WO1991000109A1

WO1991000109A1 - Carrier having antibody immobilized thereto, process for its production and its use

Info

Publication number: WO1991000109A1
Application number: PCT/JP1989/000678
Authority: WO
Inventors: Hiroshi Nakajima
Original assignee: Kukita, Takeshi
Priority date: 1989-07-05
Filing date: 1989-07-05
Publication date: 1991-01-10

Abstract

An antibody-immobilizing carrier having immobilized thereto a polyclonal antibody or monoclonal antibody capable of selectively removing harmful substances or colored substances from blood or serum is disclosed. This carrier having the antibody immobilized thereto may be provided in the route of blood circulation to selectively remove said harmful substances from blood or serum. This may also be used in clinical inspection to effectively remove, particularly, colored substances unfavorable for the inspection.

Description

Description Antibody-immobilized carrier, its production method and its use

The present invention relates to an antibody-immobilized carrier, a method for producing the same, and a use thereof, and more particularly, to immobilizing a polyclonal antibody or a monoclonal antibody capable of selectively removing harmful or colored substances from blood or serum. TECHNICAL FIELD The present invention relates to an immobilized antibody-immobilized carrier, a method for producing the same, and a method for disposing the same in a blood circulation path to selectively remove harmful or colored substances from blood or serum. Background technology

A healthy person circulates waste substances in the body by venous flow, absorbs them by the kidneys in the circulation path, and effectively works the system power that is excreted from the kidneys to the outside of the body. Patients who do not work properly due to the power of the excretion system due to some obstacles need to artificially remove such waste substances in the body. Kidney dialysis is a powerful system. However, there are harmful substances that cannot be easily removed even by such a kidney dialysis system, and a system that can easily and effectively remove such harmful substances has not yet been developed. In addition, the removal of toxic substances such as pyrilrubin is a very difficult and troublesome problem even in artificial livers after liver resection or liver transplantation until the liver detoxifies and excretes the liver. When blood or serum is used in a clinical test, colored substances in the blood or serum interfere with various measurements during the test, and it is effective to remove it before the test. However, there is still no effective means for removing such colored substances.

In view of the current state of the art, the present inventors have found that the use of antibodies having specificity for harmful substances in blood or serum thereof, especially polyclonal antibodies or monoclonal antibodies, makes such harmful substances or colored substances effective. As a result of diligent efforts to develop a system that can be easily and easily removed, such polyclonal antibodies and Alternatively, the use of an antibody-immobilized carrier obtained by immobilizing a monoclonal antibody to an antibody-immobilized carrier makes it possible to effectively remove such harmful substances or colored substances, and to eliminate the pain for patients. It has been found that a very effective excretion system that can be significantly reduced can be developed, and at the same time, colored substances that are an obstacle to clinical examination can be effectively and simply removed, thus completing the present invention.

Disclosure of the invention

An object of the present invention is to provide an antibody-immobilized carrier characterized in that a polyclonal antibody or a monoclonal antibody capable of selectively removing harmful substances from blood or serum is immobilized on an antibody-immobilizing carrier. .

Another object of the present invention is to provide a method for producing an antibody immobilization carrier, which comprises immobilizing a polyclonal antibody or a monoclonal antibody on an activated antibody immobilization carrier to obtain an antibody immobilization carrier. And

Furthermore, the present invention relates to a method for immobilizing an antibody-immobilized carrier obtained by immobilizing a polyclonal antibody or a monoclonal antibody capable of selectively removing harmful substances or colored substances from blood or serum onto an antibody-immobilizing carrier. Another object of the present invention is to provide an antibody-immobilized carrier characterized by selectively removing said harmful substance or colored substance from blood or serum.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. In addition, blood sac can be used for the excretion system using the antibody-immobilized carrier according to the present invention. Examples of harmful or colored substances in serum include toxins such as pyrilrubins, bile acids, and toxins; There are certain powers ^ In the following, specific examples will be described using pyrirubin as an example, but it should be understood that the same applies to other harmful substances or colored substances unless otherwise specified. .

When the term `` bilirubin '' is used, unless otherwise specified, it also includes free pyrilvin, its various isomers, mono- and di-conjugates with glucuronic acid, degradation metabolites, etc. Should be understood.

The antibody-immobilized carrier according to the present invention can be obtained by immobilizing a polyclonal antibody or a monoclonal antibody on an antibody-immobilized carrier. Polyclonal antibodies or monoclonal antibodies that can be used in the present invention can be of any desired origin in blood or serum. When a harmful substance can be selectively and specifically recognized, and it is immobilized on a carrier for immobilizing antibodies and placed in the blood circulation path, it does not have any adverse effect on blood or serum, etc. A polyclonal antibody or a monoclonal antibody capable of removing the antibody can be immobilized, and any antibody can be used as long as it does not have any adverse effect on the immobilized carrier for antibody immobilization.

In addition, the use of the antibody-immobilized carrier according to the present invention makes it possible to efficiently remove various types of colored substances that are obstructive to clinical examination.

Examples of powerful polyclonal or monoclonal antibodies include those capable of specifically recognizing pyrirubin. Such a polyclonal antibody or monoclonal antibody is, for example, an albumin-conjugated bilirubin obtained by covalently binding pyrirubin to bovine serum albumin by an acid anhydride method, and immunizing a mouse with the resulting antibody. The cells were fused by the polyethylene glycol method and cultured in an MT selection medium. A powerful polyclonal or monoclonal antibody is an indispensable force, a component of serum that has the property of not reacting to the heme protein required by the human body, that is, pyrilvin, a metabolite of heme protein. Have. The polyclonal or monoclonal antibody used in the present invention is preferably one that can recognize not only free pyrilvin but also a site common to various isomers, conjugates, degraded metabolites, and the like of pyrilrubin. This makes it possible to remove various bilirubins only by using a polyclonal antibody or one type of monoclonal antibody, and it is possible to remove pyrirubin very efficiently. However, it is natural that monoclonal antibodies having selective specificity for various bilirubins can also be used.

As a carrier for immobilizing an antibody used in the present invention, a polyclonal antibody or a monoclonal antibody can be immobilized, has no adverse effect on the polyclonal antibody or the monoclonal antibody, and has no effect on blood. Anything that has no adverse effect on blood or serum, particularly useful components such as blood cells in blood when placed in the circulation circuit 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, etc., particularly those used as a carrier for affinity chromatography are preferable. However, high-performance liquid chromatography resins and the like can also be used. Further, a carrier for immobilizing an antibody, such as magnetic protein A, in which a magnetic substance such as iron is incorporated into the carrier for immobilizing an antibody having the matrix structure, also adsorbs the polyclonal antibody or the monoclonal antibody according to the present invention. Alternatively, it can be used as a binding carrier. The carrier for immobilizing an antibody incorporating such a magnetic substance is particularly suitable for use in a clinical test for removing a colored substance from blood or serum.

Immobilization of a polyclonal antibody or a monoclonal antibody as a ligand on a powerful carrier can be performed according to a conventional method. Preferably, the ligand and the carrier are coupled via, for example, an amide group, a sulfur bond, a tresyl bond, or the like.

For example, an outline of the immobilization method using agarose as an example is as follows. To use agarose as a carrier for immobilizing antibodies, agarose must be activated first. Even in this activation method, a conventional method can be used.For example, agarose can be activated using a condensing agent, a cyanogen halide, a periodic acid, a crosslinking reagent, an epoxide, or the like. it can. For example, in the case of a matrix having an amino group or a carboxyl group such as AH-type agarose and CH-type agarose, a polyclonal antibody or a monoclonal antibody as a ligand is converted to an amide by using a condensing agent such as carbodiimide. It can be immobilized via a group. In the case of a matrix having an epoxy group such as epoxy-activated hyagarose, a polyclonal antibody or a monoclonal antibody as a ligand is coupled via an S bond. Further, in the case of matrices and socks such as active lignin CH agarose having a succinimide group or the like, a polyclonal antibody or a monoclonal antibody as a ligand can be immobilized via an amide group. Furthermore, in the case of a tresyl-activated matrix, for example, immobilization can be achieved by rubbing the relifluoromethyl group with the amino group of a polyclonal antibody or a monoclonal antibody as a ligand.

(Effect)

The antibody-immobilized carrier according to the present invention obtained as described above can be, for example, incorporated in an existing kidney dialysis system as a part of the system, and can be arranged. For patients who do not need renal dialysis, it can be used as an analysis system such as a renal dialysis system. It can also be incorporated into an artificial liver that can be used until liver function is restored for patients whose liver function is weakened due to liver resection or liver transplantation, which is extremely useful from a therapeutic perspective. is there.

As described above, the antibody-immobilized carrier according to the present invention can selectively and specifically eliminate only harmful or colored substances from blood or serum, and can easily and efficiently eliminate or alleviate patient's pain. It is extremely useful and is also very effective in removing colored substances from blood or serum that may interfere with the test during clinical tests.

(Example)

Hereinafter, the present invention will be described with reference to examples.

Example 1

(Preparation of antigen)

A serum albumin (BSA) solution was obtained by dissolving 10 mg of serum albumin in 2.Oml of dioxane.

Separately, 10 mg of pyrilirubin was dissolved in 1.0 ml of dimethyl sulfoxide and activated with 3.0 l of tri-n-butylamine and 2.0 uI of isobutyl chloroformate. After 50 μl of 0.1N sodium hydroxide was added thereto, the solution was added dropwise to the serum albumin solution obtained as described above. After the pH of this solution was adjusted to about 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 pyrilvin_BSA (8 mol of pyrilvin / 1 mol of protein) was obtained by gel filtration using Sephadex G-25.

(Preparation of monoclonal antibody)

BALBA: mice were immunized by intraperitoneal injection of 100 l (60 "g) of the emulsion prepared above, in which the bound pyrilvin-BSA was milked with an equal volume of Freund's adjuvant. Mice were given an additional injection of 200, 400 μ ^, 400 ^ 1 and 400 l per day from the day before.The spleen cells of the immunized mice and mouse bone marrow cells (P3-X63Ag-Ul) were Cell fusion was performed by the polyethylene glycol method.

After culturing the fused cells in HAT medium and selecting them, The monoclonal antibody was treated with the hybridoma in a conventional manner to obtain the desired monoclonal antibody.

(Fixation processing)

Pirirubin and Kore of monochromator port one monoclonal antibody obtained as described above, papermaking seminal the I Takeno globulin (IgG) using clones showed binding characteristics _{^{(K D = 1.33 X 10- 7}} Μ), It was immobilized on activated CNBr-Sepharose (the amount immobilized was 20 mg / l Sepharose).

The immobilization was performed as follows. Washing and swelling of lg dry weight of CNBr-Sepharose on a glass filter were repeated. The thus swelled gel, and washed with cutlet pulling buffer the antibody was dissolved 3 mg of a coupling (0. 1M NaHC0 ₃ containing 0.5M NaCl _(P H8.3) solution). This solution was mixed with the gel suspension and stirred at 4 overnight. The gel thus obtained was transferred to 1M ethanolamine (pH 8.0) to block the remaining active groups. Then, it was washed with a coupling buffer, 0.1 M acetate buffer (pH 4.0) containing NaCl (0.5), and further washed with a force coupling buffer to wash away excess adsorbed antibody. Next, the coupling buffer was washed away to complete the immobilization treatment. As controls, sepharose in which human serum albumin (HSA) and mouse per-globulin (MGG) were immobilized in the same amounts were prepared.

And protein immobilization sepharose this three, filled untreated Sepharose in a 2 ml Dzu' column respectively, about 5 X 10 ⁴ dpni this as human Bok serum (G, single mono adjusted to Pirirubin value 5 mg / dl Of ³ H-pyrilvin) was added to each lml, and the mixture was eluted with a phosphate buffer. As a result, 61.3% of the added amount of リル .5 fi g (per gram of Sepharose) of bilirubin was adsorbed on the IgG-Sepharose column. This amount was 3.5 times that of the HSA-Sepharose column and several tens of times that of the other columns. Furthermore, during the monitoring, no change was observed in the total protein, GOT, GPT, and fibrinogen values before and after passage through all columns. Example 2

(Production of pyrilvin antigen)

Unconjugated pyrilvin (UCB) KalOmg was dissolved in 1.0 ml of dimethyl sulfoxide, and after ensuring that no crystals were formed in this solution, tri-n-butylamine was added. 3.0 ul and 2.0 ul of isobutyl chromate formate were added.

Separately, 10 mg of serum albumin was dissolved in 0.024 M NaOH, 2.0 ml of dioxane was added to this solution, and this mixture was centrifuged at lO. OOOG for 5 minutes to remove insoluble substances.

Then, 50 wl of Na0H was added to the pyrilrubin solution obtained above, and then added directly to the separately prepared albumin solution. The mixture was adjusted with NaOH or HC1 so that 1)} 1 was 9.5, and then the solution was stirred at 4 ° C overnight.

The resulting conjugate was purified by gel filtration on Sephadex G-25. Unbound pyrilvin was absorbed by 450 ran, and albumin was analyzed by protein assay. As a result, it was confirmed that the obtained conjugate had 8 mol of bilirubin and 1 mol of bilirubin per 1 mol of albumin.

(Manufacture of monoclonal antibodies)

BALB / c mice were immunized by intraperitoneal injection of 100 μl (60 g) of an emulsion prepared by emulsifying the bilirubin antigen thus obtained with the same amount of Freund's complete adjuvant. Four days prior to cell fusion, mice were additionally injected with 200 ^ 1, 4001, and 400 uL / 400 uL / day, respectively.

10 pairs of spleen cells and mouse bone marrow cells (Ρ3-Χ63-Ag8-U1)

The cells were mixed at a ratio of 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 treated in this manner were suspended in hypoxanthine-thymidine (HT) medium, injected into a tissue culture plate well, and cultured at 37 ° C. in a 5% CO ₂ incubator. After 24 hours, when this medium was changed to HAT medium to select for hybridomas, colony development was visible after 2-3 weeks. The supernatant of the colony thus obtained was removed, and it was examined by ELISA whether or not the desired monoclonal antibody was produced. Next, colonies positive for monoclonal antibody production by this method were further cultured together with mouse spleen cells.

(Screening)

Put an antigen solution of UCB-HSA (1 μl in phosphate buffered saline (PBS) 100 / X1) in a polystyrene plate well, leave overnight at 4 and wash the well 3 times with PBS To remove unadsorbed antigen, then add 1% gelatin (PBS) solution Note, and left at room temperature for 1 hour. Subsequently, unbound protein was removed by washing 3 times with PBS containing 0.05% Tween 20. Next, the antibody-containing supernatant was diluted with a 10-fold amount of 1% HSA-PBS, and 10 l of an aliquot was added to the well. The wells were cultured at 37 for 30 minutes, washed three times with PBS containing 0.05% Tween 20, and then washed with anti-mouse IgG (Fab ') _2-ゥ lease conjugate (0.25¾B SA-PBS in advance). (10Q dilution). This well is cultured at 37 ° C for 30 minutes, washed three times with PBS containing 0.05% Tween 20, then 100 ul of the substrate solution is added to each well, and the mixture is incubated at room temperature for 30 minutes. After standing, the absorbance at 590 nm was measured using an ELISA reader (manufactured by Toyo Sokki). As a result of the measurement, it was found that 64 clones did not bind to the carrier protein but produced specific antibodies bound to UCB.

Among the clones thus obtained, 16 clones showing the highest reactivity to UCB antigen by Competitive Saturation Analysis were selected. Subclasses of these clones were examined by a conventional method. As a result, it was confirmed that 7 IgGl, 3 IgG2a, 4 IgG2b, 1 IgG3, and 1 IgGM were obtained. (Fixation processing)

The monoclonal antibody obtained above was treated substantially as in Example 1 and coupled to an agarose matrix.

When this matrix was used and treated in the same manner as in Example 1, it was confirmed that pyrylvins in serum were removed in the same manner as in the matrix of Example 1, and that other values were not adversely affected. .

Example 3

(Method of producing antigen)

Pyrirubin was dissolved in chloroform (1 mg / ml) containing 5% p-hydroxybenzaldehyde, and several crystals of P-toluenesulfonic acid were added to this solution. When this mixed solution was allowed to stand in a dark place for about 8 hours, a bilirubin derivative in which a formylphenoxy group was bonded to an exo-vinyl group of pyrirubin was obtained.

The resulting pyrylvin 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 that had a strong S bond with the pyrilbin derivative and albumin. Generated. Next, this Schiff base was stirred at room temperature using sodium borohydride, and then reduced to obtain a corresponding Schiff salt-reduced compound power S.

Using this antigen, the same treatment as above was performed to produce a monoclonal antibody. The antibody thus obtained was treated as described below and coupled to an agarose matrix. That is, this antibody was dissolved in water to adjust the pH to 4.5 to prepare a ligand solution. On the other hand, ω-carboxylhexylamino-agarose-sgel dry weight lg was washed with 200 ml of 0.5 M NaCl to prepare agarose matrix. Further, 10 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is dissolved in water to adjust the pH to 4.5, and this solution is mixed with the above-mentioned ligand solution. Stirred at night. At the end 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 thus-coupled monoclonal antibody, good results were obtained as in the above example.

Example 4

Using the antigen obtained in Example 1, screening was performed in the same manner as in Example 1 to prepare a polyclonal antibody. This antibody was treated with immobilized lignin in the same manner as in Example 1, and the removal rate of bilirubins from the serum was examined. The results were the same as those in the above Example.

Example 5

Samples 500-600 μl of phosphate buffered saline (PBS) plus bilirubin at the concentrations shown in Table 1 below were used for magnetic protein A 500 u1 and IgG. After mixing with 500 u1 (1.1 mg), the mixture was incubated at room temperature for 5 minutes, and the amount of pyrilvin removed from the sample was determined from the absorbance at 448 nm. Table 1

Examples 6 to 9

As a sample, in Example 6, 1% human serum albumin plus bilirubin, in Example 7, 0.1% human serum albumin plus pyrirubin, and in Example 8, human serum (sample Nol: pyrilirubin concentration) : 0.1 mg / dl) and Example 9 except that human serum (sample No.2: pyrirubin concentration: 0.4 mg / dl) was used. I asked. The results are shown in Table 2 below.

Table 2

Example 10

500-600 μl of a sample obtained by adding bilirubin to phosphate buffered saline (PBS) to the concentration shown in Table 3 below was used for magnetic protein A 5001, sodium salicylate and IgG 500 μl. (1.1 mg), the mixture was incubated at room temperature for 5 minutes, and the amount of pyrilvin removed from the sample was determined from the absorbance at 448 nm. Table 3

Example 11-: 13

As a sample, in Example 11, 1% human serum albumin was supplemented with pyrilvin, and in Example 12, human serum (sample Nol: pyrirubin concentration: 0.1 mg / dll and in Example 13, human serum (sample After culturing in the same manner as in Example 9 except that No. 2 (pyrirubin concentration: 0.4 mg / dl) was used, the removal rate of pyrirubin was determined, and the results are shown in Table 4 below.

Table 4

Industrial availability

The antibody-immobilized carrier in which a polyclonal antibody or a monoclonal antibody against bilirubin according to the present invention is immobilized on two solid carriers can remove harmful substances such as bilirubin or colored substances by adsorbing or binding thereto. Not only that, it is extremely useful in clinical tests. Further, if such an antibody-immobilized carrier is used as a test kit, it can be used as an extremely simple test kit.

Claims

The scope of the claims

(1) An antibody-immobilized carrier obtained by immobilizing a polyclonal antibody or a monoclonal antibody capable of selectively removing harmful or colored substances from blood or serum on an antibody-immobilizing carrier.

(2) The antibody-immobilized carrier according to claim (1), wherein the polyclonal antibody or the monoclonal antibody is capable of specifically recognizing a harmful substance or a colored substance in blood or serum.

(3) The antibody-immobilized carrier according to (2), wherein the harmful substance or the colored substance is bilirubin, bile acids, toxin, or a drug.

(4) The antibody-immobilized carrier according to claim (1), wherein the polyclonal antibody or the monoclonal antibody has specificity for bilirubin.

The antibody-immobilized carrier according to claim 1, wherein the antibody-immobilized carrier is capable of immobilizing a polyclonal antibody or a monoclonal antibody.

(6) A method for producing an antibody-immobilized carrier, characterized in that an antibody-immobilized carrier is obtained by standardizing a polyclonal antibody or a monoclonal antibody to an activated antibody-immobilized carrier.

(7) A polyclonal antibody or monoclonal antibody capable of selectively removing harmful or colored substances from blood or serum is immobilized on an antibody immobilizing carrier. Use of the antibody-immobilized carrier, wherein the harmful substance is selectively removed from blood or serum.

(8) Using an antibody-fixed carrier obtained by immobilizing a polyclonal antibody or monoclonal antibody capable of selectively removing harmful substances or colored substances from blood or serum onto an antibody-immobilizing carrier, using the above-mentioned method from blood or serum. Use of an antibody-immobilized carrier characterized by selectively removing harmful substances or colored substances.