but the stromylysin 1 band was not observed, indicating that it was sufficiently decomposed.

(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 the 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.

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.

  The reactivity of 296-33F3 and 296-12H8 almost disappeared due to the degradation of stromlysin 1 by lysyl endopeptidase. Therefore, since these antibodies do not react with degradation products by lysyl endopeptidase, these antibodies are considered to recognize steric structures such as the secondary structure and tertiary structure of the thromtomlysin 1 protein. In addition, although 296-21C4 and 296-29C1 remained reactive to strome lysin 1 after decomposition, they significantly decreased compared to before decomposition. In addition, 296-23A8 and 296-7B10 remained reactive to strome lysin 1 before decomposition at a high rate after decomposition. Regarding 55-2A4, 80% of the reactivity with respect to stromalicin 1 before decomposition remained after decomposition. With each antibody, the reactivity after degradation by lysyl endopeptidase decreased, suggesting that at least higher-order structures were recognized.

(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-A12, B1-B12.

(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 (still). After washing the well 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 the mixture 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.

(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). Peptides contained in C10 were estimated. The peptide was estimated to be QIAEDFPPGIDSK (No. 425 to 436 of SEQ ID NO: 1). 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.

(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.

  From Table 9, 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. A large number of peptides were mixed in C10, and it was shown that stromalysin 1 peptide QIAEDFPPGIDSK was not a peptide to which 296-7B10 reacts. In addition, 296-21C4, 296-23A8 and 55-2A4 showed reactivity to this peptide, but since these monoclonal antibodies were found to be cross-reactive with stromelysin 2, there was no presence of cross-reactivity. Suggests that this peptide and other peptides and structures are involved.

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 Biological Deposit) 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, deposit agency: National Institute of Advanced Industrial Science and Technology, Patent Biology) Deposit Center, Tsukuba Center Central, 1-1-1 Tsukuba City, Ibaraki Prefecture 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, Tsukuba Center Central, 1-1-1 Tsukuba City, Ibaraki Prefecture 6)

Claims (4)

A monoclonal antibody produced by a mouse-derived monoclonal anti-human stromal lysin 1 antibody-producing hybridoma 296-29C1 (International Deposit Number: FERM BP-11199) or 296-23A8 (International Deposit Number: FERM BP-11198). An immunostaining method using the monoclonal antibody according to claim 1 . A method for immunological measurement of strome lysin 1, characterized in that strome lysin 1 is quantified using the monoclonal antibody according to claim 1 . A immunoassay using two kinds or more monoclonal antibodies against stromelysin 1, at least one of said monoclonal antibody is a monoclonal antibody according to claim 1, immunology of claim 3 Measurement method.

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