JPH05184359A - Anti-superoxide dismutase monoclonal antibody and hybridoma producing the antibody - Google Patents

Abstract

PURPOSE:To provide a new monoclonal antibody useful as an immunoabsorbent, etc. CONSTITUTION:An anti-superoxide dismutase monoclonal antibody (subclass: IgG1), e.g. anti-Cu,Zn-SOD monoclonal antibody. The exemplified monoclonal antibody can be produced from hybridoma 1-9-2.

Description

Detailed Description of the Invention

This invention relates to a novel monoclonal antibody produced by a hybridoma. More specifically, the present invention relates to a novel anti-superoxide dismutase antibody produced by a hybridoma and a hybridoma producing the antibody. Hereinafter, superoxide dismutase will be referred to as SOD, anti-superoxide dismutase antibody will be referred to as anti-SOD antibody, and anti-superoxide dismutase monoclonal antibody will be referred to as anti-SOD monoclonal antibody.

The anti-SOD monoclonal antibody of the present invention is a cell culture of a hybridoma produced by fusing spleen cells collected from a mammal immunized with a mammal-derived SOD and mammalian myeloma (myeloma) cells. Can be prepared by Fusion of splenocytes and myeloma cells is achieved by bringing them into contact with each other in the presence of a fusion promoter (eg, polyethylene glycol). A small proportion of splenocytes and myeloma cells are fused to produce hybridomas. Furthermore, the hybridomas thus obtained produce different antibodies depending on each of the different fused splenocytes. However, it is possible to isolate a hybridoma producing a desired antibody from the thus obtained hybridoma by cloning.

The hybridoma thus isolated can be grown in a nutrient medium or in the abdominal cavity of a mammal, and the produced antibody is natural or artificial from the culture supernatant or the ascites or serum of the mammal. Proteins can be purified by standard methods commonly used to isolate or purify proteins from various protein sources. Such methods include centrifugation, dialysis, salting out with ammonium sulfate, DEAE.
Column chromatography using cellulose, gel filtration using gel [eg Sepharose 4B, Sephadex, etc. (Pharmacia Fine Chemicals)],
Examples include isolation and purification methods such as affinity column chromatography using immunoglobulin-bound polysaccharide, lyophilization and the like.

The advantage of this method is that SO used for immunization
Specific anti-SOD uncontaminated by an antibody that reacts with any of the other antigenic determinants to D or by the antigenic impurities contained in the SOD preparation used
To provide a source of antibodies. Another advantage of this method is that large quantities of the desired anti-SOD antibody are readily provided. The anti-SOD of the present invention thus obtained
The monoclonal antibody has the ability to bind to SOD,
It can be used as an immunoadsorbent in the immunoadsorption purification of SOD. More specifically, the anti-SOD monoclonal antibody of the present invention can be bound to a polysaccharide by reacting with an active polysaccharide [eg, bromocyan-activated Sepharose 4B (Pharmacia Fine Chemicals)] by a conventional method.
Furthermore, the anti-SOD monoclonal antibody is also useful as a reagent for the enzyme linked immunosorbent assay (enzyme immunoassay) of SOD. The method of the present invention will now be described with experimental details, which are intended to be illustrative and not limiting.

Example 1 Preparation of anti-Cu, Zn-superoxide dismutase monoclonal antibody (hereinafter referred to as anti-Cu, Zn-SOD monoclonal antibody) (I) Preparation of immune splenocytes Cu, Zn-superoxide dismutase (hereinafter anti-C)
u, Zn-SOD) (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 / ml. This Cu, Zn-SOD solution 10
0 μl of diphtheria / tetanus toxoid / pertussis vaccine mixture (manufactured by Osaka University Institute for Microbiology, 1 ml pertussis 2
X 10 ¹⁰ containing) B by intraperitoneal injection with 100 μg
It was administered to female ALB / c mice. 28 days later, C
100 μl of u, Zn-SOD solution was intravenously administered. Four days after the second administration, the mice were sacrificed, their spleens were collected,
Used for cell fusion. Four mice were used for this purpose.

(II) Preparation of hybridoma The spleen cells immunized as described above are subjected to the method of Koehler and Milstein [Nature, 256, 495-497 (1975).
Year)] by mouse myeloma cells P3 × 63Ag8U
fused with l. That is, splenocytes were suspended in Dulbecco's modified Eagle's minimum essential medium (minimum essential medium, hereinafter referred to as D-MEM). The red blood cells in the suspension were mixed with 0.83% ammonium chloride solution (9
Volume) and 0.17 M trisaminomethane-hydrochloric acid buffer solution (pH 7.65, 1 volume) for 5 minutes at 4 ° C. to destroy and to remove by centrifugation. Mouse myeloma cells and splenocytes cultured in D-MEM supplemented with 15% fetal bovine serum were washed with D-MEM three times each.

Mouse myeloma cells (6 × 10⁷Pieces)
Splenocytes (3 x 10⁸Individual) suspension. Mixed
Use a 50 ml plastic tube (Corning
In a Las Works 50 ml Corning centrifuge tube)
Mixed well. The medium was removed by centrifugation. Water cells
Warmed to 37 ° C in bath. Shake this cell
45% polyethylene glycol (manufactured by Sigma, average
Gradually add 4,000) solution (1 ml) over 1 minute
In addition, the mixture was left at 37 ° C. for 7 minutes. Reaction mixture
15 ml of D-MEM was added dropwise to the mixture at 37 ° C for 5 minutes.
To stop the cell fusion reaction. Add a large amount of D-MEM
After that, the mixture was centrifuged to remove the supernatant. To the remnant
15% fetal bovine serum (Centour, Lot 75)
7), glutamine 2 mM, 2-mercaptoethanol 2
× 10^-FiveM, streptomycin sulfate 100 μg / m
1, penicillin G100U / ml, gentamicin sulfate
80 μg / ml and Fungizone 0.25 μg / ml
Complete medium that becomes D-MEM supplemented with (hereinafter referred to as CM)
Was added. After mixing the mixture a little, the resulting fused cell suspension
Spleen cells into 8 24-well plates (Nunc)
1 x 10 per well⁶1 ml for each well
We dispensed each. The cells were cultured at 37 ° C in a 5% carbon dioxide gas atmosphere. 1
A day later, aminopterin (4 × 10^-7M), thymidine
(1.6 x 10^-FiveM) and hypoxanthine (1 x 10
^-Four1 ml of CM (HAT medium) containing M) in each well
Was added to. After another day, add half of the medium from each well.
Remove by suction and HA in each well every 2 or 3 days for 8 days
T medium was added and the medium was exchanged. Next, the medium of each well
Half the amount of hypoxanthine (1 x 10^-FourM) and thymidine
(1.6 x 10 ^-FiveCM containing (M) and (HAT medium)
I replaced it with. After 2 more days, add half of the medium in each well to C
Exchanged for M. 13 days after cell fusion
Proliferation was observed in almost all wells.

(III) Assay of anti-Cu, Zn-SOD antibody Cu, Zn-S was added to each well of a 96-well plate (M-174, cup U rigid immulon, manufactured by Cook).
100 μl of a solution of OD (50 μg / ml) was added and the plate was kept at 37 ° C. for 3 hours to allow the wells to be attached with Cu, Zn—SOD. Then, a bovine serum albumin (10 mg / ml) solution was added to completely block the unattached portion of the wells with the above-mentioned protein. After removing the blocking solution, 100 μl of the hybridoma culture supernatant obtained above was added to each well and the mixture was incubated at 37 ° C. for 90 minutes.
Hold for a minute. After washing three times with PBS, 100 μl of ¹²⁵ I-labeled goat anti-mouse F (ab ′) ₂ solution (10,000 cpm, specific activity 5 μCi / μg IgG) purified by affinity column chromatography was added, and the mixture was kept at 4 ° C. overnight. The radioactivity in each well was measured with a gamma scintillation counter. Anti-Cu, Zn-SOD activity is
It was detected in 96 cultures in the measured 173 cultures.

(IV) Cloning of hybridoma producing anti-Cu, Zn-SOD antibody Ten hybridoma cultures showing high binding ability for Cu, Zn-SOD were fed to thymocytes of BALB / c mice in a feeder layer (5 x). 10 ⁶ cells / ml),
Cloning was performed by a limiting dilution method in a 96-well flat bottom microplate (manufactured by Nunc).

(V) Purification of Anti-Cu, Zn-SOD Monoclonal Antibody The hybridoma obtained above was transplanted into the abdominal cavity of a BALB / c mouse to which tetramethylpentadecane was administered one week before. After 1 to 3 weeks, ascites was collected from the abdominal cavity of the mouse, and anti-Cu, Zn-SOD antibody was isolated from the ascites by a precipitation method using a 50% saturated ammonium sulfate solution.
That is, the hybridoma cells were removed from the ascites by centrifugation, and while stirring the supernatant, ammonium sulfate was gradually added thereto until the final concentration reached 50% saturation. The mixture was stirred under ice cooling for 30 minutes and allowed to stand for 30 minutes. After centrifugation, remove the residue with a small amount of 10 mM PBS (pH
8.0) and dialyzed against 10 mM PBS, 1
It was subjected to DEAE cellulose (DE52, Whatman Chemical Separation Co., Ltd.) column chromatography equilibrated with 0 mM PBS (pH 8.0). The solution passed through the column was collected and used as an immunosorbent in the Cu, Zn-SOD purification process, the details of which will be described in Example 2.

(VI) Identification of Antibody Immunoglobulin Class To the culture supernatant (500 ml), an equal amount of saturated ammonium sulfate was added, and the mixture was allowed to stand on ice for 1 hour. The precipitate obtained by centrifugation was dissolved in 1/10 amount of PBS (50 μl). The immunoglobulin of the antibody thus obtained was identified by the double immunodiffusion method of Octalony. Goat polyclonal antibody (Miles) was used to identify the subclass of monoclonal antibody. Anti-Cu, Z obtained in this way
The n-SOD monoclonal antibody is as follows. (1) Anti-Cu produced by hybridoma 1-9-2,
Zn-SOD monoclonal antibody 1-9-2: (a) Subclass: IgG ₁ (2) Anti-C produced by hybridoma 1-66-18
u, Zn-SOD monoclonal antibody 1-66-18: (a) Subclass: IgG ₁ (3) Anti-C produced by hybridoma 1-41-10
u, Zn-SOD monoclonal antibody 1-41-10: (a) Subclass: IgG ₁

Example 2 Cu, Zn of anti-Cu, Zn-SOD monoclonal antibody
-Application to SOD purification (1) Binding of anti-Cu, Zn-SOD monoclonal antibody to Bromocyan-activated Sepharose 4B (Pharmacia Fine Chemicals) 1 Bromocyan-activated Sepharose 4B (1.3 g)
mM hydrochloric acid, then 0.1M sodium bicarbonate (pH
8.0) and 0.5M sodium chloride in the coupling buffer, and the suspension was sequentially washed to prepare a suspension of bromocyan-activated sepharose in the coupling buffer (9 ml). Anti-Cu, Zn-SO prepared by dialyzing 2 ml of this solution against the above coupling buffer in Example 1.
Each 1 ml of a solution of a coupling buffer of D monoclonal antibodies 1-9-2, 1-66-18 and 1-41-10 (5 mg of protein) was added. The resulting mixture was shaken overnight at 4 ° C until the coupling was complete. After centrifugation, 0.2 M glycine (pH 8.0, 2 ml) was added thereto, and antibody-bound Sepharose 4B was shaken at room temperature for 2.5 hours to block the remaining active site. After blocking, the resulting antibody-bound Sepharose 4B is washed successively (twice) with a solution of 0.1 M acetate buffer (pH 4.0) containing 0.5 M sodium chloride and the above coupling buffer, 0.5
Equilibration was carried out with 25 mM sodium phosphate buffer (pH 7.4) containing M sodium chloride. The bromocyan-activated sepharose 4B-conjugated anti-Cu, Zn-SO thus obtained
D monoclonal antibody (hereinafter referred to as antibody-bound Sepharose, particularly column 1-9-2, 1-66-18 or 1-4, respectively)
1-10) was used to prepare the column for affinity column chromatography.

(II) Adsorption and elution of Cu, Zn-SOD using antibody-bound Sepharose 4B Antibody-bound Sepharose 4B (0.6 ml) was added to 0.5M.
The column equilibrated with 25 mM sodium phosphate buffer (pH 7.4) containing sodium chloride is packed. C
u, Zn-SOD (manufactured by Sigma, lot 52F-934)
5. Specific activity: 2235 cytochrome units or 13,000 sulfite units per mg of protein (1,900 sulfite units / 148 μg / 0.2 ml) were applied to the column, and then 0.
25 mM phosphate buffer containing 5 M sodium chloride (pH 7.
The sample was washed with 4) at a flow rate of 5 ml / hour and eluted with a 0.2 M glycine hydrochloride solution (pH 2.5). Cu, Zn-SO
D was measured by a modified nitrite method based on Elstner et al. (Analytical Biochemistry, Vol. 70, p. 616, 1976). According to this method, 5
Cu, Zn required to achieve 0% inhibition (1 unit)
-The amount of SOD is the cytochrome method (McCord et al., Journal of Biological Chemistry, 2nd
Volume 1/6 of 44, 6049, 1969) was sufficient. That is, 1 cytochrome unit equals 6 nitrite units. The xanthine-xanthine oxidase system was used at pH 8.2 to generate superoxide radicals. Since no Cu, Zn-SOD activity was detected in the flow-through fraction of each column, most of Cu, Zn-S was detected.
The OD was considered bound to the column. Eluate 0.1
It was dialyzed overnight at 4 ° C. against 25 mM sodium phosphate buffer (pH 7.4) containing 5 M sodium chloride.

[Table 1]

Example 3 Human red blood cells Cu, Z using antibody-bound Sepharose 4B
Adsorption and elution of n-SOD Antibody-bound Sepharose 4B prepared in Example 2 (I)
(That is, columns 1-9-2, 1-66-18,
Column 1-41-10) (0.6 ml) in each column,
Cu, Zn-S from human erythrocytes prepared by the Tsuchihashi method
OD extract (100 nitrite unit / 260 μg protein / m
1, specific activity: 280 nitrite units per mg of protein). In this case, Cu, Zn-SOD was completely bound to the column. For elution, 0.2 M glycine-hydrochloric acid buffer (pH
2.5) was used. The specific activity of the activity peak was measured.

[Table 2]

Example 4 Human serum Cu, Zn using antibody-bound Sepharose 4B
-SOD adsorption and elution Antibody-bound Sepharose 4B prepared in Example 2 (I)
(That is, columns 1-9-2, 1-66-18,
Column 1-41-10) (0.5-0.6 ml) was applied to each column of commercially available human serum, 0.5 mM sodium chloride-containing 25 mM sodium phosphate buffer (pH 7.0), and commercially available human Cu, Zn-. SOD (manufactured by Sigma, lot 52
F-9345, 1 mg / ml) (10: 10: 1) was applied. Total protein: 185,000 μg Total activity: 2,650 Nitrite unit Specific activity: 14 Nitrite unit per mg of protein Column is a 25 mM sodium phosphate buffer solution (pH 7.4) containing 0.5 M sodium chloride, flow rate 5 ml / hour Then, it was washed until the optical density at 280 nm reached 0.05 or less. Then, at an elution rate of 10 ml / hour, 0.2M
It was eluted with a glycine hydrochloride (pH 2.5) solution. The eluate was diluted with the same amount of 10 mM phosphate buffer (pH 7.4) containing bovine serum albumin (200 μg / ml) and dialyzed against 5 mM phosphate buffer at 4 ° C. overnight.

[Table 3]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // C12P 21/08 8214-4B (C12P 21/08 C12R 1:91) (72) Inventor Osawa Shigeyoshi 1-4-18 Anokaji-cho, Takatsuki-shi

Claims (2)

[Claims] 1. An anti-superoxide dismutase monoclonal antibody. 2. A hybridoma that produces an anti-superoxide dismutase monoclonal antibody.