JPH054076B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a collagenase inhibitor monoclonal antibody that is extremely useful in the medical and physiochemical fields. [Background Art] Collagenase inhibitors (TIMPs) are effective for human and other animal bones, skin, dental pulp, amniotic fluid,
Its presence is recognized in blood, joint fluid, articular chondrocytes, synovial fluid cells, fibroblasts derived from various tissues, and fibrosarcoma cell culture fluid, and its molecular weight is approximately 30KD.
(kilodalton) glycoprotein.
(Murphy et al., Biochem. J. 195, 167-170, 1981;
Welgus et al., J. Biol. Chem. 258, 12259-12264,
1983; Kishi et al., J.Biochem.96, 395-404, 1984
(see et al.). On the other hand, increased collagenase activity has been observed in corneal tumors, periodontal disease, third-degree burn granulation, congenital epidermolysis bullosa, chronic rheumatoid arthritis, and intractable skin tumors (Kishi et al., Biomedical Res. 5, 149).
156, 1984), it is known that increased collagenase activity delays the healing of these diseases. Therefore, by measuring the activity of a collagenase inhibitor in relation to this increase in collagenase activity, it becomes possible to apply the method to the diagnosis of various disease conditions and their treatment methods as described above. On the other hand, the present inventors have discovered that a large amount of collagenase inhibitor is produced when bovine unerupted wisdom tooth root pulp is cultured in Eagle's MEM medium. Purification could not be easily carried out using a method using graphie, and a sufficient purified product could not be obtained. Affinity purification methods using antibodies have been reported for purifying enzymes or proteins easily and in high yields, but from the point of view of specificity, it is more advantageous to use monoclonal antibodies.
In conventional technology, antibody-producing B cells or their precursor cells are fused with bone marrow cells (myeloma cells) capable of continuous division and proliferation to obtain homogeneous fused cells (hybridoma) that have the characteristics of both these cells. Attempts have been made to create clones of the same (Ko¨hler et al.
et al., Nature, 256, 495-497, 1975) described a method for continuously producing large amounts of homogeneous and highly specific monoclonal antibodies against bovine collagenase inhibitors, and a method for affinity purification of collagenase inhibitors using these antibodies. has not been previously reported. As a result of various studies regarding collagenase inhibitor monoclonal antibodies, the present inventors succeeded in providing a novel collagenase inhibitor monoclonal antibody that is extremely useful for measuring the activity of the collagenase inhibitor described above or for purifying the collagenase inhibitor. [Disclosure of the Invention] The present invention provides monoclonal antibodies of the IgG ₁ type that have immunological cross-reactivity with bovine collagenase inhibitors and have immunological cross-reactivity with human collagenase inhibitors. The novel collagenase inhibitor according to the present invention can be obtained by the following method. First, animals are immunized with a bovine collagenase inhibitor, hybridomas are formed using anti-bovine collagenase inhibitor-producing cells and myeloma cells from various animals, and the hybridomas are cloned. Then, clones producing anti-bovine collagenase inhibitor antibodies are selected and cultured. then aim
Obtain an IgG ₁ type monoclonal antibody. The novel monoclonal antibody according to the present invention plays an extremely useful role in measuring collagenase inhibitor activity, purifying collagenase inhibitor, and other purposes, and is important for medical or physiochemical purposes. Hereinafter, the present invention will be specifically explained with reference to Examples and Reference Examples. However, the present invention is not limited to these. Example 1 Preparation of anti-bovine collagenase inhibitor monoclonal antibody (a) Preparation of antigen-bovine collagenase inhibitor Root of bovine unerupted wisdom tooth according to the method of the present inventors as described in J.Biochem, 96, 395-404 (1984) Con A-Sepharose,
Collagenase inhibitors were purified using Ultrogel AcA44 and DE-52 cellulose columns. Purified collagenase inhibitor
J.Mol.Biol. 80 , 579-599 (1973).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS) according to the method of Laemmli et al.
-PAGE) showed a single band with a molecular weight of about 32,000 Daltons (D). (b) Preparation of antibody-producing cells Two 6-week-old Belb/c female mice are first immunized with the purified bovine collagenase inhibitor described in (a) above in Freund's complete adjuvant. Each mouse is given 48 μg of bovine collagenase inhibitor intraperitoneally in a 0.4 ml solution. Further, on day 30, a booster dose of 84 μg of bovine collagenase inhibitor dissolved in saline is given. As a final immunization, supplementary immunization is performed by intraperitoneal administration (95 μg/500 μg physiological saline) on day 58, and 3 days later, the mouse spleen is removed and splenocytes are prepared. (c) Cell fusion (1) Use the following materials and methods. RPMI1640 medium: RPMINo.1640 (Difco
Laboratories) and sodium bicarbonate (12 m
M), sodium pyruvate (1mM), L-
Glutamine (2mM), penicillin G potassium (50U/ml), streptomycin sulfate (50μg/ml), and amikacin sulfate (100μg/ml)
g/ml), adjust the pH to 7.2 with dry ice, and sterilize with a 0.2 μm Toyo membrane filter. NS-1 medium: Fetal calf serum (MABioproducts) sterilized in the above PRMI1640 medium
to a concentration of 15% (v/v). PEG4000 solution: Polyethylene glycol 4000 (PEG4000, Merck &
CO., Inc.) Prepare a 50% (w/w) serum-free solution. 8-Azaguanine-resistant myeloma cells NS
-1 (P3-NS-1) fusion is Selected
Method in Cellular Immunology (ed.BB
Michelle and SMShiigi), WH Freeman
and Company (1980), 351-372.
The method was carried out with some modifications. (2) Nucleated spleen cells prepared in (b) above (viable cell rate
100%) and myeloma cells (viable cell rate 100%)
and are fused at a ratio of 5:1. The spleen cells and myeloma cells are washed separately with the RPMI1640 medium described above. Next, suspend in the same medium and mix at the above ratio for fusion. Capacity 50ml
Conical styrene resin test tube (Iwaki
400 in 40 ml of RPMI1640 medium.
Centrifuge at xg for 10 minutes and aspirate the supernatant completely. Precipitate cells with 1.3 ml of PEG4000 solution heated at 37°C.
was added dropwise over 1 minute with gentle stirring, and stirred for an additional 1 minute to resuspend and disperse the cells. Next, add 1.3 ml of RPMI1640 medium warmed at 37°C.
Drip in minutes. After repeating this operation once more, 9 ml of the same medium is added dropwise over 2 to 3 minutes with constant stirring to disperse the cells. this
Centrifuge at 400 xg for 10 minutes and aspirate the supernatant completely. Next, warm the precipitated cells at 37°C.
Immediately add 12.9 ml of NS-1 medium and carefully pipette to disperse large clumps of cells using a 10 ml pipette. Furthermore, add 26 ml of the same medium to dilute it and use polystyrene 96
Add 6.0 × ¹⁰ cells/0.1 ml per well to microwells (Iwaki Glass). In addition, 0.2 ml of NS-1 medium was added to the 96-well microwell used at this time as a pretreatment, and the medium was kept overnight in a carbon dioxide gas incubator (37°C), and the medium was removed by suction before use. The microwells containing the cells are cultured in 7% carbon dioxide gas/93% air at a temperature of 37° C. and a humidity of 100°C. (d) Selective growth of hybridomas using selective media (1) The media used are as follows. HAT medium: Hypoxanthine (100 μM), aminopterin (0.4 μM), and thymidine (16 μM) are further added to the NS-1 medium described in (c) above. HT medium: Same composition as the above HAT medium except that aminopterin was removed. (2) The next day (day 1) after starting the culture in (c) above, add 2 drops of HAT medium (approx.
0.1ml). On days 2, 3, 5, 8, and 11, half of the medium (0.1 ml) was replaced with fresh HAT medium, and on day 14, half of the medium was replaced with fresh HT medium.
Replace with medium. Replace half of the medium with fresh HT medium every 3-4 days thereafter. Normal 2
Sufficient hybridoma growth is observed in ~3 weeks. All wells in which hybridomas are grown are checked for positive wells using the solid-phase antibody binding test method (ELISA) described in the next section (e). Next, as a feeder, 1 ml of HT medium containing ¹⁰⁷ mouse thymocytes was placed in a polystyrene tube.
Using a 24-well cell well (Iwaki Glass), transfer the entire contents of each positive hybridoma detected above. This is cultured at 37° C. for about 1 week in the presence of 7% carbon dioxide as in (c) above. During this time, replace 0.5 ml of supernatant of each well with 0.5 ml of fresh HT medium once or twice. Once the hybridoma has grown sufficiently
Reconfirm the positivity using ELISA, and perform cloning using the limiting dilution method described in (f) below. In addition, the remaining liquid after being used for cloning is transferred to a 25 cm ² polystyrene tissue culture flask (Iwaki Glass) to prepare a sample for cryopreservation. (e) Search for anti-bovine collagenase inhibitor antibody-producing hybridomas by solid phase-antibody binding test (ELISA) A slightly modified method of Rennard et al. described in Anal.Biochem.104, 205-214 (1980) is used. This method is suitable for detecting hybridoma antibodies. 96-well microtitration plate (Flow Laboratories, Inc.) with 0.5–1.0μ
g of bovine collagenase inhibitor and then block the uncoated areas with 1% bovine serum albumin (BSA). A portion of the supernatant of the hybridoma growth well obtained in step (d) above is added to this and incubated at room temperature for about 1 hour. Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (cappel) was used as a secondary antibody.
Lab.) and incubate for about 1 hour at room temperature. Next is hydrogen peroxide and the substrate.
- Qualitatively judge the degree of brown color produced by adding phenylenediamine with the naked eye, or use a corona two-wavelength microplate photometer (MTP-22,
The absorbance at 500 nm is measured using a camera (Corona Electric Co., Ltd.). (f) Cloning After the operation in (d) above, since there is a possibility that two or more types of hybridomas are growing in each well, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. 10 ⁷ as a feeder per 1ml of NS-1 medium
Prepare a cloning medium containing 36 mouse thymocytes and add 5, 1 and 0.5 hybridomas per well to 36, 36 and 24 wells of a 96-well microwell. 5th day,
On day 12, add approximately 0.1 ml of NS-1 medium to all wells. For groups in which sufficient hybridoma growth is observed 14 to 15 days after the start of cloning, and 50% or more of wells are negative for colony formation.
Perform ELISA method. If all the wells tested are not positive, check the number of colonies in the antibody-positive wells, and select 4 to 6 wells in which one colony is confirmed for recloning. Finally, 17 hybridoma strains producing monoclonal antibodies against bovine collagenase inhibitor were obtained. (g) In vitro and in vivo proliferation of monoclonal antibodies Monoclonal antibodies are propagated by conventional methods. That is, monoclonal antibodies can be obtained from the culture supernatant by culturing each hybridoma obtained in an appropriate culture medium such as NS-1 medium (in vitro growth) (the concentration of monoclonal antibodies is 10 to 100 μg/ ml). On the other hand, in order to obtain antibodies in large quantities, the tumorigenic agent pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chemical
0.5ml per mouse was administered intraperitoneally.
After ~3 weeks, 1 x 10 ⁷ of each hybridoma is similarly administered intraperitoneally to obtain in vivo, and after 1 to 2 weeks, ascites with a monoclonal antibody concentration of 4 to 7 mg/ml can be obtained. (h) Isotype of monoclonal antibody Each of the ascites obtained in the above (g) is first bound to a microtitration plate coated with bovine collagenase inhibitor according to the ELISA method described above. After washing with PBS,
Add isotype-specific rabbit optical mouse Ig antibody (Zymed Laboratories). After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) antibody was added, and detection was performed using 2,2'-azinodi (3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide as substrates. The results are summarized in Table 1 below. Of the monoclonal antibodies against bovine collagenase inhibitor obtained, 15 had immunoglobulin chains γ1/κ, 1 had γ2a/κ, and 1 had γ2b/κ. (i) Purification of monoclonal antibodies Each of the ascites obtained in above (g) was fractionated with ammonium sulfate (40% saturation), and DEAE-
The non-adsorbed fraction of Sephacel (Pharmacia) was separated, and this IgG fraction was further equilibrated with Sephacryl S-300 Superfine (Pharmacia
FCS and mouse-derived proteins in the culture medium were separated and removed by gel filtration using a Co., Ltd. column. (j) Cross-reactivity with human collagenase inhibitor (1) Preparation of human collagenase inhibitor Human collagenase inhibitor is human gingival fibroblast cells, Gin-1 (manufactured by ATCC) and Dulbecco's modified Eagle containing 10% fetal calf serum (FCS). After culturing to confluence in medium (manufactured by Nissui Pharmaceutical Co., Ltd.), Ultrogel AcA54 was cultured in the same culture medium without FCS.
Partial purification was performed using a column. (2) Cross-reactivity test The cross-reactivity between each of the purified monoclonal antibodies of the 17 strains obtained in the above (i) and the human collagenase inhibitor obtained in the above (1) was investigated.
The results are shown in Table 2 below. As shown in Table 2, clones 7-6C1, 7-19F6 and 7-21B12 crossed with human collagenase inhibitor. These results suggest that there may be some similarity between the antigen recognition sites of bovine collagenase inhibitor and human collagenase inhibitor. Example 2 Immunohistochemical staining for bovine collagenase inhibitor Bovine dental pulp and bovine aortic smooth muscle cells were frozen with ethanol and dry ice, and 5μ sections were prepared using a cryostat. Next, the plate was fixed with cold acetone for 5 minutes, the purified monoclonal antibody (clone 7-15E8) obtained in Example 1(i) was added, and further stained using Funakoshi ABC kit (manufactured by Vector Lab.). Both bovine dental pulp (see Figure 5) and aortic smooth muscle cells (see Figure 6) showed clear positive findings. Example 3 Affinity purification of bovine collagenase inhibitor (a) Preparation of affinity column As described in Nature 214 , 1302-1304 (1967)
Axen et al. and Proc. Matl. Acad. Sci. USA, 61 ,
The purified monoclonal antibody obtained in Example 1(i) was immobilized as a ligand on the carrier Sepharose 4B via cyanogen bromide according to the method of Cuatrecasas et al., described in 636-643 (1968). Next, 0.3 ml of antibody-bound Sepharose 4B gel was filled into a glass tube, equilibrated with 30 mM Tris-HCl buffer containing 0.1 M sodium chloride and 5 mM calcium chloride, and used. (b) Collagenase activity measurement method The method of Nagai et al. described in Inflammation 4 , 123-130 (1984) was modified. i.e. 1.0mg/ml
Fluorescein isothiocyanate (FITC)-collagen (manufactured by Collagen Technology Workshop) 0.05ml with a concentration of 50ml containing 0.1M sodium chloride and 5mM calcium chloride activated with 1mMp-aminophenylmercuric acetate (APMA).
0.15 ml of a collagenase solution dissolved in M Tris-HCl buffer (PH7.5) was added, and the mixture was reacted at 35°C for a certain period of time. Then 80 mMo-phenanthroline or 200
Add 0.01 ml of mM sodium ethylenediaminetetraacetate (EDTA) to stop the reaction, and then
0.4ml of 70% ethanol and 10mM Tris-HCl buffer (PH7.5) containing 30mM sodium chloride was added and stirred. Thereafter, the mixture was centrifuged at 3000 rpm for 10 minutes to extract degraded collagen fragments. The amount of collagen decomposition (amount of collagen fragments produced) was determined by adding only the buffer solution instead of the collagenase-containing solution, assuming that the fluorescence intensity of the extract was 0%, and heating the reaction mixture at 80°C for 10 minutes. FITC-denatures collagen, total FITC-
100% fluorescence intensity when collagen is extracted
It was determined from the fluorescence intensity when In other words, collagenase activity (U) = total sample volume (ml) x sample fluorescence intensity x 50μg/reaction time (minutes) x sample addition amount One unit (U) of collagenase activity degrades 1μg of collagen in 1 minute at 35°C. It is expressed as the amount of oxygen required to (c) Collagenase inhibitor activity measurement method Add collagenase inhibitor-containing sample to 1mM APMA-activated collagenase solution.
After pre-incubation at ℃ for 10 minutes, see (b) above.
Collagenase activity was measured by the method described in section 2.
It was determined by using collagenase activity in the absence of collagenase inhibitor as a control. That is, 1 U of collagenase inhibitor activity was expressed as the amount of oxygen required to inhibit 1 U of collagenase activity. (d) Selection of optimal monoclonal antibody-binding affinity column Bovine dental pulp culture solution (protein amount 1.4 mg) was passed through the various antibody-binding columns prepared in the previous section (a), and 0.1M
Contains sodium chloride and 5mM calcium chloride
The cells were washed with 2.5 ml of 30 mM Tris-HCl buffer (PH8.0), and the extra-culture solution and washing solution were combined to obtain a non-adsorbed fraction. Next, it was eluted with a Tris-HCl buffer (PH7.5) containing 8M urea (eluted fraction) and dialyzed against a Tris-HCl buffer (PH8.0) containing 0.1M sodium chloride and 5mM calcium chloride.
As shown in Table 3 below, collagenase activity in both fractions was observed in the non-adsorbed fraction in both columns. On the other hand, the collagenase inhibitor activity of clones 7-3F1, 7-4F2, and 7-
In the antibody binding column from 20C2 and 7-21B12, it was observed in the elution fraction (recovery rate 60-70
%). Furthermore, the collagenase activity in the non-adsorbed fraction increased about 10 times compared to that in the extra-culture fluid, whereas the collagenase inhibitor activity disappeared. In the case of antibody-bound columns from clones 7-6C1 and 7-15E8, the proportions of collagenase activity and collagenase inhibitor activity in the non-adsorbed fraction were approximately the same as the proportions of those activities in the extra-culture fluid, and these columns It has become clear that collagenase inhibitors are not adsorbed to (e) Stability of collagenase inhibitor The stability of collagenase inhibitor in various eluents when eluting collagenase inhibitor from an antibody-bound affinity column was investigated. That is, bovine dental pulp culture solution (A) 0 ~
30mM Tris-HCl buffer containing 8M urea (PH
7.5), (B) 30mM Tris-HCl buffer containing 0-4M Rhodankali (PH7.5), (C) Tris-HCl buffer containing 0-6M guanidine hydrochloride (PH7.5) and 0
After incubation at 35°C for 30 minutes in 0.2M glycine-hydrochloric acid buffer (PH2.0) containing ~6M guanidine hydrochloride, each incubation was performed in 30mM Tris-hydrochloric acid buffer (PH8. 0) and examined for residual collagenase inhibitor activity. As is clear from the accompanying drawings and FIG. 1, no deactivation of the collagenase inhibitor activity was observed under any of the conditions (A) to (D). (f) Examination of elution conditions for collagenase inhibitor Bovine dental pulp culture fluid was subjected to antibody-binding affinity columns from clones 7-20C2 and 7-21B12, which had high recovery rates in section (a) above, and various elutions were performed. Collagenase inhibitors were eluted using a reagent. As is clear from Table 4, the recovery rate of collagenase inhibitor activity was 60-70% when using either clone 7-20C2 or 7-21B12 column, and was not affected by the type of eluent. . In addition, looking at the SDS-PAGE patterns of fractions eluted with various eluents, the main components are a band corresponding to collagenase inhibitor with a molecular weight of 32 KD, a band with a molecular weight of 20 KD, and a band with a molecular weight of 60 to 60 KD.
A band of 70KD was observed (see Figure 2 of the attached drawings). A, B, C, and D in FIG. 2 have the following meanings, respectively. (A) 30mM Tris-HCl buffer containing 8M urea (PH7.5) (B) 30mM Tris-HCl buffer containing 3M Dankali (PH7.5) (C) 30mM Tris-HCl buffer containing 1M guanidine hydrochloride (PH7. 5) (D) 0.2M glycine-hydrochloric acid buffer (PH2.0) (g) Consideration of reusing antibody-binding affinity column Bovine dental pulp culture fluid was injected into antibody-binding affinity column from clones 7-20C2 and 7-21B12. 0.2M glycine-hydrochloric acid buffer (PH2.0)
After eluting the collagenase inhibitor with
Each column was thoroughly washed with 30mM Tris-HCl buffer (PH8.0) containing 0.1M sodium chloride and 5mM calcium chloride. Bovine dental pulp extraculture fluid was added to these columns again, and the collagenase inhibitor was eluted in the same manner as the first operation. Clone 7 as shown in Table 5 below
In the case of the -20C2 column, 19% of the collagenase inhibitor activity in the extra-culture fluid was observed in the non-adsorbed fraction. On the other hand, in the case of clone 7-21B12 column, no activity was observed in the non-adsorbed fraction, and 61% of the activity was recovered in the eluted fraction, resulting in a first recovery rate of 58% (see Table 4 below). ) almost the same results were obtained. Therefore, in the present invention, it has been revealed that the antibody from clone 7-21B12 is optimal as a ligand for the affinity column. (h) Washing effect of antibody-binding affinity column Clone 7-
After application to an antibody binding column from 21B12 and washing with various solutions, the collagenase inhibitor was eluted. 2M as shown in Table 6 below
30mM Tris-HCl buffer (PH7.5) containing sodium chloride and 0.2M containing 0.5M sodium chloride
Glycine-sodium hydroxide buffer (PH11.0)
The washed fraction does not contain collagenase inhibitors and contains some impure protein.
A cleaning effect was observed. On the other hand, washing with 30mM Tris-HCl buffer (PH7.5) containing 0.5% Torin Nakatsuta. In addition, acetate buffer containing 0.5M sodium chloride (PH4.0) and 30mM Tris-HCl buffer containing 1M guanidine hydrochloride (PH7.5)
When washed with 29% and 21% of the collagenase inhibitor, respectively, it became clear that the detergent was unsuitable as a detergent. (i) Purification by DE-52 column chromatography The collagenase inhibitor obtained from the antibody-binding affinity column from clone 7-21B12 in section (f) above has a molecular weight of 20 KD and 60-70 KD.
Furthermore, DE-
Purified with 52 column. As shown in Figure 3 of the accompanying drawings, most of the collagenase inhibitor was eluted with (B) 10mM Tris-HCl buffer (PH8.0) containing 50mM sodium chloride, and impure proteins were also removed. Also, 10mM containing 2M sodium chloride
Collagenase inhibitor was eluted in Tris-HCl buffer (PH8.0), but at the same time most of the impure proteins were also eluted. The impure protein-containing collagenase inhibitor obtained from the antibody-binding affinity column is Ultrogel.
Purification into a single band was also possible by gel filtration using an AcA54 column. Example 4 Affinity purification of human collagenase inhibitor 750 ml of the extra-cultured human gingival fibroblast (Gin-1) described in Example 1 (j) (1) was subjected to high pressure using a Toyo Ultra Filter, UK-10. The mixture was concentrated to 25 ml and dialyzed against 30 mM Tris-HCl buffer (PH8.0) containing 0.1 M sodium chloride and 5 mM calcium chloride. It was then equilibrated with the above buffer to an antibody-binding column from clone 7-21B12 (5
ml), washed with 10 ml of the above buffer (non-adsorbed fraction), and then the column was washed with 30 mM Tris-HCl buffer containing 2 M sodium chloride (PH
7.5) and 10 ml each of 0.2 M glycine-sodium hydroxide buffer (PH11.0) containing 0.5 M sodium chloride (washing fraction), and finally 0.2 M glycine-sodium hydroxide buffer (PH11.0).
Collagenase inhibitor was eluted from the column with 10 ml of hydrochloric acid buffer (PH2.0) (eluted fraction). As shown in Table 7 below, 42 μg of protein was eluted in the elution fraction. In addition, in SDS-PAGE of both fractions, a band with a molecular weight of 32K, which is the same as bovine collagenase inhibitor, and impure proteins of 60 to 70 KD and 20,000 or less were observed. Next, each fraction of these two types of samples was subjected to SDS-PAGE, and then Fab′(7-21B12)-horseradish peroxidase was extracted according to the method of Tabe described in Cell Engineering 1 & 2, 1061-1068 (1983). Western blotting was performed using the complex. The above complex was developed by J.
Antibody from clone 7-21B12 according to the method of Ishikawa et al. as described in Immunoassay 4, 209-327 (1983).
Prepared using IgG1 and horseradish peroxidase. As is clear from the Western blotting pattern shown in the accompanying drawings and Figure 4, human collagenase inhibitor that reacts with the monoclonal antibody against bovine pulp collagenase inhibitor exists in the human gingival fibroblast culture solution, and its molecular weight is similar to that of bovine pulp collagenase inhibitor. pulp collagenase inhibitor
It was the same as 32KD.

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[Table] 0.5 μg of human collagenase inhibitor was coated per well, and 1.0 μg of each purified mouse anti-bovine collagenase inhibitor antibody was added and incubated.

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【table】 [Brief explanation of drawings]

Figure 1 is a graph showing the stability of bovine collagenase inhibitor in various eluent solutions, and Figure 2 is an SDS-PAGE of bovine collagenase inhibitor eluted from a monoclonal antibody-binding affinity column with various eluents. Figure 3 shows the SDS-SDS of each fraction when bovine collagenase inhibitor was purified using an antibody (clone 7-21B12)-bound affinity column and then subjected to DE-52 column chromatography. It is a figure showing a PAGE pattern, (A) elution fraction from the antibody-binding affinity column, (B) DE-52 column non-adsorbed fraction,
(C) 0-50mM sodium chloride solution elution fraction, (D) 50
~100mM sodium chloride solution elution fraction, (E)100~
The 2000mM sodium chloride solution elution fractions are shown in Figure 4 (A) human gingival fibroblast culture solution, (B)
Each antibody (clone 7-
21B12) A Western blotting diagram of the elution fraction from the binding affinity column is shown; Figure 5 is a photograph of stained bovine dental pulp cells under a microscope attached instead of a diagram; The figure is a photograph of stained bovine aortic smooth muscle cells taken under a microscope in place of the drawing.

Claims (1)

[Claims] 1. An IgG ₁ type monoclonal antibody that has immunological cross-reactivity with bovine collagenase inhibitors and immunological cross-reactivity with human collagenase inhibitors.