JP3155961B2 - Anti-bilirubin monoclonal antibody-immobilized carrier and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an anti-bilirubin monoclonal antibody-immobilized carrier and a method for producing the same, and more particularly, to an anti-bilirubin monoclonal antibody-immobilized antibody having an antibody against bilirubin immobilized thereon and a method for producing the same. It is. Healthy people circulate waste substances in the body by venous flow,
A system that is absorbed by the kidneys in the circulation pathway and excluded from the body outside the kidneys is working effectively. But,
Patients whose elimination system does not work properly due to some obstacles need to artificially remove such waste in the body. Renal dialysis is performed as such a system. However, there are harmful substances that cannot be easily removed even by such a kidney dialysis system, and at present, a system capable of easily and effectively removing such harmful substances has not yet been developed. In addition, it is very difficult and troublesome to remove bilirubin and other harmful substances from blood or serum even in artificial livers that are used after liver resection or liver transplantation until liver detoxification and excretion function is restored. It is a problem. In view of this situation, the present inventor has proposed a system capable of effectively and easily removing such harmful substances by using an antibody having specificity for harmful substances such as bilirubin in blood or its serum, particularly using a monoclonal antibody. As a result of our utmost efforts, we can immobilize an anti-bilirubin monoclonal antibody on a carrier for immobilizing an anti-bilirubin monoclonal antibody and use the resulting anti-bilirubin monoclonal antibody-immobilized carrier to identify bilirubin in such harmful substances. It has been found that an effective and simple excretion system can be developed which can be effectively and simply removed, and which can remove or significantly reduce the pain for the removal from the patient, and completed the present invention. . Hereinafter, the present invention will be described in detail. In the present specification, when the term "bilirubin" is simply used,
Unless otherwise specified, it is to be understood that the term includes free bilirubin, various isomers thereof, mono-, di-conjugates with glucuronic acid, decomposed metabolites, and the like. The anti-bilirubin monoclonal antibody that can be used in the present invention can selectively and specifically recognize bilirubin, which is a harmful substance in blood or serum, and can be immobilized on a carrier for in vivo fixation and disposed in a blood circulation path. And when placed in such a route, it can remove the harmful substances without any adverse effect on blood or serum, that is, on the patient. The carrier for immobilizing an anti-bilirubin monoclonal antibody according to the present invention can be obtained by immobilizing an anti-bilirubin monoclonal antibody on a carrier for immobilizing an anti-bilirubin monoclonal antibody. That is, an anti-bilirubin monoclonal antibody is obtained, for example, by covalently linking bilirubin and bovine serum albumin by an acid anhydride method to obtain albumin-conjugated bilirubin, immunizing a mouse with this as an antigen, spleen cells of the mouse and mouse bone marrow. The cells can be produced by fusing cells with the polyethylene glycol method and then culturing in a HAT selection medium. The monoclonal antibody thus prepared can recognize sites common to free bilirubin, various isomers of bilirubin, sutures, degraded metabolites, and the like. When such a monoclonal antibody is used, various types of bilirubin can be simultaneously recognized by only one type, and such various types of bilirubin can be simultaneously removed,
An extremely efficient, effective and simple bilirubin removal system can be constructed. Similarly, a monoclonal antibody capable of selectively specifically recognizing each specific site such as free bilirubin, various isomers of bilirubin, sutures, degradation metabolites and the like can be naturally produced. In this case, each bilirubin can be individually recognized and removed. Furthermore, the monoclonal antibody thus obtained has a property that it does not react with heme protein, a serum component required for the human body, in particular, a metabolite of bilirubin heme protein. As the antibody-immobilizing carrier used in the present invention, an anti-bilirubin monoclonal antibody can be immobilized,
It has no detrimental effect on the monoclonal antibody and, when placed in the blood circulation, has no adverse effect on blood or serum, especially on useful components such as blood cells in blood. Any one may be used as long as it is suitable for the purpose of the present invention. As such a carrier, those having a matrix structure are preferable. For example, cellulose, dextran, agarose, polyacrylamide and the like, particularly those used as a carrier for affinity chromatography are preferable. Also,
High-performance liquid chromatography resins and the like can also be used. Immobilization of a monoclonal antibody as a ligand on such a carrier can be performed according to a conventional method. The ligand and the immobilizing antibody are preferably coupled via, for example, an amide group, a sulfur bond, a tresyl bond, or the like. The outline of the immobilization method will be described using agarose as an example. That is, in order to use agarose as a carrier for immobilizing antibodies in the present invention, first, agarose must be activated. This activation can be performed according to conventional methods. Examples of the agarose activator include a condensing agent, a halogenated cyanide, a periodic acid, a crosslinking reagent, and an epoxide. For example, when using agarose having a matrix structure having an amino group or a carboxyl group such as AH-type agarose and CH-type agarose, a monoclonal antibody as a ligand is converted to an amide group using a condensing agent such as carbodiimide. Can be immobilized via. When agarose having a matrix structure having an epoxy group such as epoxy-activated agarose is used, a monoclonal antibody as a ligand can be coupled via an S bond. Furthermore, when using agarose having a matrix structure such as activated CH agarose having a succinimide group or the like, it can be immobilized via an amide group. Furthermore, when agarose having a tresyl-activated matrix structure is used, for example, the trifluoromethyl group and the amino group of a monoclonal antibody as a ligand can be mapped and immobilized. (Effect) The anti-bilirubin monoclonal antibody-immobilized antibody of the present invention obtained as described above can also be incorporated into an existing renal dialysis system as part of an existing renal dialysis system, for example. For patients whose liver function is weak due to liver resection or liver transplantation, it can also be used by incorporating it into an artificial liver that can be installed until the liver function is restored, which is extremely useful from a therapeutic perspective. is there. As described above, the anti-bilirubin monoclonal antibody-immobilized antibody according to the present invention can selectively and specifically remove only harmful substances such as bilirubin from blood or serum, and can easily and efficiently remove or alleviate patient pain. It is a very useful thing that can be done. (Example) Hereinafter, this description will be further described with reference to an example. Example 1 (Preparation of antigen) Bovine serum albumin (BSA) solution was obtained by dissolving 10 mg of bovine serum albumin in 2.0 ml of dioxane. Separately, 10 mg of bilirubin was dissolved in 1.0 ml of dimethyl sulfoxide and activated with 3.0 μl of tri-n-butylamine and 2.0 μl of isobutyl chloroformate. After adding 50 μl of 0.1N sodium hydroxide to this,
This solution was added dropwise to the bovine serum albumin solution obtained above. After adjusting the pH of this solution to about pH 9.5 with 1N hydrochloric acid or sodium hydroxide, the mixture was stirred at 4 ° C. for 12 hours to complete the coupling. From this reaction solution, bound bilirubin-BSA (8 mol of bilirubin / 1 mol of protein) was obtained by gel filtration using Sephadex G-25. (Preparation of Monoclonal Antibody) 100 μl of an emulsified liquid obtained by emulsifying the bound bilirubin-BSA obtained above with the same amount of Freund's adjuvant.
(60 μg) was immunized by intraperitoneal injection into BALB / c mice. 4 days before cell fusion, 200μ / day
, 400 μl, 400 μl, and 400 μl were each injected intraperitoneally for booster immunization. The spleen cells of the mice thus immunized and the mouse bone marrow cells (P3-X63Ag-U1) were fused by the polyethylene glycol method. After the cells obtained by the cell fusion were cultured in a HAT medium and selected, the hybridoma producing a bilirubin-specific monoclonal antibody was treated by a conventional method to obtain a desired monoclonal antibody. (Immobilization treatment) Among the monoclonal antibodies obtained as described above, a large amount of immunoglobulin (IgG) was purified from a clone exhibiting a high binding characteristic to bilirubin (K _D = 1.33 × 10 ⁻⁷ M), and the activity was purified. Immobilized on immobilized CNBr-Sepharose (the amount of immobilization was 20 mg / g of dry Sepharose). The immobilization was performed as follows. 1g dry weight
The CNBr-Sepharose was repeatedly washed and swelled on a glass filter. The swollen gel thus obtained was washed with a coupling buffer (0.1 M NaHCO ₃ (pH 8.3) solution containing 0.5 M NaCl) in which 3 mg of the antibody to be coupled was dissolved. This solution is mixed with the gel suspension and
And stirred overnight. The gel thus obtained was transferred to 1 M ethanolamine (pH 8.0) to block the remaining active groups. Next, it was washed with a coupling buffer, 0.1 M acetate buffer (pH 4.0) containing NaCl (0.5 M), and further washed with a coupling buffer to wash away excess adsorbed antibody. Thereafter, the coupling buffer was washed away to complete the immobilization treatment. As a control, Sepharose was prepared by immobilizing human serum albumin (HSA) and mouse γ-globulin (MGG) in the same amounts. Each of the three types of protein-immobilized sepharose and untreated sepharose was packed in a column of 2 ml each, and human serum adjusted to a bilirubin value of 5 mg / dl was added thereto (about 5 × 10 ⁴ dpm of 3H-bilirubin was added as a tracer). Each 1 ml was added and the mixture was eluted with a phosphate buffer. As a result, 17.5 corresponding to 61.3% of the added amount was applied to the column of IgG-Sepharose.
It was found that μg (per 1 g of Sepharose) had been adsorbed. This amount corresponds to 3.5 times of the HSA-Sepharose column and several tens of times of the other columns. In addition, the total amount of protein, GOT
The values, GFT values and fibrinogen values did not change before and after the passage in all columns. Example 2 (Preparation of bilirubin antigen) 10 mg of unconjugated bilirubin (UCB) IX α was dissolved in 1.0 ml of dimethyl sulfoxide, and after ensuring that no crystal was formed in this solution, 3.0 μl of tri-n-butylamine was added.
1 and 2.0 μl of isobutyl chloroformate were added. Separately, 10 mg of bovine serum albumin was dissolved in 0.024 M NaOH, 2.0 ml of dioxane was added to the solution, and the mixture was centrifuged at 10,000 G for 5 minutes to remove insoluble substances. Next, 50 μl of NaOH was added to the bilirubin solution described above, and immediately added dropwise to the separately prepared albumin solution. After adjusting this mixture to pH 9.5 with NaOH or HCI,
The solution was stirred overnight at 4 ° C. The conjugate obtained in this manner was separated into Sephadex G
Purified by gel filtration at -25. The unbound bilirubin was examined by absorption at 450 nm, and albumin was examined by protein assay. As a result, it was confirmed that 8 mol of bilirubin was bound to 1 mol of albumin. (Production of Monoclonal Antibody) 100 μl of an emulsified liquid obtained by emulsifying the bilirubin antigen obtained as described above with the same amount of Freund's complete adjuvant (6
(0 μg) was immunized by intraperitoneal injection into BALB / c mice.
4 days before cell fusion
Booster immunization was performed by injection of 200 μl, 400 μl, 400 μl, and 400 μl. Spleen cells and mouse bone marrow cells (P3
-X63-Ag8-U1) was mixed at a ratio of 10: 1 (spleen cells to bone marrow cells) and treated with 45% polyethylene glycol 6000 at 37 ° C. for 8 minutes to perform cell fusion. The cells thus obtained by cell fusion are suspended in hypoxanthene-thymidine (HT) medium, injected into a tissue culture plate well, and then placed in a 5% CO ₂ incubator in an incubator.
The cells were cultured at 7 ° C. After 24 hours, the medium was changed to HAT medium for selection of hybridomas, and colonies were visually observed after a few weeks. The supernatant of this colony was removed, and the production of the desired monoclonal antibody was examined by ELISA. Next, the colonies confirmed to be positive for monoclonal antibody production by the above method were further cultured together with mouse spleen cells. (Screening) UCB-HSA (phosphate buffered saline (PBS) 100μ)
(1 μl / l) of the antigen solution was placed in a polystyrene plate well, left at 4 ° C. overnight, and the well was washed three times with PBS to remove unadsorbed antigen. Then
A 1% gelatin (PBS) solution was injected and left at room temperature for 1 hour. Subsequently, it was washed three times with PBS containing 0.05% Tween 20 to remove unbound protein. Next, the supernatant containing the antibody was diluted with 10 volumes of 1% HSA-PBS, and then 10 μl aliquots were added to the wells. At 37 ° C
After culturing for 30 minutes and washing three times with PBS containing 0.05% Tween 20, a rabbit anti-mouse IgG (Fab ') 2-urease conjugate (diluted 100-fold with 0.25% BSA-PBS in advance) Was added. The wells were cultured at 37 ° C. for 30 minutes, washed three times with PBS containing 0.05% Tween 20, then 100 μl of the substrate solution was added to each well, and left at room temperature for 30 minutes. Thereafter, the absorbance at 590 nm was measured using an ELISA reader (manufactured by Toyo Sokki). As a result of this measurement, 64 clones did not bind to the carrier protein,
It turned out that it produced a specific antibody bound to B. Competitive Satu
Analyzed by ration Analysls, 16 clones with highest reactivity to UCB antigen were selected. As a result of examining the subclasses of these clones by a conventional method, it was confirmed that there were 7 IgG1, 3 IgG2a, 4 IgG2b, 1 IgG3, and 1 IgGM. (Immobilization treatment) The monoclonal antibody obtained above was treated in substantially the same manner as in Example 1, and coupled to an agarose matrix. Using the matrix thus obtained, the same treatment as in Example 1 was carried out, and the bilirubins were removed in the same manner as in the matrix of Example 1, and other values were not adversely affected at all. Was confirmed. Example 3 (Production of antigen) Bilirubin was dissolved in chloroform (1 mg / ml) containing 5% p-hydroxybenzaldehyde, and p-
Several crystals of toluenesulfonic acid were added. When this mixture was allowed to stand in a dark place for about 8 hours, a bilirubin derivative in which a formylphenoxy group was bonded to an exovinyl group of bilirubin was obtained. The resulting bilirubin derivative was then reacted with calf serum albumin in the presence of 0.33 M dipotassium hydrogen phosphate adjusted to pH 9 with potassium hydroxide to form a Schiff base in which the bilirubin derivative and albumin were bound. . Next, this Schiff base was stirred at room temperature using sodium borohydride, and then reduced to obtain a compound in which the corresponding Schiff base was reduced. Using this reducing compound as an antigen, a monoclonal antibody was obtained in the same manner as in Example 1. The monoclonal antibody thus obtained was treated as described below and coupled to an agarose matrix. That is, this antibody was dissolved in water and adjusted to pH 4.5 to prepare a ligand solution. On the other hand, ω of 1g dry weight
-0.5 M carboxyhexylamino-agarose gel
After washing with 200 ml of NaCl, an agarose matrix was prepared. Further, 10 g of 1-ethyl-3- (3-ditylaminopropyl) carbodiimide hydrochloride was dissolved in water to adjust the pH to 4.5, and this solution was mixed with the above-mentioned ligand solution.
Stirred overnight at room temperature. After completion of the reaction, excess ligand, unreacted carbodiimide and by-products were washed away with water. As a result of examining the effect of removing bilirubins from serum using the monoclonal antibody coupled as described above, good results were obtained in the same manner as in the above Examples.

Claims (1)

(57) [Claims] An anti-bilirubin monoclonal antibody-immobilized carrier, wherein the anti-bilirubin monoclonal antibody is immobilized on an antibody-immobilizing carrier. 2. Activating the antibody-immobilizing carrier, the anti-bilirubin monoclonal antibody-immobilized carrier characterized in that the antibody-immobilizing carrier is coupled with an anti-bilirubin monoclonal antibody using a coupling agent to obtain an anti-bilirubin monoclonal antibody-immobilized carrier. Production method. 3. 3. The method for producing an anti-bilirubin monoclonal antibody-immobilized carrier according to claim 2, wherein the anti-bilirubin monoclonal antibody-immobilized carrier is a carrier having a matrix structure. 4. 3. The method for producing an anti-bilirubin monoclonal antibody-immobilized carrier according to claim 2, wherein the coupling agent is a condensing agent, a cyanogen halide, a periodic acid, a crosslinking reagent, or an epoxide.