JPS6216499A - Antirheumatism-specific protein monoclonal antibody and cell strain - Google Patents

Abstract

NEW MATERIAL:The monoclonal antibody against rheumatism-specific protein, and having a molecular weight of 150,000-170,000 (measured by polyacrylamide electrophoresis using sodium dodecylsulfate), an ioelectric point of 7.3-7.8 (by two-dimensional electrophoresis without using a protein denaturation agent) and a mobility of 0.30-0.45 (taking the mobility of albumin as 1.0). USE:An immunoassay reagent for the diagnosis of rheumatoid arthritis. PREPARATION:For example, a BALB/C mouse is immunized with Freund's complete adjuvant together with a rheumatism-specific protein separated and purified from the blood plasma of a patient of rheumatism, and the spleen is extracted from the immunized mouse to obtain a lymphatic cell. The cell is fused with a myeloma cell, cultured in HAT medium, and cloned by limiting dilution method. The obtained monoclonal hybridoma is cultured to obtain the objective monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a monoclonal antibody against a rheumatoid-specific protein, that is, an anti-rheumatoid-specific protein monoclonal antibody and a cell line that produces an anti-rheumatoid-specific protein monoclonal antibody.

(Conventional techniques and their problems) Diagnosis of rheumatoid arthritis is currently performed according to the diagnostic criteria of the American Rheumatology Association, but these diagnostic criteria rely heavily on the doctor's findings and patient complaints, and are not objective or effective.
It cannot be said that the results are satisfactory in quantitative terms. Therefore, there are high expectations for the development of diagnostic methods with excellent objectivity and quantitative properties.

By the way, rheumatoid-specific protein is a novel protein with a molecular weight of 150,000 to 170,000 that was first reported by Koen et al. By dimensional electrophoresis, the isoelectric point (the pH corresponding to the electrophoresis position of the protein is taken as the isoelectric point of the protein) is 7.3 to 7.8, and the mobility (the mobility of the leading edge of albumin is 1.0). ) 0.30-0.4
The present inventors have already succeeded in isolating a rheumatism-specific protein (Japanese Patent Application No. 59-191754). Although this rheumatoid arthritis-specific protein is found very frequently in the plasma of patients with rheumatoid arthritis, it is not found in the plasma of healthy people or patients with other diseases (cancer, liver disease, kidney disease, etc.). is known and is considered to be a disease-specific substance for rheumatoid arthritis. Furthermore, rheumatoid factors (
Reumatoid f actor: RF)
, Immune Complex:
IC) or C-reactive protein (IC) or C-reactive protein
Since it is an abnormal protein different from e protein (CRP), new clinical information different from RF, IC, and CRP can be obtained by measuring rheumatoid-specific protein.

By the way, immunological methods are currently considered to be excellent methods for specific detection of proteins. This is because immunological methods have excellent sensitivity, specificity, reproducibility, and quantitative performance, can measure a large number of samples in a short time, and have simple measurement procedures, so they can be widely used in general laboratories. Because you can. Therefore, in order to objectively, quantitatively, and specifically measure rheumatoid arthritis-specific proteins in a general laboratory, it is important to establish an immunoassay method.

Since this immunoassay method utilizes an antigen-antibody reaction, establishing an immunoassay method for rheumatoid-specific proteins requires antibodies specific to rheumatoid-specific proteins.

On the other hand, after injecting a foreign substance (antigen) that stimulates the animal's immune system into an animal, antibodies obtained from the animal's serum (i.e., antiserum) have high specificity for the foreign substance used for immunization. In addition to antibodies with low specificity,
Alternatively, it may also contain antibodies specific to other foreign substances that are mixed in the foreign substance used for immunization and are different from the target.

Further, according to the results of subsequent research by the present inventors, it was found that the rheumatoid-specific protein is a protein that has some common antigenicity with human IgG. Therefore, when obtaining antibodies by immunizing animals with rheumatoid-specific proteins, human
Antibodies against common antigenic regions with gG are also present, and the specificity of the resulting antibodies against rheumatoid arthritis-specific proteins becomes low.

Therefore, anti-rheumatism-specific protein antibodies obtained by collecting an immunoglobulin fraction by subjecting antiserum to ion exchange chromatography or the like have insufficient specificity and are not suitable for practical use.

Furthermore, if you immunize an animal and purify specific antibodies from its antiserum, you will need a rheumatoid-specific protein that constantly acts as an antigen to immunize the animal, and if the quality of the rheumatoid-specific protein changes in parentheses, it will naturally result in immunity. Antibodies obtained from treated animals also have a different quality. Furthermore, the titer of antibodies obtained varies among individual animals, and therefore it is difficult to obtain antibodies of stable quality.

By the way, whereas antibodies obtained from antiserum are a mixture of various antibodies, monoclonal antibodies consist of only one type of antibody (i.e. monoclonal antibody), so they always have a certain antigen specificity. show. This monoclonal antibody was manufactured by Milstein
et al [Nature.

256, pp. 495-497, 1975], it is known that an antibody-producing strain can be newly formed and obtained from this antibody-producing strain.

Or, some viruses (Epstein-B a
rr V 1rus), etc., to mutate normal cells capable of producing antibodies into an antibody-producing strain that can be cultured for a long period of time,
It is also possible to obtain monoclonal antibodies from the antibody-producing strain.

To explain Milstein et al.'s method in more detail, for example, immunizing an immunizable animal such as a mouse with an antigen,
After establishment of immunity, cells capable of producing antibodies are obtained by surgically removing the spleen or the like from the animal.

These cells capable of producing antibodies (lymphoid B cells) and an infinitely proliferating cell line (hereinafter simply referred to as parent strain) having a certain type of marker are fused in the presence of a fusion promoter or in the presence of a certain type of virus. merge below. As a marker for the parent strain used here, in general, in a culture medium lacking certain components,
Alternatively, the fact that they cannot survive in culture medium containing certain components is often used. For example, hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in DNA synthesis in the DNA synthesis circuit (salvage circuit)
PhosphoribosylTransfera
se: HGP RT) or thymidine kinase (Thymidine K1nase: TK
) being defective, etc. Because HGPRT
In cells with TK and TK enzymes, the DNA synthesis circuit (de
A culture solution containing aminopterin, which is a DNA synthesis inhibitor in the no-vo circuit)
Aginopterin Thymidine: HA
When cultured in a selection culture medium (HAT medium) containing T), normal cells will not produce HGP even if the DNA synthesis cycle (de novo cycle) is inhibited by aminopterin.
The rescue circuit (
The salvage circuit (rescue pathway) operates, and DNA synthesis is performed. On the other hand, H.G.
In cells lacking enzymes such as PRT or TK,
Since the salvage circuit by enzymes such as HGPRT or TK does not work, DNA synthesis becomes possible when the DNA synthesis cycle (de novo cycle) is inhibited by aminopterin, and therefore they cannot survive in the HAT medium.

In this way, after fusion of the parent strain with normal cells capable of producing antibodies, the parent strain and the fused cells can be separated using the marker possessed by the parent strain, and only the fused cells can be selected (non-fused cells). (Since cells that have the ability to produce antibodies are normal cells, they will die if the culture is continued.) The fused cells thus obtained are a group of cells that have divided and proliferated more than a single cell that produces the desired antibody. Monoclonal antibodies can be obtained from this cell group.

(Object of the invention) The present invention applies monoclonal antibody technology to
The aim is to establish a new cell line that produces an anti-rheumatoid-specific protein monoclonal antibody that specifically reacts with rheumatoid-specific protein without cross-reacting with gG, and to obtain an anti-rheumatoid-specific protein monoclonal antibody from this cell line. It is said that

(Structure of the Invention) The antibody-producing strain that the present inventors finally succeeded in establishing after intensive research can be obtained as follows, to give a specific example. That is, a BALB/C mouse is immunized with a rheumatoid-specific protein together with Freund's complete adjuvant, etc., and after the immunization is established, the lungs are surgically removed from the mouse to obtain cells capable of producing antibodies.

These cells are P3-X63-A, an HGPRT-deficient cell line derived from myeloma cells of BAL B/C mice.
G8-U1 strain (parent strain) is subjected to cell fusion in the presence of a fusion promoter. Many other strains have been reported as parent strains, and fusion is also possible by combining these parent strains with mouse strains suitable for them. As a fusion promoter, polyethylene glycol (PEG) of various molecular weights is used.
is commonly used. Alternatively, liposomes, which are artificial lipid vesicles, can be used as fusion promoters.
) or Sendai virus (HVJ) can also be used for cell fusion. Further, an electric fusion method is also known in which cells are fused by applying voltage to the cells without using these fusion promoters. P3-X63-A as parent strain
When cells are fused using the g8-U1 strain, H
By culturing in AT medium, only fused cells consisting of cells capable of producing antibodies (normal cells) and P3-X63-Ag 8-U 1 can be selected.

A fused cell that produces the antibody of interest (ie, an antibody-producing strain) can be selected by an immunoassay method for measuring a specific antibody, using the culture supernatant of the fused cell. However, since this rheumatoid-specific protein is immunologically very similar to human IgG, many of the fused cells obtained by the previous cell fusion produce antibodies that have cross-reactivity with human RGC. Therefore, it is necessary to carefully examine the specificity of antibodies, especially regarding cross-reactivity with human IgG.

For the measurement of specific antibodies, for example, a polystyrene microplate or the like is used as a solid phase to adsorb human plasma proteins or purified human IgG other than rheumatoid-specific protein as a specific antigen or rheumatoid-specific protein as a control antigen, and then This can be carried out by an immunoassay method in which a fused cell culture supernatant is added and reacted. To give an example and explain in detail, first, protein is adsorbed onto a polystyrene microplate. At this time, as a buffer for protein adsorption,
Generally, sodium carbonate/sodium hydrogen carbonate buffer solutions are often used. Alternatively, it is also possible to use a phosphate buffer or the like. In the experience of the present inventors, a concentration of the specific antigen or control antigen during adsorption of 1 to 10 μg/me was sufficient. Even below this concentration, sufficient measurement was possible by changing the antibody concentration and the following reaction conditions. A fixed amount of the specific antigen or control antigen prepared at an optimal concentration using this sodium carbonate/sodium hydrogen carbonate buffer is added to a polystyrene microplate and left to stand for a fixed period of time. This is most commonly done in one apparatus at 4°C.
Alternatively, it is also possible to leave it at room temperature for about 2 hours.

Alternatively, it is also possible to leave it at 37° C. for 1 hour. After washing the polystyrene microplate sensitized with the antigen with, for example, a phosphate buffer containing a surfactant, the antibody in the fused cell culture medium is allowed to react for a certain period of time. After washing the polystyrene microplate in the same manner as above, an enzyme-labeled anti-mouse immunoglobulin (Ig) antibody diluted to a predetermined dilution ratio was added to perform an antigen-antibody reaction on the polystyrene microplate. React with antibody. Furthermore, after washing the polystyrene microplate in the same manner as above, an enzyme substrate is added and enzyme activity is measured. The enzyme activity measured here indirectly indicates the amount of antibody in the fused cell culture solution that reacted with the antigen adsorbed on the polystyrene microplate. This allows the specificity of the antibody in the fused cell culture solution to be determined. Furthermore, although an enzyme immunoassay method using an enzyme-labeled anti-mouse antibody has been described here, it is also possible to perform the same procedure using an anti-mouse Ig antibody labeled with a radioisotope.

Alternatively, after two-dimensional electrophoresis of rheumatoid patient plasma containing rheumatoid-specific proteins without protein denaturing agents, the electrophoretic proteins are transferred to a nitrocellulose membrane, and the electrophoretic proteins and fused cell culture medium are transferred onto the nitrocellulose membrane. It is also possible to perform the immunoblotting method, which involves reacting with antibodies in the blood. At this time, the isoelectric point (p corresponding to the electrophoretic position of the protein)
H is the isoelectric point of the protein) 7.3-7.8, mobility (assuming the mobility of the leading edge of albumin is 1.0) 0
．． What is necessary is to select a fusion cell that produces an antibody that reacts only with the rheumatoid arthritis specific protein existing in the range of 30 to 0.45 and does not react with other proteins.

To explain the nature in more detail by giving an example, in the two-dimensional electrophoresis method that does not include a protein denaturing agent, plasma etc. containing rheumatoid arthritis-specific proteins are first subjected to isoelectric focusing using a polyacrylamide tube gel. Next, the isoelectrically focused plasma and the like are run on a polyacrylamide slab gel with a gel concentration gradient. Plasma or the like subjected to two-dimensional electrophoresis is transferred from the polyacrylamide slab gel to a nitrocellulose membrane or the like by overlaying the nitrocellulose membrane or the like on the polyacrylamide slab gel and applying a voltage. The nitrocellulose membrane onto which the electrophoresed plasma etc. were transferred was immersed in the fused cell culture supernatant, and each protein of the plasma etc. transferred onto the nitrocellulose membrane,
React with antibodies likely to be contained in the fused cell culture supernatant. Next, an enzyme-labeled anti-mouse antibody or the like prepared in advance at an optimal concentration is added and further reacted with the antibody in the fused cell culture supernatant that has reacted with each protein such as plasma transferred onto the nitrocellulose membrane. Thereafter, by measuring the enzyme activity retained on the nitrocellulose membrane, antibodies in the fused cell culture supernatant that have reacted with each protein in the plasma or the like transferred onto the nitrocellulose membrane are detected.

In addition, it can also be performed by commonly used antibody specific detection methods. By combining these screening methods with cloning methods such as the limiting dilution method and the method using soft agar, an antibody-producing strain (i.e., a single cell clone that ultimately produces the desired antibody) Anti-rheumatism specific protein monoclonal antibody producing strain)
can be established.

From the anti-rheumatoid-specific protein monoclonal antibody-producing strain thus established, the anti-rheumatoid-specific protein monoclonal antibody can be obtained as follows. The anti-rheumatism-specific protein monoclonal antibody-producing strain established as described above is injected into the peritoneal cavity of a mouse that has been previously stimulated with, for example, pristane, and after a certain period of time, ascites collected in the peritoneal cavity of the animal is collected. Alternatively, culture the antibody-producing strain and collect the culture supernatant. The solution containing the anti-rheumatoid specific protein monoclonal antibody thus collected can be purified by a commonly used antibody purification method to obtain the anti-rheumatoid specific protein monoclonal antibody.

For example, to purify an anti-rheumatoid specific protein monoclonal antibody from ascites, a salt commonly used for salting out, such as sodium sulfate, is added to the ascites for salting out, and the resulting precipitate is collected by centrifugation. This precipitate is dissolved in a neutral buffer such as phosphate buffer. After removing the salts used for salt precipitation, such as sodium sulfate, by dialysis or the like, a fraction containing the desired anti-rheumatoid-specific protein monoclonal antibody is collected by a commonly used antibody purification method such as ion exchange chromatography. It can be done by

Alternatively, an anti-rheumatoid specific protein monoclonal antibody can be obtained from the culture supernatant by performing the above purification method after concentration, or by affinity chromatography using an anti-mouse Ig antibody or protein A.

The anti-rheumatoid-specific protein monoclonal antibodies thus obtained are often of the immunoglobulin G (IgG) or immunoglobulin M CIgM) class.

For IgG class, sodium dodecyl sulfate (S
It shows a molecular weight of approximately 150.000 to 160.000 by polyacrylamide electrophoresis using SDS, or the molecular weight is determined by polyacrylamide electrophoresis using SDS after reduction treatment with a reducing agent such as 2-mercaptoethanol. Approximately 25 when measured
．． Two types of subunits, 000 and 50.000, are shown.

(Example) Next, a more detailed explanation will be given with reference to examples.

Example 1 Cell fusion Purification 0.3 mg of RASP was added to Dulbecco's phosphate buffered saline (Dulbeacos' PBS).
(-) After dissolving in 30.5+wji', add an equal amount of Freund's complete adjuvant (Freund con+p
Lete adjuvant, manufactured by Gibco,
(USA) and mixed thoroughly. A complete water-in-oil emulsion was subcutaneously injected into the back of BALB/C mice to immunize them. Furthermore, the animals were immunized twice in the same manner every three weeks. 10 days later, 0.5mj of Dulbecco's phosphate buffered saline solution was applied. Purified RASP dissolved in 0.
Immunity was boosted by intraperitoneally injecting 1 g of 3I.

Three days after the final immunization, the lungs were removed and lysed.
ng buffer: 0.16M ammonium chloride solution and 0.17M) Liss buffer (pH 7.65) mixed at a ratio of 9:1, and an appropriate amount of hydrochloric acid added to adjust the pH to 7.2] After hemolysis, Hank's liquid
The cells were thoroughly washed with a solution) and the obtained cells were used as antibody-producing cells.

Cell fusion was performed using antibody-producing cells (2X
10”) and hypoxanthine guanine phosphoribosyl transferase (Hypoxanth
ine guanine phosphorybosy
p3-X63-Ag8-Ul (2X1
After sufficiently mixing polyethylene glycol (PE) with
G (MW 4,000), Sigma (Sign+a)
fused in the presence of the company, USA]. That is, to a mixture of antibody producing cells and P3-X63-Ag8-U1,
PEG2 per Hank's liquid 2°8IIll. 1.5 ml of a solution containing Og was added in a water bath at 37°C to fuse. After the fusion procedure, the cells were thoroughly washed and treated with hypoxanthine for 10-'Ms.
Aminopterine 4
Xl0-7M% thymidine 1.6
X10-'M, RPMI-1 with 10% fetal bovine serum
Only fused cells were selected by culturing in 640 culture medium (hereinafter referred to as HAT medium).

Cells producing the desired antibody were selected by enzyme immunoassay as follows.

(1) Microplate antigen sensitization Purified rheumatoid-specific protein (specific antigen) used for immunization, commercially available purified human albumin, or purified human immunoglobulin G (Ig
G) (control antigen: all manufactured by Miles, USA) at a protein concentration of 10 μg/n/n in 0.02 M sodium carbonate/sodium hydrogen carbonate buffer (pH 9.6).
! It was prepared as follows. 100 μl of this solution was added to each well of a polystyrene microplate, and allowed to stand overnight at 4°C to prevent evaporation to adsorb proteins.

(2) -Next reaction A polystyrene microplate (hereinafter simply referred to as a microplate) on which the specific antigen or control antigen was immobilized by physical adsorption was soaked in phosphate buffered saline (sodium chloride 8.0 g/7?). Potassium phosphate 0.2g/l, disodium phosphate heptahydrate 2.17g
/A', chloride power') Lo, 2g/l, Tween 20
(Tween 20) 0.5m (! / Il
, solution containing 0.2 g/l of sodium azide, pH 7.4
, hereinafter referred to as PBST). Into this microplate, culture supernatant of fused cells (10oμl/well)
was added and reacted [at room temperature (22-25°C),
2 hours]. At this time, if an antibody having reactivity with the specific antigen or control antigen immobilized on the microplate is present in the culture supernatant, the antibody is retained on the microplate by the antigen-antibody reaction.

(3) Secondary reaction After the reaction, the microplate was washed with PBST. Next, the optimal dilution factor determined in advance (in this case, 2
Add 100 μβ of alkaline phosphatase-conjugated anti-mouse IgG antibody (Miles, USA) diluted with PBST to each well of the microplate at room temperature (22-25
℃) for 2 hours. This alkaline phosphatase-conjugated anti-mouse IgG antibody reacts with mouse IgG in the culture supernatant maintained on the microplate.

(4) Measurement of enzyme activity After the secondary reaction, the microplate was washed with PBST, and then the alkaline phosphatase activity retained on the microplate was measured. The enzyme substrate paranitrophenyl phosphate dicyclohexylammonium salt (p-N1
trophenyl Phosphate+Di(c
yclotiexylammonium) 5alt
), diethanolamine buffer [Di-ethanolamine> 97ml
/l s Magnesium chloride hexahydrate 100mg/
2 mg/mj! in a solution containing 0.2 g/J of sodium azide adjusted to pH 9.8 using hydrochloric acid! The solution dissolved in the following manner was used as the enzyme substrate solution. 100 μβ of this enzyme substrate solution was added to each well of the microplate and reacted for 1 hour at room temperature (22 to 25° C.). After the reaction, add 50 μl of IN sodium hydroxide solution.
of each well to stop the enzyme reaction. The enzyme activity was determined by measuring the absorbance of the enzyme substrate solution in each well at a wavelength of 405 nm. This enzyme activity indirectly indicates the amount of antibody in the culture supernatant that has reacted with the specific antigen or control antigen on the microplate.

By repeating the above measurement method and cloning by limiting dilution method, six clones of a single cell-derived cell population (ie, new monoclonal hybrid cells) that produced the desired antibody were obtained. Anti-rheumatoid specific protein monoclonal antibody-producing new hybrid cells of these 6 clones (hereinafter referred to as
5S-1,5
S-3゜5s-4A, 5s-14, 5s-15, 5s
-16, and the monoclonal anti-θ-specific protein antibodies produced by each of these new hybrid cells were injected into S-16, respectively.
1, S-3, 5-4A, 5-14.3-15.3-16
I decided to call it.

The results of the above-mentioned enzyme immunoassay using the purified anti-rheumatism-specific protein monoclonal antibody obtained in Example 3-2 are shown in FIGS. 1 to 3. Figure 1 shows 5-4A,! :
5-14, 5-15 and 5-16 in Figure 2, and S-1 and S-3 in Figure 3. The absorbance at a wavelength of 405 nm (i.e., enzyme activity) is plotted on the vertical axis, respectively.
The horizontal axis shows the concentration of anti-rheumatoid-specific protein monoclonal antibody added to the reaction (double dilution from 10 Mg/mβ).

In the figure, 1 shows the reaction between 5-4A and purified rheumatoid-specific protein.
2 is reaction of 5-4A with purified human albumin, 3 is 5-
Reaction between 4A and purified human IgG, 4 is reaction between 5-14 and purified rheumatoid-specific protein, 5 is reaction between 5-14 and purified human albumin, 6 is reaction between 5-14 and purified human IgG, 7 8 is a reaction between 5-15 and purified rheumatoid-specific protein, 9 is a reaction between 5-15 and purified human IgG, and 10 is a reaction between 5-16 and purified rheumatoid-specific protein. 11 is a reaction between 5-16 and purified human albumin, 12 is a reaction between 5-16 and purified human IgG, 13 is a reaction between 5-15 and purified rheumatoid-specific protein, 14 is S-1 and purified Reaction with human albumin, 15 Reaction of S-1 with purified human IgG, 16
17 is the reaction between S-3 and purified rheumatoid-specific protein,
-3 and purified human albumin, and 18 is the reaction between S3 and purified human 1gG.

Calculated from the absorbance. The reaction between the anti-rheumatoid-specific protein monoclonal antibody and the specific antigen showed absorbance (ie, enzyme activity) that correlated with the protein concentration of the anti-rheumatoid-specific protein monoclonal antibody added. On the other hand,
No reaction was observed between the anti-rheumatoid specific protein monoclonal antibody and the control antigen at various protein concentrations of the anti-rheumatoid specific protein monoclonal antibody. From the above,
It can be seen that the anti-rheumatism-specific protein monoclonal antibodies that were successfully established this time all react specifically with rheumatism-specific proteins, but not with human albumin or human IgG.

Example 2 Confirmation of specificity of monoclonal antibody by immunoblotting method 1. Two-dimensional electrophoresis Plasma containing a rheumatoid arthritis-specific protein was subjected to two-dimensional electrophoresis without adding a protein denaturing agent. That is, first, polyacrylamide tube gel (gel size diameter 3 mm x 6.
5 mm) was added with 6 μl of plasma containing rheumatoid-specific proteins, and wasoelectrically focused using 0.01 M phosphoric acid and 0.14 N sodium hydroxide at a pH between 3.5 and 10 (
(constant voltage 200V, 120 minutes).

Next, this tube gel was brought into close contact with a polyacrylamide slab gel (gel size: width 75 x length 60 x thickness 2.7 mm) with a concentration gradient of 4 to 17%, and then
Glycine buffer 1 (tris hydroxymethylaminomethane 0.05M, glycine 0.384M, pH 8,
3) at a constant current (30 mA per gel) of approximately 1
The electrophoresis was carried out for 80 minutes (the electrophoresis time was determined from the electrophoresis state of albumin bound to bromophenol blue (BPB)).

2. Transferring the migrating protein from the two-dimensional electrophoresis gel to the nitrocellulose membrane. Add Tris-glycine buffer 2 (Tris-Hydroxy Methylaminomethane 0.025M, glycine 0.192M,
pH 8.3) was added until the "suppressing vat" was submerged, and then a filter paper was placed on top of the pressing vat. On this filter paper,
The polyacrylamide slab gel (two-dimensional electrophoresis gel) that was subjected to two-dimensional electrophoresis in step 1 was placed on top of the polyacrylamide slab gel (two-dimensional electrophoresis gel), and a nitrocellulose membrane [Schreiber and Schuell (
Schleicher & 5chuell),
West Germany, cut to size 75 x 551]. These were energized at a constant voltage (20 V) for 18 minutes, and the serum proteins migrated and fractionated by two-dimensional electrophoresis were transferred from the two-dimensional electrophoretic gel to a nitrocellulose membrane.

3. Reaction of monoclonal antibody on transferred nitrocellulose membrane The nitrocellulose membrane onto which proteins were transferred in step 2 (hereinafter referred to as transferred nitrocellulose membrane) was diluted with Tris-HCl buffer (10 nM Tris hydroxymethylamino acid) containing 2% bovine serum albumin. Methane/hydrochloric acid buffer, pH 7,2,0,8
%NaCj! , 0.01% NaN:+) and left at 4° C. overnight. Next, the transferred nitrocellulose membrane was immersed in a solution in which the culture supernatant containing the monoclonal antibody was diluted 5 times with Tris-HCl buffer, and shaken (20 times/1 hour) at room temperature (20-25°C). minutes) to react. After the reaction, the transferred nitrocellulose membrane was washed by immersing it in Tris/HCl buffer and shaking (20 times/min) at room temperature.

At this time, the Tris/hydrochloric acid buffer was exchanged 5 times at 5 minute intervals. After washing, peroxidase-labeled anti-mouse IgG antibody [
Miles, Israel] using a Tris-HCl buffer containing 1% bovine serum albumin.
Dilute to a predetermined water concentration (usually 100%
The transferred nitrocellulose membrane was immersed in the solution (secondary antibody solution) which had a concentration of 1:0 and was allowed to stand at room temperature for 2 hours to react.

Next, after washing the transferred nitrocellulose membrane in the same manner as above, an enzyme substrate solution was added and left to stand at room temperature for 10 minutes.
Peroxidase activity on the transferred nitrocellulose membrane was measured. Here, the enzyme substrate solution contains 0.2mM 3.3'
- Diaminobenzidine, 30% hydrogen peroxide solution 0.02
A Tris-HCl buffer solution containing %.

4. Results were observed directly. S-1, S-3, 3-4A,
5-14. 5-15. In any of the anti-rheumatoid-specific protein monoclonal antibodies 5-16, the position on the nitrocellulose membrane corresponding to the migration position of the rheumatoid arthritis-specific protein, that is, the isoelectric point on the two-dimensional electrophoresis gel (corresponding to the migration position of the protein) A brown spot within the range of pH 7.3-7.8 (as the isoelectric point of the protein) and mobility (assuming the mobility of the leading edge of albumin as 1.0) of 0.30-0.45. Peroxidase activity was observed as
Anti-rheumatism specific protein monoclonal antibody was detected. However, peroxidase activity was not observed at positions on the nitrocellulose membrane other than those corresponding to the electrophoresis positions of rheumatoid arthritis-specific proteins on two-dimensional electrophoresis gels, and therefore, the anti-rheumatoid-specific protein monoclonal antibody was detected to be associated with plasma proteins other than rheumatoid arthritis-specific proteins. was shown to be unresponsive. The results of 5-4A are shown in FIG. Isoelectric point on two-dimensional electrophoresis gel 7.3-7.8, mobility 0.30-0.
It can be seen that the anti-rheumatoid specific protein monoclonal antibody reacted only to the position on the nitrocellulose membrane corresponding to the range of 45. Although not shown here, S-1, S-3
, 5-14, 3-15, and 5-16. For reference, plasma containing rheumatoid arthritis-specific proteins was subjected to two-dimensional electrophoresis using method 1 above.
Brilliant Blue (Coomass 1eBril
The results of staining with liant Blue R-250 are shown in FIG. Isoelectric point 7.3-7.8, mobility 0.
A spot of rheumatism-specific protein is observed at the position of 30 to 0.45.

Example 3 Preparation of anti-rheumatism-specific protein monoclonal antibody 1. Method by culturing Novel hybrid cells were cultured in a commercially available serum-free medium [Product name: HBlol]
, manufactured by ANA-media, USA] at a cell concentration of 0.5 to 2 x 10 cells/ml,
The culture supernatant was collected every 24 hours. The collected culture supernatant was diluted with 0.1M phosphate buffer (pH 8,0) at 4%
The pH was adjusted to 8.0 by dialysis overnight at °C. This solution was added to Protein A-bound Sepharose CL-4B (Pr-o
tein A-5epharose CL-4B
, manufactured by Pharmacia, Sweden], and the anti-rheumatism-specific protein monoclonal antibody in the culture supernatant was transferred to Sepharose C.
It was bound to protein A on L-4B. After thorough washing with 0.1M phosphate buffer (pH 8.0), glycine-hydrochloric acid buffer (a solution containing 0.1M glycine and 0.1M sodium chloride, adjusted to pH 3.0 by adding hydrochloric acid) was run, and the eluted protein fraction was collected. The recovered protein fraction was immediately neutralized by adding 0.5M phosphate buffer (pH 7.2), and this was used as a purified anti-rheumatism specific protein monoclonal antibody solution. Table 1 shows the amount of this specially purified anti-rheumatism specific protein monoclonal antibody.

Table 1 (Calculated from absorbance at wavelength 280n- with E=14.0) 2. Method using mouse peritoneal cavity Stimulate by injecting 0.5 ml of Pristane (Sigma, USA) into the peritoneal cavity in advance. 5 x 10 new hybrid cells were injected into the peritoneal cavity of a kept mouse (6 weeks old, BALB/C, female).

Approximately one week later, ascites began to accumulate. Ascites collected in the abdominal cavity was collected using a syringe as appropriate.

The ascites thus obtained was purified as follows. The ascites was first centrifuged at 3.00 rpm for 20 minutes to remove the precipitate. Next, 1.8 g of sodium sulfate per 1011 IIl was added to the obtained supernatant, and the mixture was shaken at room temperature for 2 hours.
Salting out was carried out by standing at room temperature for 1 hour. The precipitate produced by salting out was collected by centrifugation at 8,000 g for 20 minutes. The collected precipitate was diluted with 0.02M phosphate buffer (pH 6, 8, 0, 0
(containing 5M sodium chloride and 0.02% sodium azide) and dialyzed overnight. This solution was then added to a DEAE-Sephadex column (DEAE-5ephadex A-50) equilibrated with the above phosphate buffer.
, Pharmacia, Sweden] for fractionation. The anti-rheumatoid specific protein monoclonal antibody activity of each fraction was measured by enzyme immunoassay using purified rheumatoid arthritis specific protein, and fractions having anti-rheumatoid specific protein monoclonal antibody activity were collected. This fraction matched the IgG elution position. This was used as a purified anti-rheumatism specific protein monoclonal antibody solution.

Example 4 Biochemical properties 1 of anti-rheumatism-specific protein monoclonal antibody, molecular weight of anti-rheumatism-specific protein monoclonal antibody The purified anti-rheumatism-specific protein monoclonal antibody solution obtained in Example 3-1 was used. Sodium dodecyl sulfate (
Sodium dodecyl sulfate.

SDS) was added to a concentration of 1%, and 100
The mixture was warmed at ℃ for 3 minutes. After heating, return to room temperature (15-25
The apparatus was incubated overnight at 10°C. A mixture of this SDS-treated anti-rheumatoid-specific protein monoclonal antibody and an equal volume of 10 M urea solution was used as a sample, and electrophoresis was performed using a polyacrylamide tube gel (gel size: 4 mm in diameter x 70 mm in length). As the running buffer, 0.1 MIJ acid buffer (pH 7.2) containing 1% SDS was used. At the same time, Pharmacia (P har
A molecular weight marker for electrophoresis manufactured by Macia, Sweden) was used for electrophoresis.

After electrophoresis, staining was performed as follows. A polyacrylamide tube gel on which a SO3-treated anti-rheumatoid specific protein monoclonal antibody or a molecular weight marker for electrophoresis was run was coated with Coomassie Brilliant Blue (Coom
assie brilliant blueR-250
) A solution containing 0.025% methanol, 50% methanol, and 7% acetic acid was immersed in the staining solution and shaken at room temperature for 8 hours. Thereafter, the mixture was shaken for 3 days to decolorize it with a decolorizing solution containing 10% methanol and 7% acetic acid.

The molecular weight was measured in the same manner as above using a purified anti-rheumatism specific protein monoclonal antibody solution that had been reduced using 2-mercaptoethanol.

Furthermore, the isoelectric point of each anti-rheumatism-specific protein monoclonal antibody was also measured by the isoelectric focusing electrophoresis shown in Example 2-1. The measurement results are shown in Table 2.

2. Determination of the subclass of the anti-rheumatoid specific protein monoclonal antibody The anti-rheumatoid specific protein monoclonal antibody was subjected to two-dimensional electrophoresis in the same manner as shown in Example 2, and then the subclass was determined by immunoblotting. The antiserum is
Commercially available anti-mouse IgG, anti-mouse IgGz, anti-mouse IgG2, anti-mouse IgG3, anti-mouse χ chain, anti-mouse λ chain [all manufactured by Miles, USA, rabbit serum, listed on the manufacturer's insert] Used at dilution ratio]
was used. After the reaction, judgment was made under direct vision.

Both anti-rheumatoid specific protein monoclonal antibodies
Since it reacted with the anti-mouse χ chain, the L chain was a χ chain. In the H chain, S-1 and S-3 are anti-mouse IgG, and 5-
4A, 5-14. 5-15. 5-16 reacted with anti-mouse IgG, T2 and T2, respectively.
,Met. The results are shown in Table 2.

(Effects of the Invention) The most useful point of the present invention is that a cell line that produces an anti-rheumatoid-specific protein monoclonal antibody that is specific to rheumatoid-specific protein and does not react with other human plasma proteins, especially human IgG antibodies ( i.e., antibody-producing strains) are established;
The success in obtaining anti-rheumatoid-specific protein monoclonal antibodies has made it possible to establish an immunoassay method for rheumatoid-specific protein in serum.

Furthermore, the antibody-producing strain that we have successfully established this time is an antibody that always has a certain antigen specificity and binding strength with the antigen (i.e.,
Monoclonal antibodies) and the antibodies obtained by immunizing animals are a mixture of a wide variety of antibodies, whereas the monoclonal antibodies are a single antibody and can be easily adjusted to a constant titer. It is.

Furthermore, it is possible to obtain antibodies by culturing.
By selecting an appropriate culture medium, it is also possible to dramatically increase the purity of the obtained anti-rheumatism-specific protein monoclonal antibody. In other words, it is industrially useful because antibodies of stable quality can be stably supplied.

[Brief explanation of the drawing]

Figures 1 to 3 are graphs showing dilution curves of purified anti-rheumatoid specific protein monoclonal antibodies, and Figure 4 is a graph showing dilution curves of purified anti-rheumatoid specific protein monoclonal antibodies.
FIG. 5 is a chart showing the results of immunoblotting for -4A, and FIG. 5 is a chart showing a two-dimensional electrophoresis image of human plasma containing rheumatoid-specific proteins. Motsuclonal) Nine-body concentration (×10 cycles And) Seven clonal antibodies; Agriculture (×10μ9/7111) Procedural amendment September 17, 1985

Claims (2)

[Claims] (1) Molecular weight 150,000-170, determined by polyacrylamide electrophoresis using sodium dodecyl sulfate,
000, the isoelectric point in two-dimensional electrophoresis without using a protein denaturing agent (the pH corresponding to the electrophoresis position of the protein is taken as the isoelectric point of the protein) is 7.3 to 7.8, and the mobility (of albumin) is 7.3 to 7.8. (assuming the mobility at the leading edge is 1.0) is 0.30~
Monoclonal antibodies against rheumatoid-specific proteins in the 0.45 range. (2) Molecular weight 150,000-170, determined by polyallylamide electrophoresis using sodium dodecyl sulfate;
000, the isoelectric point in two-dimensional electrophoresis without using a protein denaturing agent (the pH corresponding to the electrophoresis position of the protein is taken as the isoelectric point of the protein) is 7.3 to 7.8, and the mobility (of albumin) is 7.3 to 7.8. (assuming the mobility at the leading edge is 1.0) is 0.30~
A cell line that produces monoclonal antibodies against rheumatoid-specific proteins in the range of 0.45.