JPS60109528A - Antisuperoxide dismutase monoclonal antibody and hybridoma producing the same - Google Patents

Abstract

NEW MATERIAL:An antisuperoxide dismutase monoclonal antibody. USE:An immunological adsorbent. PREPARATION:An antisuperoxide dismutase (SOD) antibody obtained by subjecting a splenic cell collected from a mammal immunized with a superoxide dismutase (SOD) originating from mammals and a myelomatous cell of a mammal to the fusion, and cultivating the resultant hybridoma cells. The fusion of the splenic cell with the myelomatous cell is achieved by bringing both into contact in the presence of a fusion promoter, e.g. polyethylene glycol. The advantage of this method is as follows; A supply source for a specific antiSOD antibody uncontaminated by an antibody reactive with any of other antigen determining groups against SOD to be used, and by antigen impurity containied in the SOD pharmaceutical solution to be used. A large amount of a desired anti SOD antibody is easily obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to novel monoclonal antibodies produced by hybridomas.

More specifically, this invention provides novel anti-superoxide dismukosis antibodies produced by hybridomas,
The present invention relates to a hybridoma that produces the antibody, and a method for purifying superoxide di-sumkuse using the antibody.

Hereinafter, superoxide dismutase will be referred to as SOD, anti-superoxide dismutase anti-[=anti-SOD antibody], and anti-superoxide dismutase monoclonal antibody will be referred to as anti-SOD seven clonal antibodies.

The anti-SOD monoclonal antibody of the present invention can be prepared by cell culture of a hybridoma produced by fusing a mammalian myeloma cell with a spider cell collected from a mammal immunized with mammalian-derived SOD. .

Fusion of spider cells and myeloma cells is achieved by bringing them into contact in the presence of a fusion promoter (eg, polyethylene glycol). A small percentage of spider cells and myeloma cells are fused to produce a hybridoma. Furthermore, the hybridoma thus obtained produces various antibodies depending on the various fused spider cells.

However, it is possible to isolate hybridomas producing the desired antibody from the hybridomas thus obtained by cloning.

Hybridomas isolated in this manner can be grown in nutrient medium or intraperitoneally in mammals, and the antibodies produced can be isolated from culture supernatants or ascitic fluid or serum of the mammal by natural or artificial proteins. Purification can be achieved using conventional methods commonly used to isolate or purify proteins from sources. Such methods include centrifugation, dialysis,
Salting out with ammonium sulfate, DEAE-t! Isolation methods such as column chromatography using lurose, gel filtration using gels [e.g. Sepharose 4B, Sephadex, etc. (manufactured by Pharmacia Fine Chemicals)], affinity column chromatography using immunoglobulin-binding polysaccharides, freeze-drying, etc. Examples include purification methods.

The advantage of this method is that specific anti-SOD antibodies that are reactive with any of the other antigenic determinants against SOD used for immunization are not yet contaminated by antigenic impurities contained in the SOD preparation used. The objective is to provide a source of antibodies. Another advantage of this method is that
Most desired anti-S'OD antibodies are readily provided.

The anti-SOD monoclonal antibody of the present invention thus obtained has the ability to bind to SOD and can be used as an immunoadsorbent in immunoadsorption purification of SOD.

More specifically, the anti-80D monoclonal antibody of the present invention can be bound to a polysaccharide by reacting it with an active polysaccharide (for example, Bromcyan-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals)) using a conventional method.

Furthermore, anti-SOD monoclonal antibodies are useful as reagents for enzyme-linked immunosorbent assays for SOD.

The method of the present invention will now be described in experimental detail, which is intended to be illustrative and not limiting.

Example 1 Tuclonal antibody (hereinafter referred to as anti-Cu, Zn-8OD mono (1
) Preparation of immunized lung cells Cu, Zn-superoxide dismutase (hereinafter referred to as Cu
, Zn-8OD) (manufactured by Sigma, lot 101
F-9325) was prepared from human red blood cells and dissolved in phosphate buffered saline (hereinafter referred to as PBS) to a concentration of 1 mg/me. This Cu.

Zn-8OD solution 100/lj? diphtheria/tetanus toxoid/pertussis vaccine combination (Osaka University Microbiology Research Institute required, contains 2 x 1010 Bordetella pertussis in 1me) 1
B A L B / by intraperitoneal injection with 00/Iy.
Four days after administration of c.m., the mice were sacrificed and the lungs were collected.
Used for cell fusion.

Four mice were used for this purpose.

(11) Preparation of hyperdomas The lung cells immunized as above were prepared by the Cooler and Milsphine method [Nature, Vol. 256, pp. 495-497 (1975
Mouse myeloma cells P3
8 fused with U 1.

That is, lung cells were suspended in Eagle's Minimum Essential Medium (minimum essential medium, hereinafter referred to as D-MEM) modified by Mr. Grupecco.

The red blood cells in the suspension were dissolved in 0.83% ammonium chloride solution (
9 volumes) and 0.17 M) lisaminomethane-hydrochloric acid buffer (pH 7,65, 1 volume) at 4°C for 5 minutes, and the cells were removed by centrifugation. Maxmieloma cells and lung cells cultured in D-MEM supplemented with 15% fetal comb serum were washed three times each with D-M and EM.

A suspension of lung cells (3×10 8 cells) was added to a suspension of Maxmieloma cells (6×10′ cells). 50ml of mixed liquid
The mixture was thoroughly mixed in a plastic tube (50me Corning centrifuge tube manufactured by Corning Glass Works). The medium was removed by centrifugation. Cells in water bath 37
Warmed to ℃. Add 45% polyethylene glycol (manufactured by Sigma, average molecular weight 4.0) to the cells while shaking.
00) Gradually add the solution (1 me) over 1 minute,
The mixture was left at 37°C for 7 minutes. 37° to the reaction mixture
D-MEM and 15me were added dropwise at C for 5 minutes to stop the cell fusion reaction. After adding a large amount of D-MEM, the mixture was centrifuged to remove the upper groove. The residue contains 15% fetal bovine serum (manufactured by Centrus, lot 757), glutamine 2mM, and 2-mercaptoethanol 2xlσ5.
M1 streptomycin sulfate 100 pi'/ml! ,
Penicillin G 100 U/me, '&L acid'y'
tammycin 80 pf/me and fungizone 0
．． 25) Complete medium (hereinafter referred to as CM) to become D-'MEM supplemented with 19/me was added. After mixing the mixture briefly, the resulting fused cell suspension was transferred to a 24-well plate (
(manufactured by Nunc) 8 sheets with lung cells per quel of I x 10
1me each was dispensed into each well. The cells were cultured at 37°C in a 5% carbon dioxide atmosphere. One day later, aminopterin (
4X I0-7M), thymidine (1,6X10-5
1 mt' of CM (HAT medium) containing M) and hypoxanthine (IX10'M) was added to each well. After another day, half of the medium was removed from each well by suction, and HAT medium was added to each well for 8 days every 2 days and every 3 days of I/i, and the medium was replaced. Next, half of the medium in each well was mixed with hyboxanthin (1 x 10-4M) and thymidine (1,6X
The medium was replaced with CM (HAT medium) containing 10-5 M). After another 2 days, half a liter of medium in each well was CM
exchanged with.

Thirteen days after cell fusion, proliferation of hybrid cells was observed in almost all wells.

Assay of anti-Cu, Zn-8OD antibodies Cu, Zn-8OD (
100 μe of a solution of 50 At/me) was added and the plate was kept at 37° C. for 3 hours to allow the wells to adhere with Cu, Zn-8OD. Next, a solution of comb serum albumin (10 my/me') was added to completely block the unattached portions of the wells with the above protein. After removing the blocking solution, 100 ml of the hybridoma culture supernatant obtained above was added to each well and incubated at 37°C for 90 ml.
It lasted for a minute. After washing three times with PBS, 100 pl of 125-labeled goat anti-Max B'(ab') 2 solution (10,000 cpm, specific activity 5 μC1/) 'f IgG ) purified by affinity column chromatography was added and incubated at 4°C. I kept it overnight. Radioactivity in each well was measured using a gamma scintillation force Kunter. Anti-Cu, Zn
-8OD activity was detected in 96 out of 173 cultures measured.

(1v) Cloning of anti-Cu, Zn-8OD antibody-producing hybridomas Culture medium of 10 hybridomas showing high binding ability to Cu, Zn-8OD was added to a feeder layer of BALB/c mouse thymocytes (5 x 106 cells/ me') and was cloned by the limiting dilution method in a 96-well flat-bottomed microplate (manufactured by Nunc).

(v) Purification of anti-Cu, Zn-8OD monoclonal antibodies The hybridoma obtained above was intraperitoneally transplanted into BALB/c macus to which tetramethylpentadecane had been administered one week earlier. One to three weeks later, ascitic fluid was collected from the abdominal cavity of the mouse, and anti-Cu and Zn-8OD antibodies were isolated from the ascitic fluid by a precipitation method using a 50% saturated ammonium sulfate solution.

That is, hybridoma cells were removed from ascites by centrifugation, and ammonium sulfate was gradually added to the supernatant while stirring until the final concentration reached 50% saturation. The mixture was stirred for 30 minutes under water cooling and allowed to stand for 30 minutes. After centrifugation, the residue was added to a small amount of 10mM PBS (pH
8,0), dialyzed against 10mM PBS,
D equilibrated with 0 mM PBS (pH 8,0)
EAE cellulose (DE52, Whatman Chemical
Separation Co., Ltd.) column chromatography. The solution passed through the column was collected and used as an immunoadsorbent in the Cu, Zn=SOD purification process, the details of which are described in Example 2.

(Identification of immunoglobulin class of vtl antibody Add saturated ammonium sulfate to the culture supernatant (500me),
It was left on ice for 1 hour. The precipitate obtained by centrifugation was dissolved in Japanese PBs (50M).

The immunoglobulin of the antibody thus obtained was identified by Ophthalony's double immunodiffusion method.

A goat polyclonal antibody (manufactured by Miles) was used to identify subclasses of monoclonal antibodies.

The anti-Cu, Zn-SOD monoclonal antibodies thus obtained are as follows.

(1) Anti-Cu produced by hybridoma 1-9-2.

Zn-8OD monoclonal antibody 1-9-2: (a) Subclass: IgG.

(2) Anti-Cu produced by hybridoma 1-66-18
, Zn=SOD hepclonal antibody 1-66-18: (a) Sag class: IgG.

(3) Anti-Cu produced by hyglidoma 1-41-10
, Zn-8OD monoclonal antibody 1-41-10: (a) subclass HIg ().

Example 2? (1) Binding of anti-Cu, Zn-8OD monoclonal antibody to Bromcyan-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals) Bromcyan-activated Sepharose 4B (1.3 g) was mixed with 1 mM hydrochloric acid and then with 0.1 M bicarbonate. Sodium (px+s, 0) and 0.5M
A suspension of bromcyan-activated sepharose in coupling buffer (9me) was prepared by washing sequentially with coupling buffer containing sodium chloride. This solution 2
me, anti-Cu, Zn-8OD seven clonal antibodies 1-9-2.1-66-18 and l-41-10 prepared by dialysis against the above coupling buffer in Example 1.
(5 my protein) of coupling buffer solution 1 ml each
Added e. The resulting mixture was shaken at 4° C. overnight until coupling was complete. After centrifugation, 0.2M glycine (pH 8, 0, 2m(')) was added to this, and the antibody-bound Sepharose 4B was shaken at room temperature for 2.5 hours to block the remaining active sites. After blocking, the resulting antibody The bound Sepharose 4B was added to a 0.1M acetate buffer (pH 4,0) containing 0.15M sodium chloride and the above coupling buffer.
) and 25 mM sodium phosphate buffer (pH 7.4) containing 0.5 M sodium chloride.
). The thus obtained bromocyan-activated Sepharose 4B-conjugated antibody (hereinafter referred to as antibody-conjugated Sepharose, particularly columns 1-9-2, 1-66-181 and 1-41-10, respectively) was used for affinity lamb chromatography. Used for column preparation.

(If) Adsorption and elution of Cu, Zn-SOD using antibody-bound Sepharose 4B.
．． Pack into a column equilibrated with 25mM sodium phosphate buffer (pH 7.4) containing 5M sodium chloride.

Cu, Zn-8OD (manufactured by Sigma, lot 52F-93
45. Specific activity: 2235 cytochrome units or 13,000 nitrite units per mg of protein (1,
900 nitrite units/148μy10. , 2me) on a column and 0.5M chloride) containing IJ cum.
The cells were washed with M phosphate buffer (pH 7.4) at a flow rate of 5 me/hour, and eluted with 0.2M glycine hydrochloride solution (pH 2.5). Cu, Zn-8OD was prepared by Elstner et al. (Analytical Biogmistry, Vol. 70, p. 616,
(1976) by a modified nitrite method. According to this method, the amounts of Cu, Zn-, and SOD necessary to achieve 50% inhibition (1 unit) are determined by the cytochrome method (Matsukhood et al., Journal of Biological Chemistry, Vol. 244). No. 6049
Page, 1969) was sufficient. That is,
One nitrite unit is equal to 6 nitrite units. Superoxide radicals were generated using a xanthine-xanthine oxyguse system at pH 8.2. Cu, Zn −
Since SOD activity was hardly detected in the flow-through fractions of each column, most of the Cu, Zn-SOD activity
was thought to be bound to the column. Eluate 0, 15M
25mM sodium phosphate buffer containing sodium chloride (p
Dialyzed against H7,4) overnight at 4°C.

41-10 61 82 Example 3 Antibody-conjugated Sepharose 4B (prepared in Example 2 (1)) was applied to the column.
That is, each column (column 1-9-2, column 1-66-18, column l-41-10) (0,6me) contains Cu, Zn-3O from human red blood cells prepared by the Dobashi method.
D extract (100 nitrite units/260/ly protein/me
, specific activity: 280 nitrite units per mg of protein). In this case, Cu, Zn-SOD was completely bound to the column. A 0.2M glycine-hydrochloric acid buffer (pH 2,5) was used for elution. The specific activity of the activity peak was measured.

Column Specific Activity Yield Column 1-. 9-2 6.700 45 column 1-66
-18 4.600 31 Column 1-41-10 4.
100 22 Example 4 Human serum Cu using antibody-conjugated Sepharose 4B.

Antibody-conjugated Sepharose 4B (
Each column (column 1-9-2, column 1-66-18, column 1-41-10) (0,5-0,6me) was filled with commercially available human serum containing 0.5M sodium chloride. 25mM sodium phosphate buffer (pH 7,0) and commercially available human Cu, Zn-SOD (manufactured by Sigma,
Lot 52F-9345, 1mg/me) (10:
A solution consisting of 10:1) was applied.

Total protein: 185,000 μ2 Total activity: 2,650 nitrite units Specific activity: 14 nitrite units per mg of protein 0.
280 n of 25 mM sodium phosphate buffer (pH 7.4) solution containing 5 M sodium chloride at a flow rate of 5 me/h.
Cleaning was performed using a knife that reached an optical density of 0.05 or less. Then, elution was performed with a 0.2 M glycine hydrochloride (pH 2,5) solution at an elution rate of 10 me1 hours. The eluate was diluted with 10 m containing the same amount of bovine serum albumin (200 py/me).
The mixture was diluted with M phosphate buffer (pH 7.4) and dialyzed against 5mM phosphate buffer at 4°C overnight.

Column 1-66-18 230 2.300 87 1
0.000 710 column 1-41-10 230 1
．． 600 60 7.000 500 applicants Fujisawa Pharmaceutical Co., Ltd. Continued from page 1

Claims (1)

[Claims] (1) Anti-superoxide dismutase mo7 clonal antibody. (2) A hybridoma producing an anti-superoxide dismutase monoclonal antibody. (3) A method for purifying superoxide dismutase, which is characterized by using anti-superoxide dismutase as an immunoadsorbent.