JP2567664B2 - Monoclonal antibody against human Mn superoxide dismutase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a monoclonal antibody having high specificity only for human Mn-SOD (hereinafter, abbreviated as “human Mn-SOD”) and a monoclonal antibody thereof. Regarding manufacturing method.

[Conventional technology]

Human Mn-SOD is an enzyme (molecular weight of one domain is about 25,000 and consists of its dimer or tetramer) existing in the matrix part of mitochondria,
As shown below, it catalyzes a reaction that disproportionates the superoxide anion radical (O ₂ ) which is a major molecular species of toxic oxygen.

_{^{2O - 2 + 2H + → H}} 2 O 2 + O 2 By the way, the measurement of human Mn-SOD concentration in serum in liver disease by study using polyclonal antibodies, it is believed that there are high diagnostic significance ( Pharmaceutical Journal; Inagaki, Sawaki, 20 , 1, 1984. 5th Tumor Marker Study Group; Iizuka, Arai et al., 1985). Taniguchi et al. Also showed that human Mn-SOD concentration was high in lung cancer by an immunological method using a polyclonal antibody prepared by immunizing goat (Journal of National Cancer Insti).
(Tute, 72 , 5, 1984), the importance of measuring human Mn-SOD concentration in serum has been pointed out.

However, human Mn-SO in serum in each of these diseases
Since a polyclonal antibody is used to measure the D concentration, the monoclonal antibody is used to further enhance the specificity of the reaction to human Mn-SOD and to always obtain an antibody having the same high specific reactivity. Although it has been necessary to prepare and establish a method for producing the same, there are still no reports regarding a monoclonal antibody having a very high specific reactivity with human Mn-SOD.

[Problems to be solved by the invention]

The object of the present invention is to provide a monoclonal antibody having a very high specific reactivity to human Mn-SOD in human serum.

[Means for solving problems]

The present inventors have conducted extensive studies to solve the above problems, as a result of immunizing mice with human Mn-SOD, and culturing the cell line obtained by cell fusion to obtain human Mn-SOD. It was found that a monoclonal antibody having a very high specific reactivity with can be produced, and the present invention has been completed.

That is, the present invention is highly specific only to human Mn-SOD produced by a cell line obtained by immunizing a mouse with human Mn-SOD and fusing lymphocytes obtained from the mouse with mouse myeloma cells. The present invention relates to a monoclonal antibody characterized by having a property.

 Hereinafter, the present invention will be described in detail.

The monoclonal antibody of the present invention is produced by a cell line obtained by immunizing a mouse with human Mn-SOD and cell fusion, and has a very high specific reactivity to human Mn-SOD. I have.

The immunogen used for immunizing mice in the present invention includes human
It is not particularly limited as long as it is possible to obtain a monoclonal antibody having a very high specific reactivity to Mn-SOD, for example, a monomer constituting human Mn-SOD, human Mn-SOD, and human. A peptide obtained by synthesizing a part of Mn-SOD can be mentioned, but human Mn-SOD is preferably used. The monoclonal antibody having high specificity only to the human Mn-SOD of the present invention,
Human Cu, Zn superoxide dismutase (hereinafter human C
u, Zn-SOD), human albumin, and human globulin have no reactivity. Monoclonal antibodies having such specificity include, for example, human Mn-SO
NI8 strain of hybridoma strain obtained by fusing lymphocytes obtained from mice immunized with D and myeloma cells of mouse (Microtech Lab Article 1606), PE9 strain (Microtech Lab Article 1607)
No.), the PG11 strain (Microtechnology Research Institute No. 1608) and the like.

Such a hybridoma can be produced by a conventionally known method, for example, the method of Milstein and Kholer [Nature, 256 , 495].
(1976)]. The outline of a preferable method for producing such a hybridoma strain will be sequentially described below.

Preparation of Monoclonal Antibody-Producing Hybridoma Strain (i) Preparation of Immunized Animal Lymphocytes Mice were immunized by human Mn-SOD (10 to 400 μg) dissolved in PBS (phosphate buffered saline) once or for several weeks. It can be performed by administering several times at intervals.

The first immunization can be performed without administering an adjuvant (immunity-promoting substance including alum, killed tuberculosis cells, nucleic acid, etc.), but it is preferable to administer an emulsion prepared using an adjuvant.

Lymphocytes are blood, lymph nodes, several days after the final immunization, after confirming a sufficient antibody titer in the immunized mouse.
It can be obtained from the spleen or the like, but is preferably obtained from the spleen.

(Ii) Preparation of myeloma cells For cell fusion, MPC-11, P3-X63-Ag8 derived from mouse was used.
・ 653 (653), P3-X63-Ag8-U1 (P3U1), P3-NS-1
(NS-1), SP2 / 0-Ag14 (SP2 / 0) and the like, and rat-derived myeloma cells such as 210.RC Y3.Ag1.2.3 (Y3) can be used, but 653, P3U1, NS- It is preferable to use myeloma cells that do not produce and secrete extracellular antibodies such as 1, SP2 / 0.

(Iii) Cell fusion Cell fusion is a cell suspension in which the cell number of lymphocytes and myeloma cells of the mouse immunized as described above is (3 to 20): 1 and does not interfere with cell fusion. Solution, for example, commonly used lymphocyte culture medium components (MEM, D
HVJ (Sendai virus) or PEG (polyethylene glycol) solution is added to the pellet (cell mass) after thorough mixing using MEM, McCoy, RPMI1640, etc.) solution, isotonic buffer, etc., and centrifugation. However, it is preferable to use a PEG solution, more preferably 30 to 60 with an average molecular weight of 1,000 to 8,000.
It is advisable to use a weight% PEG solution. At this time, colchicine, dimethyl sulfoxide, poly-L-arginine and the like can be added to promote cell fusion.

As the myeloma cells used for cell fusion, those derived from an animal different from the immunized mouse can be used, but considering the antibody production amount and stability of the resulting monoclonal antibody-producing hybridoma strain, It is better to use allogeneic myeloma cells with
It is more preferable to use the same type.

(Iv) Selection of hybridoma Hybridoma is selected by selecting cells after the operation of cell fusion.
It can be carried out by culturing in a HAT medium (medium containing hypoxanthine, aminopterin, thymidine and fetal calf serum. As the medium component, a commonly used lymphocyte culture medium component can be used).

Hybridomas are cultivated by inserting a number of cells suitable for searching antibody-producing wells into each well of the culture plate (culture well), and at this time, the hybridoma growth-promoting substance or cells producing it (eg, thymus, spleen). , Lymph nodes-derived lymphocytes) can be used as feeder cells as required.

Hybridomas selected by growing in HAT medium were used as HT medium (medium containing hypoxanthine, thymidine, fetal calf serum, generally used as a component of this medium) until the number of cells suitable for searching antibody-producing wells was reached. The lymphocyte culture medium component can be used) for several days, and further, it is further cultured in a commonly used lymphocyte culture medium containing fetal bovine serum.

(V) Selection of Antibody-Producing Hybridoma The test whether or not the hybridoma obtained in (iv) produces the target antibody can be carried out by, for example, ELISA (enzyme-linked immunosorbent assay), plaque formation, aggregation. Reaction method, RI
A (method using radioisotope), indirect fluorescent antibody method (IFA) and the like can be performed, but when the number of assays is very large, the ELISA method is preferable.

 This ELISA method is performed as follows.

The hybridoma culture supernatant is added to each well (measurement well) of an ELISA plate on which human Mn-SOD is immobilized, and the mixture is left standing for a certain period of time. Then, an enzyme-labeled antibody capable of binding by reacting with the mouse-derived antibody bound to each of these washed measurement wells (the enzyme used for labeling is, for example,
Peroxidase, alkaline phosphatase, β-galactosidase and the like can be mentioned. The antibody to be labeled is not particularly limited as long as it can bind only by reacting with the antibody of animal origin bound to the measurement well, and for example, serum obtained from mouse, rat, rabbit, goat, etc., or mouse cell, etc. The monoclonal antibody produced by the hybridoma strain produced by using ) Is added to these measurement wells and left for a certain period of time. Next, these measurement wells are washed, and a substrate solution corresponding to the enzyme used is added to measure the enzyme activity. If the enzyme activity is observed, it can be seen that the hybridoma producing the desired antibody was present in the culture well from which the culture supernatant was collected.

In this way, a hybridoma in which cell proliferation is recognized and which produces an antibody can be obtained.

(Vi) Hybridoma Straining (Cloning) The hybridomas in the culture wells in which antibody production was observed were the limiting dilution method, the single cell manipulation method (under an inverted microscope, one hybridoma was placed in each well). , Picking colonies using soft agar,
It can be cloned by a method using FACS (Fluorecent Activated Cell Sorter). At this time, the antibody-producing hybridoma found in (v) was cultured by any of the above-mentioned cloning methods, and the supernatant of the culture well in which the growth was observed was used to select the antibody-producing hybridoma in (v). The antibody producing wells are searched in the same manner as the ELISA method.

In this way, a hybridoma strain that produces a monoclonal antibody with high specificity for human Mn-SOD and high antibody titer can be selected.

Production of Monoclonal Antibody High specificity for human Mn-SOD, and production of a high antibody titer can be performed by culturing the hybridoma strain obtained in (vi) above in a flask, or in the abdominal cavity of an animal. It can be performed by culturing.

The production of the monoclonal antibody in the flask culture of the hybridoma strain obtained in (vi) above is, for example, 0 to 20%.
It can be carried out by culturing in a commonly used lymphocyte culture medium containing fetal bovine serum (for example, a medium containing medium components such as MEM, DMEM, McCoy and RPMI1640) until the cell concentration reaches the upper limit. At this time, the monoclonal antibody is contained in the culture supernatant obtained by centrifugation.

On the other hand, the production of the monoclonal antibody by intraperitoneal culture of the hybridoma strain obtained in (vi) above can be performed using an animal of a different type from the animal from which the cells used for cell fusion are derived. It is preferable to use the animal of 1st line, and more preferable to use the animal of the same line.

The production of the monoclonal antibody having high specificity for human Mn-SOD and high antibody titer by such a method reduces the immunocompetence of an appropriate animal such as mouse, rat or hamster in the abdominal cavity of the animal. Substance, for example, mineral oil such as pristane, is administered, and several weeks later, 10 ⁶ to 10 ^{7 of} the hybridoma cell line obtained in (vi) above are administered, and this cell line is injected intraperitoneally for several weeks. This can be done by growing to density. At this time, the monoclonal antibody is contained in the ascites supernatant obtained by centrifugation.
The antibody concentration is 10 to 1000 times the antibody concentration of the culture supernatant when it is obtained by in-flask culture.

The monoclonal antibody obtained by culturing the hybridoma strain in a flask or in the abdominal cavity of an animal undergoes salting out, dialysis, ion exchange chromatography, affinity chromatography, etc., which are applied to general protein purification methods. By doing so, it becomes a highly pure monoclonal antibody.

The monoclonal antibody obtained as described above is human
It has a very high specific reactivity to Mn-SOD.

〔Example〕

Hereinafter, examples of the present invention will be specifically described. It should be noted that these examples do not limit the scope of the present invention.

Example 1 [Production of monoclonal antibody-producing hybridoma strain against human Mn-SOD] (a) Immunization of mouse and preparation of spleen lymphocytes 1 ml of PBS (phosphate buffer, pH7.4) in which 80 μg of human Mn-SOD was dissolved ) And 1 ml of Freund's complete adjuvant were thoroughly mixed and 0.5 ml of the emulsion obtained was mixed with BALB / c.
It was intraperitoneally administered to mice (♀, 8 weeks old).

An emulsion prepared in the same manner as above except that Freund's incomplete adjuvant was used instead of Freund's complete adjuvant two weeks after the first immunization.
5 ml was intraperitoneally administered to the mouse.

Furthermore, after 2 weeks, the above-mentioned human Mn was used as the final immunization.
0.2 ml of PBS containing 20 μg of SOD was administered to the tail vein of the mouse.

From the mice immunized in this way, the spleen extracted on the 4th day after the final immunization was cooled to 10 ml with RPMI1640 solution (a medium in which distilled medium for lymphocyte culture was dissolved in distilled water).
It was washed in a petri dish containing the mixture, transferred into a newly prepared RPMI1640 solution, divided into 4 equal parts with scissors, and loosened with tweezers.

The floating lymphocytes thus obtained were suspended in RPMI1640 solution, centrifuged (rotation speed; 1000 rpm, time; 5 minutes), resuspended in RPMI1640 solution, and mouse spleen lymphocytes used for cell fusion. did.

(B) Cell fusion 1.82 × 10 ⁷ 8-azaguanine-resistant mouse myeloma cells (NS-1) in the logarithmic growth phase and 1.39 × 10 ⁸ spleen lymphocytes of the mouse in a 50 ml plastic conical centrifuge tube The mixture was placed in a tube, mixed, and then the supernatant was centrifuged (rotation speed; 1500 rpm, time; 5 minutes), and then the same centrifuge tube was tapped to loosen the pellet.

While shaking the pellet vigorously,
% PEG4000 solution (37 ° C.) was added in 1 ml over 1 minute, and the mixture was vigorously shaken for 1 minute.

While gently shaking the centrifuge tube, gradually add 10 ml of RPMI1640 solution (37 ° C) over 3 minutes, and further add 40 ml of RP.
MI1640 solution (37 ° C) was added, and the mixture was allowed to stand at room temperature for 20 minutes. Then, centrifuge at room temperature (rotation speed; 1000 rpm, time; 5 minutes)
Then, the supernatant was removed by suction.

Tap the same centrifuge tube to loosen the pellet, and add 75 ml of HA.
Suspend in T medium (RPMI1640 medium containing 1 × 10 ^-4 M hypoxanthine, 4 × 10 ^-7 M aminopretene, 1.6 × 10 ^-5 M thymidine and 20% fetal calf serum, kept at 37 ° C.), 100 μl was dispensed into each of the 7 wells of a 96-well culture plate and cultured using a CO ₂ incubator (5% C
O ₂ , 95% air, 37 ° C, 100% humidity).

On the 4th and 6th days after cell fusion, 50 μl of the above HAT medium was used.
50 μl HT medium (1 x
The same amount was exchanged with RPMI1640 medium containing 10 ⁻⁴ M hypoxanthine, 1.6 × 10 ⁻⁵ M thymidine and 20% fetal bovine serum, kept at 37 ° C.).

(C) Selection of hybridoma Is the antibody against human Mn-SOD contained in the culture supernatant of each well of the culture plate in which cell proliferation was observed from 1 to 3 weeks after the start of culture in (b) above? Whether or not it was examined by the following ELISA method.

First, to each analysis well of a 96-well flat bottom ELISA plate, a polyclonal antibody solution against human Mn-SOD (manufactured by Goat, 100 μg / ml, dissolved in 0.05M carbonate buffer of pH 9.8) was added 5
It was dispensed in 0 μm aliquots and allowed to stand at room temperature for 2 hours.

Then wash each assay well of the ELISA plate with wash solution (0.0
After washing twice with PBS containing 5% Tween20, 0.1% OV
Aliquot A (ovalbumin) solution (dissolved in PBS) into each analysis well by 100μ, leave at room temperature for 30 minutes, wash twice with washing solution, and add human Mn-SOD solution (1μg / ml) by 50μ It dispensed and left still at 4 degreeC overnight. After washing each analysis well twice with the same washing solution, 50 μl of the culture supernatant of each culture well of the culture plate was dispensed into each of the analysis wells and allowed to stand at 37 ° C. for 1 hour (negative control). The supernatant obtained by similarly culturing mouse myeloma cells was used as the positive control, while the positive control was prepared by diluting the mouse serum used for cell fusion of the present invention 10-fold with a washing solution. I was there.).

Next, each assay well of the ELISA plate was washed three times with a washing solution, and a horseradish peroxidase-labeled antibody solution having reactivity with mouse IgC and IgM was dispensed into each assay well in an amount of 50 µ, at room temperature for 1 hour. Let stand for hours. And
After washing each analysis well of the ELISA plate four times with the washing solution,
Substrate solution (20 mg o-phenylenediamine, 10 μ 35
% H ₂ O ₂ in 50 ml of 0.1 M citrate buffer, pH 5.0)
Dispense μ into each analysis well, shield from light and react at room temperature for 30 minutes, then dispense 50 μ of 2N sulfuric acid into each analysis well to stop the enzyme reaction and use an absorbance measuring device for a microplate to The absorbance of the wells at 490 nm was measured.

As a result of such examination, production of an antibody against human Mn-SOD was observed in 8 out of 678 culture wells in the culture plate.

(D) Establishing hybridoma strain (cloning) Using RPMI1640 medium containing 20% fetal bovine serum, one of eight culture wells in which antibody production shown in step (c) above was confirmed Hybridomas were cloned in the wells by the limiting dilution method.

For the culture, a 96-well culture plate was used, and BALB / c mouse thymocyte suspension (10 ⁷ cells / ml) was used as feeder cells, and (hybridoma 0.1-5 cells) / (thymocyte suspension). 100 μ) / well.

In cloning one culture well as described above, 10
The supernatant of the culture well observed as a single colony from around the day is collected, and the antibody-producing well is screened by the ELISA method using human Mn-SOD (the same method as the above-mentioned step (c)). It was Regarding the supernatant in which antibody production was observed against human Mn-SOD, the reactivity with human globulin, human albumin and human Cu, Zn-SOD was further determined by the ELISA method (the same method as step (c) above). ) Examined. In this way, at least one hybridoma strain that produced an antibody reactive with only human Mn-SOD was obtained in each cloning, and these were recloned.

The strains thus obtained were NI8 strains (Microtech Lab.
No. 6), and the monoclonal antibody produced by this strain is called NI8.

The class / subclass of the monoclonal antibody contained in the culture supernatant of the NI8 strain was determined by the following measurement test I, and human Mn
-The reactivity between SOD and the monoclonal antibody was confirmed in the measurement test II, and the reactivity with respect to various proteins was examined in the measurement test III.

Assay Test I [Clam of monoclonal antibody against human Mn-SOD
Determination of subclass] The class / subclass of the immunoglobulin produced by the NI8 strain is determined by an antibody solution (IgG ₁ , IgG ₂ a, IgG ₂ b, IgG ₃ , IgM, IgA) specific to each class / subclass of mouse antibody. (Antibodies against etc.) was used.

As a result, the monoclonal antibody (NI
8) was found to be an antibody belonging to IgM.

Measurement test II [Confirmation of reactivity between human Mn-SOD and monoclonal antibody] Culture supernatant containing 30 μm human Mn-SOD (10 μg / ml) and 50 ml NI8 (1 time, 10 times or 100 times dilution) Used) and reacted at 37 ° C for 30 minutes, then add 10 μl of protein A agarose solution (protein A binding amount; 1-2 mg / ml agarose gel), and further react at 37 ° C for 10 minutes After that, centrifugation was performed (10,000 rpm, 5 minutes), and the human Mn-SOD activity of the supernatant was measured (the myeloma supernatant was used as a negative control).

As a result, the human Mn-SOD activity of the supernatant was decreased (shown in FIG. 1).

Measurement test III [Study of reactivity of human Mn-SOD with monoclonal antibody and various proteins] Regarding reaction specificity with NI8, various proteins such as human albumin, human globulin, human Cu, Zn-SOD, and human Mn-SOD Was examined by Western blotting. As a result, NI8 was found to be reactive only with human Mn-SOD, and not with other proteins.

Example 2 [Preparation of monoclonal antibody-producing hybridoma strain against human Mn-SOD] (a) Immunization of mouse and preparation of spleen lymphocytes In the same manner as in Example 1, immunization and spleen lymphocytes were prepared.

(B) Cell fusion Among the procedures described in Example 1, 1.7 × 10 ⁷ 8-azaguanine-resistant mouse myeloma cells (P3U1) in the logarithmic growth phase and the above-prepared mouse spleen lymphocytes 8.
The same operation was performed except that 5 × 10 ⁷ cells were used.

(C) Selection of hybridoma The procedure described in Example 1 was performed.

As a result of such examination, production of antibodies against human Mn-SOD was observed in 20 out of 678 culture wells in the culture plate.

(D) Hybridoma Straining (Cloning) Using RPMI1640 medium containing 20% fetal bovine serum, one of 20 culture wells in which antibody production shown in the above step (c) was confirmed Hybridomas were cloned by performing the same operation as in Example 1 on the wells.

The strain thus obtained was used as PE9 strain
7), and the monoclonal antibody produced by this strain is called PE9.

Various properties of the monoclonal antibody contained in the culture supernatant of PE9 strain were examined by performing the same operation as in Example 1. As a result, the class and subclass of PE9 belonged to IgM. The human Mn-SOD activity of the supernatant obtained by reacting PE9 with a protein A agarose solution was decreased (shown in FIG. 1). PE9 was found to be reactive only with human Mn-SOD and not with other proteins.

Example 3 [Preparation of monoclonal antibody-producing hybridoma strain against human Mn-SOD] (a) Immunization of mouse and preparation of spleen lymphocytes In the same manner as in Example 1, immunization and spleen lymphocytes were prepared.

(B) Cell fusion Among the procedures described in Example 1, 1.4 × 10 ⁷ 8-azaguanine-resistant mouse myeloma cells (P3U1) in the logarithmic growth phase and the mouse spleen lymphocytes prepared as described above 5.
The same operation was performed except that 3 × 10 ⁷ cells were used.

(C) Selection of hybridoma The procedure described in Example 1 was performed.

As a result of such examination, production of antibodies against human Mn-SOD was observed in 15 out of 678 culture wells in the culture plate.

(D) Hybridoma Straining (Cloning) Using RPMI1640 medium containing 20% fetal bovine serum, one of 15 culture wells in which antibody production shown in step (c) above was confirmed Hybridomas were cloned by performing the same operation as in Example 1 on the wells.

The strain thus obtained was designated as PG11 strain
08) and the monoclonal antibody produced by this strain is called PG11.

Various properties of the monoclonal antibody contained in the culture supernatant of the PG11 strain were examined by performing the same operations as in Example 1. As a result, class-subclass of PG11 is, IgG ₂ a
Belonged to. The human Mn-SOD activity of the supernatant obtained by reacting PG11 with a protein A agarose solution is much lower than those of IgM antibodies NI8 and PE9 because they react more easily with protein A. (As shown in FIG. 1). PE11 was found to be reactive only with human Mn-SOD and not with other proteins.

Example 4 [Production of monoclonal antibody against human Mn-SOD in flask culture] It was obtained by culturing in RPMI1640 medium containing 15% fetal bovine serum.
The cultured cells of the NI8 strain were transferred to 10 ml of RPMI1640 solution (without fetal bovine serum) and cultured until just before death.

The monoclonal antibody (NI8) against human Mn-SOD is
The supernatant obtained by centrifuging the culture solution (rotation speed; 3000 rpm, time; 5 minutes) contained 35 μg / ml (measured by the one-dimensional plate immunodiffusion method).

Example 5 [Production of monoclonal antibody against human Mn-SOD in mouse peritoneal cavity] To obtain a large amount of monoclonal antibody against human Mn-SOD, cells of PG11 strain were cultured in mouse intraperitoneal cavity.

BALB / c mice (♀, 6 weeks old, 2 weeks before pristane
0.5 ml was intraperitoneally administered (5 ml) and 5 × 10 ⁶ cells of the PG11 strain suspended in RPMI1640 were administered.

The body weight of this mouse showed a remarkable increase from about 1 week, and ascites (10 ml / mouse) was collected at 2 weeks. The ascites was centrifuged (rotation speed; 3000 rpm, time; 5 minutes) to obtain an ascites supernatant.

Monoclonal antibody against human Mn-SOD (PG11)
Was contained in this ascites supernatant in an amount of 8.0 mg / ml (measured by the one-dimensional plate immunodiffusion method).

〔The invention's effect〕

The “human Mn-SO obtained by culturing a cell line of the present invention
The “monoclonal antibody having a very high specific reactivity to D” can be expected to be used as a reagent for basic research on human Mn-SOD and a measurement test used in clinical tests.

[Brief description of drawings]

Figure 1 shows human Mn-SOD and NI8 (□), PE9 (■) or P
After mixing with culture supernatant containing G11 (●) (use 1-fold, 10-fold or 100-fold diluted solution) and react at 37 ℃ for 30 minutes,
Human Mn of supernatant obtained by adding protein A agarose and further reacting at 37 ° C for 10 minutes and then centrifuging
-Indicates relative activity of SOD [Note that NS-1 is used as a control.
The culture supernatant of the strain (○) was used].

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/573 9162-4B C12N 15/00 ZNAC 33/577 9281-4B 5/00 B (C12P 21 / 08 C12R 1:91) Jury President Yoshinobu Ishida Judge Judge Naohisa Tanaka Judge Ken Ukai (56) References JP 62-167479 (JP, A) Hokkaido Medical Journal, 59 [6] (1984) Iizuka , P .; 739-749

Claims (1)

(57) [Claims] 1. A cell line obtained by immunizing a mouse with human Mn superoxide dismutase (hereinafter abbreviated as “human Mn-SOD”) and fusing lymphocytes obtained from the mouse with mouse myeloma cells ( PG11 strain) produced, human albumin, human globulin, human Cu, Zn-SOD substantially does not react, human Mn-SOD monoclonal antibody against human Mn-SOD characterized by reacting