JPS63246396A - Monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:An antihuman IV type collagen monoclonal antibody capable of specifically reacting all human IV type collagens, having 100,000-160,000 (SDS-PAGE) molecular weight and immunogloblin class of IgG2a and consisting of JK-132 specifically recognizing decomposed product of the human IV type collagen having >=5,000mol.wt. and JK-199 recognizing no decomposed product of the human IV type collagen reduced by 2-mercaptoethanol and having <=7,000 mol.wt. USE:A reagent for studying and purifying human IV type collagen. PREPARATION:For example, the aimed monoclonal antibody is obtained by fusing an immunocompetent cell collected from a mammal immobilized with a human IV type collagen with a myeloma cell, subjecting the resultant clone specific to the human IV type collagen to screening and cloning and cultivating the resultant monocloning hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an anti-type collagen monoclonal antibody that recognizes human type IV collagen. More details: Anti-type collagen monoclonal antibody JK that recognizes human type ■ collagen and recognizes human type IV collagen decomposition products
-199 and anti-type collagen monoclonal antibody JK-132, which recognizes human type IV collagen but does not recognize human type ■ collagen decomposition products,
These monoclonal antibodies can be used as reagents for histological or biochemical studies of human biological connective tissues by analyzing the structure of human type II collagen and observing the distribution and existence form of human type IV collagen. It is also useful as a purification reagent for human type II collagen, such as gel.

[Prior Art] Collagen is one of the constituent components of biological connective tissue, and different types of collagen from type RI to type XI have been discovered. These different types of collagen are thought to have different functions, and have attracted particular attention and research among biochemists. It is difficult to distinguish each type of collagen from other types of collagen, even if it is found in tissues or purified, and its existence can only be confirmed by biochemical methods or observation by experts. Ru.

[Problems to be solved by the invention] Among collagens, type IV collagen normally exists along the basement membrane of biological connective tissue, and during normal metabolism,
When tissues disintegrate for some reason, their existence forms may differ, or they may exist in the form of decomposed products in the blood. In order to conduct biochemical research, monoclonal antibodies must be able to specifically recognize degraded human type IV collagen, and recognize human type IV collagen but not human type IV collagen degraded products. it is conceivable that. However, as anti-human type IV collagen monoclonal antibodies, for example, Oderm
There is an example of a type IV collagen extracted from human kidney being created by att et al. as an antigen (Proc, Nat.
l, Δcad, Sci, Vol, 81.7343-
7347゜1984), but this product is used as a reagent for histological or biochemical studies of human biological connective tissues by analyzing the structure of human type II collagen and observing the distribution and morphology of human type IV collagen. It did not satisfy the above-mentioned conditions required for use.

[Means for Solving the Problems] The present inventors have been conducting research to obtain a monoclonal antibody that specifically binds to human tV type collagen. The present invention was completed by developing a monoclonal antibody that also recognizes type ① collagen degradation product and a monoclonal antibody that recognizes human type IV collagen but not human type IV collagen degradation product.

That is, the present invention provides an anti-human type IV collagen monoclonal antibody JK-199 that recognizes human type IV collagen and a human type IV collagen decomposition product, and an anti-human type IV collagen monoclonal antibody JK-199 that recognizes human type IV collagen but not a human type IV collagen decomposition product. The purpose of the present invention is to provide an anti-human type II collagen monoclonal antibody JK-132.

Hereinafter, the configuration of the present invention will be explained in detail.

The anti-human type IV collagen monoclonal antibody of the present invention is obtained by immunizing a mammal with type IV collagen, fusing the obtained immunocompetent cells with myeloma cells that can be subcultivated to form a hybridoma, and selecting the target hybridoma from among these hybridomas. It is produced by selecting cells that produce only monoclonal antibodies and culturing them in large quantities under conditions that allow them to produce antibodies.

Furthermore, the human type IV collagen used in the present invention can be prepared, for example, by the salt-based precipitation method described in Sekisei Kagaku Jikken Koza 6, page 465. The material for extracting human type IV collagen may be any human tissue containing a basement membrane, such as skin, kidney, placenta, etc. In the present invention, the bipridoma can be prepared using known methods such as Ha
It is carried out according to the method described in ture256, 495-497 (1975).

The animal to be immunized in the present invention may be any animal other than humans, such as pigs, cows, rabbits, mice, rats, etc., but mice are particularly preferred since the myeloma cells that serve as antibody-producing cells are derived from mice. The type ■ collagen prepared by the above method was prepared using the method of 5tahli et al. (J, Immunol,
Methods Vol, 32, 297-304.1
Mice are immunized by subcutaneous injection according to 980). The injection method is not limited to this, and may be intravenous injection or intraperitoneal injection. Mice are boosted as needed.

Three days after the final immunization, the spleens containing antibody-producing lymphocytes are removed from these mice. The tissue to be removed may be any peripheral lymphoid tissue, such as a lymph node. Lymphocytes are isolated as single cells from the obtained tissue, for example, by the method described in Immunological Experiment Procedures B (Published by the Japanese Society of Immunology, 1974, p. 1253). Next, the lymphocytes are given the ability to proliferate so that they can be subcultured semi-permanently. Methods include E, B, -virus and Δbelson-virus.
A method of infecting and transforming lymphocytes with viruses such as Sendai virus, in the presence of polyethylene glycol,
There are methods such as cell fusion with certain cancer cells. In order to continue producing stable antibodies, cancer cells derived from the same mouse or the same species of mouse, such as mouse myeloma cells, are used as a partner for the lymphocytes. The actual cell fusion method is generally described in J. Immunol, Method.
39, 285-308 (1980). That is, the above-mentioned two types of cells are fused in the presence of polyethylene glycol in HAT medium (hyboxanthin, aminopterin here) in which only hybridomas can grow.

Grow in thymidine-supplemented medium). Once hybridoma colonies are confirmed, the antibodies in the culture solution are screened. As a screening method, for example, the method of Collagen Experimental Method (Kodansha, p. 496) may be followed. That is, one drop of human type IV collagen phosphate M surgical solution solution (100 μg/ml) is added to each well of a vinyl chloride microplate and left overnight at 4°C. IO after removing human type IV collagen solution
A phosphate buffer solution containing X beef tallow serum is added to each well for blocking, and after the blocking solution is removed, a drop of the supernatant of each biplidoma colony is added to separate predetermined wells. After the reaction supernatant is removed and washed, a secondary antibody is added to confirm the hybridoma producing the desired antibody. The secondary antibody used is an anti-immunoglobulin that binds to the antibody produced by the hybridoma, and is labeled with a fluorescent substance, enzyme, radioactive isotope, etc. to show that it has bound to the antibody. This may be a commercially available product or may be prepared by yourself using a commonly used method. By repeating the limiting dilution method for hybridomas producing the antibody of interest, a colony consisting of a single hybridoma (referred to as a clone) can finally be obtained. Monoclonal antibodies produced by clones can be separated and purified from cell culture fluid, but since the antibody titer is generally high, the supernatant can be used as is, depending on the purpose. It is also possible to use monoclonal antibodies with extremely high antibody titers present in the ascites fluid and serum produced by injecting clones into the peritoneal cavity of mice pretreated with Blistane. These antibodies can be purified by an appropriate combination of general biochemical techniques such as salting out, ion exchange, gel filtration, affinity chromatography, and electrophoresis.

The human type II collagen monoclonal antibodies used in the present invention include those purified as described above, as well as preparations obtained in the state of culture supernatant, serum, and ascites. The thus obtained human type ■ collagen monoclonal antibody is directly reacted with the antigen, and as a secondary antibody, an antibody labeled with a fluorescent substance such as fluorescein isothiocyanate, or an enzyme such as alkaline phosphatase or peroxidase with a radioactive isotope such as 125■ is used. The presence of antigen can be confirmed by binding immunoglobulin to the membrane. Furthermore, the human type IV collagen monoclonal antibody itself may be used after being labeled with a fluorescent substance such as fluorescein inthiocyanate, or a radioactive isotope such as enzyme 12J such as alkaline phosphatase or peroxidase.

[Example] The present invention will be explained in more detail with reference to Examples below.

[Example 1] a) Preparation of human type II collagen as an antigen 2750 g of fresh human placenta was shredded, washed, homogenized with a positron homogenizer, and then centrifuged with cold ff (10,000 g).
Xg, 40 minutes) t, te, precipitate! After suspending in 02M IJ sodium phosphate solution, cryocentrifugation was repeated three times. The obtained precipitate was suspended in 4 L of 0.5 M acetic acid, and the pH was adjusted with hydrochloric acid.
3, added pepsin, and allowed to react at 4°C for 7 days. The supernatant was collected by centrifugation (10,000×g, 1 hour), and the pH was adjusted to 8.5 by adding Tris and sodium hydroxide to inactivate pepsin for 2 days.

After neutralization, add sodium chloride 4 times a day, add glacial acetic acid to the solution, adjust the pl (3), and leave it overnight. After centrifugation (1
The supernatant was collected, 0.5M acetic acid was added to the precipitate, and the mixture was stirred for 5 hours. After centrifugation (10,
oooxg, 1 hour) Add 1 portion of sodium chloride to the supernatant.
．． Add 2 batches and leave to stand overnight. After centrifugation (10
,000 Mg, 1 hour), the precipitate was collected, dissolved by adding 0.5 M acetic acid, and only the dissolved portion (supernatant) was collected.

The supernatant was dialyzed against water and centrifuged to remove the precipitate.

Tris-sodium chloride solution is added to the obtained supernatant, and finally Tris is 0.1)4 and sodium chloride is 1.
．． I set it to 0M. The undissolved portion was removed by centrifugation, and the supernatant was salted out with 0.005M Tris-1.5M sodium chloride. The precipitate was collected by centrifugation and purified by phosphocellulose column chromatography to obtain 235 mg of human type II collagen.

b) Preparation of immunized lung cells 100 μl of phosphate buffer containing 100 μg of human type II collagen was prepared and intraperitoneally injected into BALB/C mice. A second immunization was performed in the same manner one week later.

BACTO-Adjuban during the first and second immunization
t (DIFCO!!A) was simultaneously injected into multiple cavities at a rate of 0.1 mt. One week later, a booster immunization was performed with a human type IV collagen solution.

72 hours after the final boost, the lungs were aseptically removed from the mice, sectioned with scissors, and passed through a mesh to obtain a suspension of lymphocytes. To remove red blood cells, the cells were suspended in a hemolysis buffer, centrifuged at 1500 rpm for 5 minutes, and washed twice with RP14I-1640 culture solution. On the other hand, mouse myeloma cells (SP210) as fused cells
) were incubated in RPMI-1640 medium containing 10% calf serum (FCS) with 10% CO2,37 from two days before fusion.
The cells were grown under 0C conditions. 100 x 106 lung cells,
Mix the culture medium containing 100x106 myeloma cells, centrifuge at 1500 rpm for 30 minutes, discard the supernatant, and 50%
Polyethylene glycol 4000 (Merck & Co., Ltd.) 1
The cells were slowly loosened by adding mL drop by drop. After 1 minute, the cells were mixed while slowly dropping 1 d of RPMI-1 (540 medium) containing FCS, and the same operation was repeated for 1 minute.
7 mL of 640 culture solution was slowly added over 3 minutes while rotating the centrifuge tube. At this point, add the culture medium to a coarse solution so that 200 x 106 cells are contained in 50 mL of culture medium, and add 0 and 1 rrL each of this cell suspension to 9
Dispense into a 6-well microplate and place under 10X CO2.
Cultured at 7°C (100X relative humidity). In addition, mouse noble phagocytes were added in advance to the microplate at a ratio of 10 cells/well, and RP old-164 containing FCS was added in advance.
0 culture solution that already contained IIAT (formerly hypoxanthine Δ niaminopterin T: thymidine) at the time of dispensing was used. According to this improved method, there is no need to change the culture medium until at least the 5th day, and the chance of contamination with bacteria can be reduced, and colonies of vibridoma were observed by about 5 days. After 10 to 25 days, 0.2 microliters of the culture supernatant was taken and used for screening for the target antibody. Screening followed the method of Collagen Experimental Method (Kodansha, p. 496). In other words, human collagen type 10
One drop of 0 μg/ml phosphoric acid t1m solution was added to each well of a vinyl chloride microplate and left overnight at 4°C. After removing the human Lv type collagen i3' solution, 10XFCS-PBS solution was added to each well of 3i using a pipette, and blocking was performed for 1 hour. After removing the blocking solution, one drop of the supernatant of each hybridoma colony was added to separate designated wells. After reacting for 1 hour, the supernatant was removed and washed three times with PBS.

Anti-mouse IgG (F (A
n) 2 fragment) solution was added and left to stand for 30 minutes, and the secondary antibody solution was removed and thoroughly washed. One of the specimens of Tsuchiura specimen that emitted fluorescence was selected, four corresponding hybridomas were obtained, and these hybridomas were cloned by the limiting dilution method. First, biplidomas were diluted with a culture medium so that each well contained 0.3, 1.0, and 3 biplidomas. At this time, 1 million thymocytes were added per well to allow accurate dilution. The microplate was incubated at 37°C and 10χCO2100χ relative humidity. On the 5th day, the clones were examined under a microscope to check if each well contained one clone, and after two weeks of growth, the same limiting dilution was repeated. Repeat this operation three times to finally select two types of clones, transfer the clones to a regular culture vessel, and use 5 x 1
The cells were cultured until the volume of 0.6 cells reached 7 ml. The culture medium of one of the colonies was transferred to a centrifuge tube and centrifuged at 1500 rpm for 5 minutes to separate the supernatant and cells.
The 1640 culture solution was resuspended slowly by adding 0.5 m (l) of λ. To this cell suspension, RPMI-1 (540
A culture solution was added and subcultured. That is, 1 x 106 cells were placed in RPMI culture medium C containing 5 mL of FCS:: Soti
k Se5' diluted by adding liquid.

Cell suspension wIj, I diluted to I x 106 cells/ml
Tnt was subcultured by adding 4 mε of culture solution, and 5×10 7 cells were obtained by centrifugation from the 50rr culture solution that had been subcultured for 10 generations and suspended in 5 L of culture solution. Dispense this into 10 rotating culture bottles and collect 5χCO
The cells were cultured for 5 days at 337°C, 100X relative humidity, and 1 rpm. Cells were removed by centrifugation from the culture solution that reached 5xlO6 cells/rnt, and the mixture was filtered with suction, and ammonium sulfate was added to the filtrate to give a precipitate of 45x. The precipitate was further purified with protein A. 73m from a total of 15L of culture solution
g monoclonal antibody was obtained as a powder. This monoclonal antibody showed a single band in 5DS-polyacrylamide gel electrophoresis and was named JK-199.

[Example 2] The properties of JK-199 were investigated.

a) Reaction specificity of JK-199 Various antigens were dissolved in phosphoric acid (fj) solution to a concentration of 1 μg/ml, and these solutions were dropped into each well of a vinyl chloride microplate at an accuracy of 0 and 1 ttr. The plate was left at °C overnight. After removing the antigen solution, 0.5 ml of 10X fetal bovine serum was added to each well and blocked for 1 hour.

After removing the blocking solution, 0 and 1 ml of JK-199 prepared at 10 ug/ml was dropped into each well and reacted for 1 hour. After thorough washing with phosphate buffer, peroxidase-labeled anti-mouse IgG antibody (
Cappe 1) was added to each well in an amount of 0.2III and allowed to react for 1 hour. After removing the secondary antibody solution, the wells were washed four times with phosphate buffer, and a substrate solution containing O,0OIX hydrogen peroxide was added to each well at 0.1 tN. Substrate solution. - Phenyl diamine immediately before use 0.1) 1 citric acid - 0
, 0.4 dissolved in 214 phosphate buffer (pt14.8)
mg/mi was used. After 10 minutes, 20 μl of 8M sulfuric acid solution was added to each well to stop the reaction, and the absorbance at 492 nm was measured using a microplate photometer. The results are shown in Table 1.

(hereinafter referred to as Kinpaku) (Table 1) Anti-human type ■ collagen monoclonal antibody JK
-199 for various antigens *The numerical value is the absorbance of each sample when the absorbance at 492 nm of the phosphate buffer used is set as 0.00 *S P2 / O is the value for myeloma cells (S
As shown in Table 1, JK-199 contains other types of collagen derived from humans, namely human E type collagen and human II type collagen.
It did not recognize type I collagen, human type ■ collagen, or bovine type lv collagen, nor did it recognize human fibronectin, bovine laminin, or mouse laminin.

b) Reaction specificity to tissue sections A frozen section of normal human cervical tissue (7 μm in thickness) collected from the human cervical tissue of a 38-year-old woman was placed on a slide glass, and 0.2 ml of JK-199 solution was added dropwise. After leaving it at room temperature for 30 minutes, it became 0. IM pH 7,0 4°
Washed with C phosphate (!7 solution) three times for a total of 15 minutes. This was followed by fluorescein-labeled anti-mouse IgG antibody (manufactured by Tabo).
After adding 0.2 drops of the solution and leaving it at room temperature for 30 minutes, the plate was washed with phosphate buffer three times for a total of 15 minutes. After draining well, 50
X-glycerin was added dropwise and a cover glass was placed on top. This was observed with a fluorescent a mirror (manufactured by Olympus Optical Co., Ltd.).

As a result, JK-199 reacted with type IV collagen along the basement membrane in normal human cervical tissue, emitting band-shaped fluorescence.

Next, the specificity of the reaction to human type IV collagen degradation product was investigated.

C) Preparation of degraded human type ■ collagen: 500 μg/ml of human type ■ collagen, 0. IMh
Lis-HCl buffer solution (5Il1M calcium chloride, 0.
2M sodium chloride, 10mM N-methylmaleic acid), CollagColla Fora+lI
t (advance biofactors Co.
rporati.

After applying 100 units (manufactured by n company) at 37°C for 2 hours,
The reaction was stopped by adding one drop of 10mM EDTA. A reduced human type IV collagen decomposition product was also prepared by adding an excess of 2-mercaptoethanol in phosphate buffer to the colander, boiling for 3 minutes, and decomposing it with collagenase.

d) Reaction specificity for human type II collagen degradation product The experimental method followed the immunoblotting method described in Collagen Experimental Methods (Kodansha, page 500). 5DS-polyacrylamide gel electrophoresis was performed on the human type IV collagen degraded product prepared by the above method using collagenase using a standard method. Two nitrocellulose membrane filters were placed on top of this gel, and an AT buffer for transfer (1 oz methyl alcohol-25ffiM tris-glycine buffer (pH 8,3
)) at 40 mM for 2 hours. The filter on the side that was in contact with the gel was used for immunoreaction, and the other one was used for staining protein bands. The filter for immune reaction is 10m.
Blocking was performed with 10 pieces of FCS-phosphate buffer and placed on a rose film. 0.5 JK-199 solution
Add it on the ml filter and put a loose film on it.1
Allowed time to react. After the reaction, wash three times with phosphate buffer and use peroxidase-labeled anti-mouse IgG as a secondary antibody.
Antibody (Cappe 1 rA) was added at 0.5a+1 and reacted for 1 hour. After the reaction, dilute with phosphoric acid once and Tween.
80-Phosphoric acid! ! 2 times with surgical solution, 1 time with phosphate buffer
Washed twice. Add 3mg/ml 4- as a substrate solution to this.
Methyl alcohol solution of chloro-1-naphthol 5ml
．． Phosphate buffer 25ml 30X hydrogen peroxide 10μ
1 was mixed immediately before, and the reaction was allowed to proceed for 5 minutes, and the reaction was stopped by moving the filter into distilled water. Comparing this with the protein band staining results, JK-199 recognized fragments of human type IV collagen degraded by collagenase with a molecular weight of 5,000 or more. The results of immunoblotting are shown in FIG. When immunoblotting was performed using a similar method on a degraded product of human type IV collagen that had been degraded with collagenase and then with 2-mercaptoethanol, fragments with molecules ff of 15,000 or more were also recognized.

e) Molecular weight of JK-199 The molecular weight of JK-199 was 140,000 to 160,000 from 5DS-polyacrylamide gel electrophoresis.

f) Immunoglobulin class of JK-199 JK-19
Identification of the immunoglobulin class of 9 was performed by ophthaloni-double immunodiffusion and was found to be IgG2a.

[Example 3] JK-199 was modified with peroxidase and its recognition specificity for human type IV collagen was investigated. peroxidase
Type II (manufactured by Sigma) 5mg was dissolved in 1L of 0.3M sodium bicarbonate aqueous solution pH 8, 1, 1, 0ml, and 0.1ml of 1% fluoronitrobenzene solution dissolved in 100x ethanol was added and mixed slowly at room temperature. . Roughly 0.05H NaIO4 solution 1,0 m
0.1 was added and reacted at room temperature for 30 minutes for quenching.
1.0 me of 2M ethylene glycol solution was added and left at room temperature for 1 hour. The reaction solution is a sufficient amount of 0. OIM p
It was dialyzed against H9.5 sodium carbonate buffer at 4°C to obtain a peroxidase-aldehyde solution. Next, the monoclonal antibody JK-199,5m obtained in Example 1
Dissolve g in 1++yl sodium carbonate buffer and add 3m
The mixture was added to 3 mL of peroxidase-aldehyde solution of L and mixed at room temperature for 3 hours. Add 5mg of NaB to this solution
11+ was added and left overnight at 4°C. The reaction product was dialyzed against phosphoric acid water and purified using a Sephadex G-200 column. The desired two fractions were obtained by tracking the protein at 280 nm % peroxidase using ultraviolet absorption at 403 nm, and fractionating the two fractions in which the two values matched. Both portions were prepared using phosphate buffer solution 6 containing 4.8 mg of JK-199.
It was d.

[Example 4] The reactivity of JK-199 labeled with peroxidase to human type II collagen degradation product was measured.

Using the human type IV collagen degradation product prepared in Example 2, 5.0 mg/wt, 2.5 mg/ml, 1.0 mg/
A phosphate buffer solution of mt was prepared. This antigen solution was diluted with phosphoric acid solution (!?) to give 5000ng/++
+1, 2500ng/me, 11000n/m
l, 500ng/mt, 1100n/ml, 50ng/
A solution of 10 ng/ml of me was prepared. Accurately 0.1 ml of these solutions was dropped into each well of a vinyl chloride microplate and left overnight at 4°C. After removing the antigen solution, 10% fetal bovine serum was added to each well for 0.5+d and blocked for 1 hour. After removing the blocking solution, add 0.1 ml of JK-199 prepared in Example 3.
It was dropped into each well and allowed to react for 1 hour. After thorough washing with phosphate buffer, 0. Substrate solution containing 0.1% OOI% hydrogen peroxide was added to each well. The substrate solution was prepared by dissolving 0-phenylenediamine in 0.1M citric acid-0.2M phosphate buffer (pH 4.8) immediately before use.
The following was used. After 10 minutes, 20 μl of 8M sulfuric acid solution was added to each well to stop the reaction, and the absorbance at 492 nm was measured using a microplate photometer. As a result, it was found that at least 5 ng/- of degraded human type IV collagen was recognized.

[Test Example 1] Type 1v collagen in human cervical tissue was observed using JK-199. A frozen section of normal human cervical tissue (7 μff1 in thickness) collected from the cervical tissue of a 50-year-old woman was placed on a slide glass, and 0.2 mft of JK-1 was placed on the specimen.
99 solution was added dropwise and left at room temperature for 30 minutes. IMP
Washed three times with 117.0 4°C phosphate buffer for a total of 15 minutes.

To this was added 0.2 drops of fluorescein-labeled anti-mouse IgG antibody (manufactured by Tabo), left at room temperature for 30 minutes, and then washed with phosphate buffer three times for a total of 15 minutes. After thoroughly draining water, 50x glycerin was added dropwise and a cover glass was placed on top.

This was observed using a fluorescence microscope (manufactured by Olympus Optical Co., Ltd.).

As a result, JK-199 reacts with type IV collagen along the basement membrane in normal human cervical tissue, emitting band-shaped fluorescence, and JK-199 recognizes type IV collagen in human cervical tissue by indirect fluorescent antibody method. It was revealed that it would.

[Example 5] In Example 1, cloning was performed and two types of clones were finally obtained, one of which was a human type IV collagen decomposition product that specifically reacted with human type IV collagen and had a molecule I of 5000 or more. It has been found that these cells produce the anti-human ■type monoclonal antibody JK-199, which specifically recognizes JK-199. Here, another 1a! which reacts with human type IV collagen according to the procedure of Example 1! clones were cultured. That is, 106 cells te 5mN (7) FC
Suspended in RPMI culture medium containing S f5%C0237°
Culture was performed under conditions of C100X relative humidity. 5X after 60 hours
The cells multiplied to 10B cells, and this operation resulted in a first generation culture.

The cells were separated from the medium gI solution by centrifugation and diluted with fresh culture medium. Cells diluted to 1X106 cells/rIrt! ! Subculturing was carried out by adding a 4-ml culture solution to 1 d of the suspension, and 5 x 10' cells were obtained by centrifugation from 50 ml of culture solution that had been subcultured for 10 generations, and 5 L of culture medium was obtained. Suspended in liquid. This was dispensed into 1° rotating culture bottles and cultured for 5 days at 5% CO2, 37° C., 100× relative humidity, and 1 rpm. Cells were removed by centrifugation from the culture solution that reached 5×10 6 cells/me, and the cells were filtered with suction, and ammonium sulfate was added to the filtrate to give a concentration of 45% to obtain a precipitate.

The precipitate was further purified using protein A. Total 15L
92 mg of monoclonal antibody was obtained as a powder from the culture solution. This monoclonal antibody showed a single band in SOS-polyacrylamide gel electrophoresis, and JK-1
Named 32.

[Example 6] The properties of JK-132 were investigated.

a) Reaction specificity of JK-132 According to the method of Example 2, the reaction specificity of JK-132 to various antigens was investigated. The results are shown in Table 2.

/;, 1. 1) (Table 2) Anti-human type ■ collagen monoclonal antibody JK-132
Recognition specificity for various antigens * Values are the absorbance of each sample when the absorbance at 492 nm of the phosphate buffer used is set as 0.00 * S P 2 / O is for myeloma cells (S
As shown in Table 2, JK-132 is a sample of the culture solution of P/2). , bovine type IV collagen, and human fibronectin, human laminin, and mouse laminin.

b) Reaction specificity to tissue sections The reaction specificity was investigated using the same indirect fluorescence method as in Example 2 on a frozen section of normal human cervix tissue collected from a 51-year-old woman. In the tissue, a strip of fluorescence was emitted along the basement membrane in response to type II collagen.

Next, the specificity of the reaction to human type IV collagen degradation product was investigated.

The immunoblotting method of Example 2 was used to prepare a degraded product of human type tV collagen and to measure the specificity of the reaction to a degraded product of human type 1 collagen.

As a result, JK-132 did not recognize fragments of less than 1,170,000 molecules among human type IV collagen degradation products.

The results of immunoblotting are shown in FIG. 2.

c) Molecular weight of JK-132 The molecular weight of JK-132 was found to be 140,000 to 160,000 by 5DS-polyacrylamide gel electrophoresis.

d) Immunoglobulin class JK-13 of JK-132
Identification of the immunoglobulin class of 2 was performed by ophthaloni-double immunodiffusion and found to be IgG2a.

[Example 7] JK-132 was fluorescently labeled for use in a direct fluorescent antibody method, and it was examined whether it recognized tV type collagen in tissue sections. Using 20 mg of JK-132 obtained in Example 6, fluorescently labeled JK-132 was added to 0.0 mg according to the method of Example 2.
A solution of 18 ml of 0.0114 phosphate buffer containing 5 M +4 ac L was obtained. This solution was found to contain 11 mg of JK-199 based on ultraviolet absorption at 280 nm.

A frozen section (7 μm thick) of human cervical normal tissue collected from the cervical tissue of a 45-year-old woman was placed on a slide glass, and 0.2 mL of fluorescently labeled JK-132 solution was added dropwise and left at room temperature for 30 minutes. After that, 0. IM pH7,
Washed three times with 0 4°C lyphosphate buffer for a total of 15 minutes.

After thoroughly draining water, 50x glycerin was added dropwise and a cover glass was placed on top. This was observed using a fluorescence Wt microscope (Olympus Optical Co., Ltd.!J). In normal human cervical tissue, band-shaped fluorescence was observed along the basement membrane in response to type IV collagen. This indicates that fluorescently labeled JK-132 is effective for identifying type IV collagen in human cervical tissue.

Example 8 JK-132 was labeled with peroxidase for direct enzyme antibody method according to the method of Example 3, and it was examined whether it recognized IV pear collagen in tissue sections. J obtained in Example 5
From 5 mg of K-132, 7 mt of phosphate buffer containing 4.0 mg of JK-132 labeled with peroxidase was obtained.

A frozen section (thick 7 μl) of human gastric normal tissue collected from the stomach of a 45-year-old man was placed on a slide glass, 0.2 mL of JK-132 labeled with peroxidase was added dropwise, and left at room temperature for 30 minutes. IM pHB, 8 of 4
Washed with 00 phosphate buffer three times for a total of 15 minutes. This was immersed in the substrate solution for 30 minutes. The substrate solution contains 25 mg of diaminobenzidine and 0.005 mg of hydrogen peroxide.
0514 was prepared by dissolving it in 100 wL of Tris buffer having a pH of 7.0.

After the staining was completed, the specimen was washed three times with phosphoric acid solution for a total of 10 minutes, and 50X glycerin was dropped and covered with a cover glass.The brown-stained specimen was observed under a microscope.As a result, human stomach In normal tissues, coloration for type 1v collagen was observed in a band-like manner along the basement membrane.
JK-132 subjected to BR with peroxidase was found to be useful for confirming type IV collagen in human gastric tissue.

[Example 9] Preparation of affinity gel a) Preparation of JK-199 Fab fragment 25mg of JK-199 was mixed with 20mM HaH2PO4, 20mM cystein-HCI, 10mM EDTA-Na4
(pH 6,2) and 0.5a+1 immobilized papain was added thereto and reacted at 37°C for 5 hours. 15m1
10mM Tr 1s-11CI II W7 FH
(pH 7.5) was added and mixed, and then centrifuged. On top of this
<7125 ml was purified with protein A and 7.1 m
A Fab fragment of g was obtained.

b) Preparation of Affinity Glue 0.5g CNBr-activated 5epha
rose 4B (manufactured by Pharmacia) in 1mM HC
1. Swell with I and repeat the swelling on a glass filter.
It was made into a 1 ml gel.

The Fab fragment of JK-199 prepared in a) was dissolved in coupling buffer y-(0.1 Mpf (8, NatlCO3 buffer) containing NaCl (0,5 M), mixed with this gel, and incubated for 2 hours in a chamber). Stirred. The gel was transferred to blocking reagent (0.2 M glycine (pH 8, 7) and stirred at room temperature for 2 hours, then added to coupling buffer and acetate buffer (NaC) to wash away excess JK-199 Fab fragment and blocking reagent.
The gel was washed with 0.1 M pH 4 acetate buffer containing 1 (0.5 M). With this operation, 1.5ml of JK
An affinity gel using -199 was obtained. When this was used to purify crude collagen obtained by treating human placenta with pepsin, only human type IV collagen could be efficiently isolated.

Similarly, when an affinity gel was prepared using JK-132, it was found that only human type IV collagen could be efficiently separated.

[Effect of the invention] Anti-human type IV collagen monoclonal antibody J of the present invention
K-199 and JK-132 are useful as reagents for histological or biochemical studies of human biological connective tissues by analyzing the structure of human type IV collagen and observing the distribution and morphology of human type 1V collagen. It is. Furthermore, affinity gels prepared using these monoclonal antibodies are useful for purifying and fractionating human type IV collagen.

[Brief explanation of the drawing]

FIG. 1 shows the results of immunoblotting of JK-199 for human type IV collagen degradation products. FIG. 2 shows the results of immunoblotting of JK-132 against human type IV collagen degradation products.

Claims (1)

[Claims] (1) JK-132 and JK-19 having the following properties
Properties of anti-human type IV collagen monoclonal antibody (A) JK-199 selected from the group consisting of 9: 1) Reacts specifically with essentially all human type IV collagen. 2) Specifically recognizes human type IV collagen degradation products with a molecular weight of 5000 or more. 3) Molecular weight is 140,000 to 160,000 (SDS-polyacrylamide electrophoresis). 4) The immunoglobulin class is IgG2a. (B) Properties of JK-132 1) Reacts specifically with essentially all human type IV collagen. 2) The molecular weight reduced with 2-mercaptoethanol is 7
Does not recognize degraded human type IV collagen of less than 0,000. 3) Molecular weight is 140,000 to 160,000 (SDS-polyacrylamide electrophoresis). 4) The immunoglobulin class is IgG2a.