JPS61212600A - Monoclonal antibody to human copper, zinc superoxide dismutase and production thereof - Google Patents

Abstract

PURPOSE:To obtain the titled antibody suitable for the determination of human copper, zinc superoxide dismutase (Cu, Zn-SOD), by immunizing an animal with Cu, Zn-SOD, and using the hybridoma of the lymphocyte of said animal and a myeloma cell. CONSTITUTION:Lymphocyte prepared from the lymph node or spleen of an animal (e.g. mouse, rat, etc.) immunized with human Cu, Zn-SOD is subjected to the cell fusion with a myeloma cell (e.g. P3U1, NS-1, etc., originated from mouse), and the obtained hybridoma is cloned to obtain a clone capable of producing an anti-human Cu, Nz-SOD monoclonal antibody. The objective antibody can be produced by the tissue culture of the clone or the culture of the clone in the abdominal cavity of a small animal.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical field to which the invention pertains] The present invention relates to human Cu, Zn-
The present invention relates to a monoclonal antibody specific to SOD, a method for producing the same, and a method for measuring human Cu and Zn-9OD using the same.

[Prior art] SOD is an enzyme that is widely distributed in the biological world, and it is a reaction that disproportionates superoxide anion radical (0□-), which is the main molecular species of toxic oxygen. catalyze.

02- + o2- + 2H” -n2o□+0□S
Due to the difference in metals chelated, OD is Cu
, Zn-3OD (DiMarch molecular weight approximately 32,000)
.

NI! It is divided into three types: -SOD and Fe-5O (both are dimers and have a molecular weight of about 40,000). In each human tissue, Cu and Zn-SOD are present in the cytoplasm, and M
n-9on has been confirmed in the mitochondrial matrix.

Fe-SOD and In-SOD show high homology in their amino acid sequences between biological species, and an evolutionary relationship is inferred. On the other hand, Cu and Zn-SODs are unique to eukaryotes and have different amino acid sequences from other types of SODs. This Cu, Zn-5on has been clinically recognized to have an anti-inflammatory effect, and bovine-derived Cu, Zn-5on
SOD is being developed as a therapeutic agent for inflammatory diseases such as rheumatoid arthritis. However, as pharmaceuticals, human-derived Cu and Zn-9OD are considered to be superior to bovine-derived ones in terms of antigenicity. Therefore, highly specific anti-human Cu, Zn-9
OD antibodies in human organs (such as placenta) containing Cu, Zn-S
Human Gu produced by microorganisms through OD or genetic manipulation,
When used to purify Zn-9OD, the purification step is much easier than conventional methods, and moreover, higher purity can be obtained.

On the other hand, Sawaki, Sugiura et al. are investigating the relationship between human serum SOD measurements and various diseases using antiserum against human SOD obtained from rabbits (presented at the 55th Annual Meeting of the Japanese Biochemical Society).
, their experimental results showed that Cu, Zn-SO in human serum
The D value increases several ten times in renal failure, especially in uremia, and in cases of hepatitis,
A value of 0 or more, which increases several times in cases of complications such as diabetes and pulmonary fibrosis, indicates that Cu, Zn-300 is an indicator for the diagnosis of these diseases, and human Cu, Zn-SO
D-specific monoclonal antibodies also show the possibility of being used in the diagnosis of the above-mentioned diseases.

[Object of the Invention] An object of the present invention is to obtain a monoclonal antibody specific to human Cu, Zn-SOD, and thereby to provide a method for measuring Cu and Zn-3on values, which are indicators of various diseases.

[Summary of the Invention] The present inventors have conducted intensive research and discovered that human Cu, Zn-9
We were able to obtain a monoclonal antibody that specifically reacts with OD.

That is, the anti-human Cu, Zn-SOD monoclonal antibody of the present invention is characterized by being produced by a hybridoma obtained by fusion of mouse lymphocytes immunized with human Cu, Zn-SOD and myeloma cells. shall be.

The present invention will be explained in more detail below.

In the present invention, hybridomas are prepared by methods known per se, for example, Nature, 258°495-49? (11375).

(a) Preparation of Immunized Animal Lymphocytes The experimental small animal to be immunized with the antigen is preferably one of the same type as that used for intraperitoneal antibody production, and mice, rats, etc. are generally used. The immunization method follows a conventional method, and the antigen dissolved in physiological saline is administered several times at intervals of 2 to 3 weeks. For the first immunization, it is preferable to administer the antigen emulsified in complete Freund's adjuvant. Three to four days after the final booster, the lymph nodes or spleen are removed and the prepared lymphocytes are used for cell fusion.

(b) Preparation of myeloma cells The myeloma cells used for cell fusion are mouse-derived (7)
NPC-11, Pi-XE13-Ag8, P3-
X83-Ag8-U 1 (P2O1),, P3-M
S-1 (MS-1) or SP210-Ag14 (SP2
10), but NPC-11 and P3-X [13
-Ag3 produces its own immunoglobulin, so P3O1
, MS-1 or 5P210 is preferably used.
These cells are 8-A due to HGPRT (hyboxanthine guanine phosphoribosyltransferase) deficiency.
It is C (azaguanine) resistant and )! It has the characteristic that it cannot grow on AT medium (medium supplemented with hypoxanthine, aminopterin, and thymidine). Myeloma cells used for cell fusion are cultured in 8-AC medium in advance to eliminate those that have undergone reversion to 8-AC sensitivity.

(c) Cell fusion For fusion, the cell numbers of immunized animal lymphocytes and myeloma cells were mixed in MEN (Eagle MEN) at a ratio of (5-1G):1.
) etc., mix well, and after centrifugation, add PEG (polyethylene glycol with average molecular weight t, ooo to e, ooo, used at 30 to 60%) heated to 37 °C to the pellet after centrifugation.
Mix well.

(d) Selection of hybridomas Hybridomas can be selected by culturing the cells after the cell fusion operation in a normal HAT medium. At this time,
Cells are cultured in appropriate numbers on tissue culture plates. The culture time is usually several days to one week in order to kill cells other than the target hybridoma. After culturing the selected hybridoma in HT medium (medium supplemented with hyboxanthin and thymidine) for several days to one week, Culture in regular medium.

(e) Testing of anti-human Cu, Zn-SOD antibody-producing hybridoma Testing whether the selected hybridoma is producing the desired antibody can be performed, for example, according to the ELISA method (enzyme immunosorbent assay). .

into microtiter wells with immobilized antigen.

Add hybridoma culture supernatant and incubate.

Antibodies labeled with various enzymes (
If the lymphocytes of the immunized animal are derived from a mouse, use an anti-mouse antibody; if the lymphocytes are derived from a rat, use an anti-rat antibody) or the same protein A for incubation. The wells are washed, substrate is added and enzyme activity is measured. If enzyme activity is observed, it is understood that a hybridoma producing the antibody of interest was present in the well from which the culture supernatant was collected.

(f) Cloning of hybridomas Hybridomas in wells in which antibody production was observed were cloned using limiting dilution method or single cell fist manipulation (SI).
Cloning is carried out using the ngle cell maniplatian method, etc. At this time, antibody-producing wells were assayed according to the ELISA method described in (e) using culture supernatants from wells in which hybridoma growth was observed. Reactivity with other antigens will also be assayed. Then, a clone with high specificity and high antibody titer for the target antigen is selected.

(g) Production of monoclonal antibodies Monoclonal antibodies are produced in tissue culture or intraperitoneally in small experimental animals using the clones selected in CF.
For tissue culture, for example, OMEN (Dulbetzko's modified Eagle's medium Dulb
ecco's Modified Eagle Med
ium) medium or RPM11840 medium until the cell concentration reaches the upper limit. Monoclonal antibodies are contained in the culture supernatant obtained by centrifugation. on the other hand,
In order to obtain even larger amounts of antibodies, it is preferable to use an animal of the same breed as the myeloma cell-derived animal.Mineral oil such as Blistane is intraperitoneally administered, and after 1 to 4 weeks, 10' to
If zero hybridomas are administered, the hybridomas can be grown to a high density in 1 to 3 weeks, and the antibody concentration in the ascites will be 10 to 1000 times that of the culture supernatant.

The antibodies obtained in this way can be purified and used as necessary. It can be purified using the following methods.

The monoclonal antibodies thus obtained are human Cu, Zn
-Reacts specifically to 9OD. Therefore, it is very useful in diagnosing diseases such as uremia.

The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Example 1. Anti-human Cu, Zn-! Preparation of JOD monoclonal antibody-producing hybridoma and production of antibody (1) Immunized human Cu, Zn-SOD 1100IL dissolved in P
Mix 0.25 si of Bs (phosphate buffer) and 0.25 si of Freund's complete adjuvant to make an emulsion, and administer 0.5 tJL of the emulsion intraperitoneally to BALB/C mice (male, 7 weeks old). did. After 3 weeks, 1100 pL of the same antigen was dissolved in PBS 0.25 layer and Freund's incomplete adjuvant (0.25 layer) were mixed to make a milk/turbid solution, and the 0.5 layer was intraperitoneally administered. .

Three weeks later, the final immunization was with the same antigen 1oo 4.
A solution containing 0.5 g of PBS was administered into the tail vein.

(2) Cell fusion On the fourth day after the final immunization, the pancreas of the mouse was removed, washed in a petri dish containing Hanks' solution under water cooling, transferred to MEN, divided into three equal parts, and loosened with tweezers. . The floating lymphocytes obtained in this way were washed 3 times with MEN (1,000 rpm (5 minutes)).
Next, 8-AC resistant myeloma cells (MS-1 or 5P2) prepared in advance were suspended in 8-AC resistant myeloma cells (MS-1 or 5P2) 2X 1G? Mix 10a cells and 2x tile lymphocytes and remove the supernatant at room temperature.
000 rpm, 5 minutes) The centrifuge tube was gently tapped to loosen the pellet. 45% PE heated to 37℃ in this
04.0001-add over 1 minute and leave at 37°C for 6 minutes
RPN118 was heated to 37°C after being left undisturbed.
40 was gradually added at a rate of 5 min/min for 3 minutes, and the final volume was 50 min/min, and the mixture was centrifuged at room temperature at 10 GO rpm.
5 minutes) The supernatant was removed. This was mixed with HAT medium (IX 104M Hyboxanthin, 4X
10-71117 Minopterin, 1.8X 1G4
The cells were suspended in 200 layers (15% FIOS-RPM11840 medium containing N-thymidine) and cultured by dispensing 100 gl into each well of 986 microtiter wells.

(3) On the fourth day after HA-selected cell fusion, add 50 IL of HA-medium to each well.
! Add L at a time and hybridoma growth is observed 10
ELISA was performed on the pewter of the eyes, and wells in which antibody production was observed were treated with HT medium (IX 104M hyboxanthin,
15% FO3-R with 1.8X 104 M Thymidine
Add 50 IL of PN11840 medium, and then add 15% FO9-RP while observing the growth of the hybridomas.
The cells were adapted to M11840 medium.

(4) Anti-human Cu, Z! in the culture supernatant of wells where cell proliferation was observed over a period of 1 to 3 weeks from the start of selective culture of hybridomas. The presence or absence of l-3OD antibody was tested by ELISA.

First, 55% of human Cu, ZZn-SOD (101 L/m) was added to each well of a 98-well U-bottom ELIJA plate.
The solution was dispensed into 0 IL1 portions and allowed to stand overnight at 4°C. Wash each well with washing solution (PBS containing 0.1% Tween20) -1
? After washing three times, the above culture supernatant was dispensed in 50% liter portions and allowed to stand at room temperature for 2 hours (a mixture of tile lymphocytes and myeloma cells before fusion was used as a negative control for the supernatant). Next, these wells were washed three times and injected with peroxidase-labeled anti-mouse (IgG).
, IgM, IgA) antibody solution in 50 ILi portions,
The mixture was allowed to stand at room temperature for 2 hours. After washing these wells four times,
Substrate solution (O-phenylenediamine 2 (lag, 35
% H20 pI (dissolved in 50 μm of 0.1M citrate buffer) was dispensed into 100 μl portions, and the mixture was left standing at room temperature for 30 minutes while shielded from light with aluminum foil. Finally, 5QILl of 2N sulfuric acid was dispensed into each well to stop the enzyme reaction, and the absorbance at 492 nm was measured. Anti-human Cu, Zn was added to the well containing the supernatant that was positive for enzyme activity.
- As a result of assaying the culture supernatant in the cell proliferation wells as above 0, which was considered to be the presence of SOD antibody-producing hybridomas, (number of antibody-producing wells/number of ELISA wells) was determined to be 15-1 as myeloma cells. When used (
8571588), and when using SF3, (
541328).

(5) Monocloning! Using 5% FCJ-RPM111140 medium, hybridomas from the antibody-producing wells described in (0) were cloned by limiting dilution method. A 9B microtiter well was used for culture, and thymocytes from HALO/C mice were used as feeder cells at 10% Pybridomas were cultured at 1/100 ILi/well using ? cells/layer. After 10 days of culture, the supernatant of a well that seemed to contain a single colony was collected and subjected to ELISA using human Cu and Zn-9OD. For those in which antibodies were detected, we further examined their reactivity with other antigens (human albumin, human globulin, etc.).In this way, we selected 6 strains, 5t-1, S-2.
, S-4, S-8, N-4, N-13; 130 Weiyobun No. 189) and were recloned (described in Table 1).

(6) Determination of class and subclass of anti-human Cu and Zn-SOD monoclonal antibodies Class and subclass of immunoglobulin produced by each hybridoma
Subclass determination is performed using peroxidase-labeled antibodies (IgG, IgG2., I
gG3. The test was carried out according to the ELISA method described in Example 1 (4) using affinity-purified antibodies against IgM and IgA. Of the monoclonal antibodies produced by the 6 strains, 2 strains had the antibody M, and 4 strains had the antibody 01 (listed in Table 1).
n) were all types.

(7) Production of monoclonal antibodies Antibodies were produced in flask culture or intraperitoneally in mice. 15% FC9-RPM1184 for flask culture
The hybridoma obtained by culturing in 0 medium was RPM11B4.
0 alone until just before death (monoclonal antibody is in the supernatant obtained by centrifugation at 10-50 g/ljL)
On the other hand, production in the mouse peritoneal cavity to obtain large amounts of antibodies is BA
RPM1184 was given to LB/C mice (female, 6-10 weeks old, 0.5 tfL pristane was administered intraperitoneally 2-3 weeks before).
The test was carried out by intraperitoneally administering 106 to 107 hybridomas suspended at 0.0%. A remarkable increase in mouse body weight was observed from around the first week, and ascites was removed as appropriate from the first to third weeks.

Example 2. Specificity of anti-human Cu, Zn-SOD monoclonal antibodies The specificity of the monoclonal antibodies produced by the six strains described in Example 1 (5) was compared to human Cu, Zn-! JOD, Ushi S
The reactivity with OD, dog SOD, human albumin, human α-nigelobulin, human γ-globulin, etc. was investigated (listed in Table 2), and the reaction between these monoclonal antibodies and antigen was described in Example 1 (4). It was carried out according to the ELISA method described.

As described in Table 2, none of the monoclonal antibodies produced by the six strains reacted with albumin or globulin, which are the main components of human serum proteins.
The antibodies of one strain (Lou 6) also reacted with bovine and dog SOD.

Table 1. Class/subclass of monoclonal antibody [Effect of the invention] The novel monoclonal antibody of the present invention does not react with human albumin and human globulin, and does not react with human Cu, Zn-9O.
It reacts specifically to D. Moreover, other monoclonal antibodies except N-8 do not react with bovine or canine SOD, and are extremely specific to human Cu and Zn-9OD. Therefore, by using the monoclonal antibody of the present invention, human Cu and Zn-9OD can be specifically measured, which increases the possibility of diagnosing various diseases quickly and accurately.

Claims (3)

[Claims] (1) An anti-human Cu, Zn-SOD monoclonal antibody having specificity for human copper, zinc superoxide dismutase (hereinafter abbreviated as Cu, Zn-SOD). (2) The monoclonal antibody according to claim 1, which is used for measuring human Cu, Zn-SOD. (3) Anti-human Cu, Zn-SOD, which is characterized by culturing a hybridoma cell line capable of producing anti-human Cu, Zn-SOD monoclonal antibodies, and collecting anti-human Cu, Zn-SOD monoclonal antibodies from the culture. Method for producing monoclonal antibodies.