WO1991000109A1 - Support sur lequel est immobilise un anticorps, et procede de production et d'utilisation de ce support - Google Patents

Support sur lequel est immobilise un anticorps, et procede de production et d'utilisation de ce support Download PDF

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Publication number
WO1991000109A1
WO1991000109A1 PCT/JP1989/000678 JP8900678W WO9100109A1 WO 1991000109 A1 WO1991000109 A1 WO 1991000109A1 JP 8900678 W JP8900678 W JP 8900678W WO 9100109 A1 WO9100109 A1 WO 9100109A1
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Prior art keywords
antibody
carrier
serum
blood
immobilized
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PCT/JP1989/000678
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English (en)
Japanese (ja)
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Hiroshi Nakajima
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Priority to PCT/JP1989/000678 priority Critical patent/WO1991000109A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption

Definitions

  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • polyclonal antibodies or monoclonal antibodies make such harmful substances or colored substances effective.
  • 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.
  • 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.
  • 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.
  • blood sac can be used for the excretion system using the antibody-immobilized carrier according to the present invention.
  • 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. .
  • ⁇ bilirubin '' when 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • a 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.
  • the ligand and the carrier are coupled via, for example, an amide group, a sulfur bond, a tresyl bond, or the like.
  • an outline of the immobilization method using agarose as an example is as follows.
  • agarose 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.
  • agarose can be activated using a condensing agent, a cyanogen halide, a periodic acid, a crosslinking reagent, an epoxide, or the like. it can.
  • 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.
  • 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.
  • a polyclonal antibody or a monoclonal antibody as a ligand can be immobilized via an amide group.
  • immobilization can be achieved by rubbing the relifluoromethyl group with the amino group of a polyclonal antibody or a monoclonal antibody as a ligand.
  • 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.
  • 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.
  • a serum albumin (BSA) solution was obtained by dissolving 10 mg of serum albumin in 2.Oml of dioxane.
  • 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 ⁇ ⁇ , 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.
  • the monoclonal antibody was treated with the hybridoma in a conventional manner to obtain the desired monoclonal antibody.
  • 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.
  • HSA human serum albumin
  • 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.
  • 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.
  • mice were immunized by intraperitoneal injection of 100 ⁇ l (60 g) of an emulsion prepared by emulsifying the bilirubin antigen thus obtained with the same amount of Freund's complete adjuvant.
  • mice Four days prior to cell fusion, mice were additionally injected with 200 ⁇ 1, 4001, and 400 uL / 400 uL / day, respectively.
  • 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.
  • HT hypoxanthine-thymidine
  • 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- ⁇ lease conjugate (0.253 ⁇ 4B 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.
  • the monoclonal antibody obtained above was treated substantially as in Example 1 and coupled to an agarose matrix.
  • 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.
  • 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.
  • ⁇ -carboxylhexylamino-agarose-sgel dry weight lg was washed with 200 ml of 0.5 M NaCl to prepare agarose matrix.
  • 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.
  • Example 1 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 6 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.
  • Example 11 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.
  • 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.

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Abstract

Support sur lequel est immobilisé un anticorps polyclonal ou monoclonal permettant d'extraire de manière sélective des substances nocives ou colorées du sang ou du sérum. Ce support peut être placé dans la circulation sanguine afin d'éliminer de manière sélective lesdites substances nocives du sang ou de sérum. On peut également l'utiliser lors de contrôles cliniques, notamment pour éliminer efficacement les substances colorées affectant défavorablement lesdits contrôles.
PCT/JP1989/000678 1989-07-05 1989-07-05 Support sur lequel est immobilise un anticorps, et procede de production et d'utilisation de ce support Ceased WO1991000109A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/JP1989/000678 WO1991000109A1 (fr) 1989-07-05 1989-07-05 Support sur lequel est immobilise un anticorps, et procede de production et d'utilisation de ce support

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Application Number Priority Date Filing Date Title
PCT/JP1989/000678 WO1991000109A1 (fr) 1989-07-05 1989-07-05 Support sur lequel est immobilise un anticorps, et procede de production et d'utilisation de ce support

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WO1991000109A1 true WO1991000109A1 (fr) 1991-01-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6161234A (en) * 1999-05-10 2000-12-19 Samina Produktions - Und Handels Gmbh Lying surface with lamellar grid
EP1779926A3 (fr) * 2005-10-24 2008-06-25 Universität für Weiterbildung Krems Système pour la purification extracorporelle du sang ou de plasma

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5764059A (en) * 1980-10-07 1982-04-17 Teijin Ltd Method of removing bilirubin in blood
JPS615025A (ja) * 1984-05-23 1986-01-10 Kukida Takeshi モノクロ−ナル抗体およびその製造法
JPS61113464A (ja) * 1984-11-06 1986-05-31 宇部興産株式会社 血液の浄化方法
JPS61181966A (ja) * 1984-12-06 1986-08-14 ユ−オ−ピ−　インコ−ポレイテツド カルボキシル基でつながれた固定化抗体

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5764059A (en) * 1980-10-07 1982-04-17 Teijin Ltd Method of removing bilirubin in blood
JPS615025A (ja) * 1984-05-23 1986-01-10 Kukida Takeshi モノクロ−ナル抗体およびその製造法
JPS61113464A (ja) * 1984-11-06 1986-05-31 宇部興産株式会社 血液の浄化方法
JPS61181966A (ja) * 1984-12-06 1986-08-14 ユ−オ−ピ−　インコ−ポレイテツド カルボキシル基でつながれた固定化抗体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JPN. J. ARFIF. ORGANS, 18 (1), (15.02.89), (Tokyo), Y. IZUMI et al., "Selective Removal of Low Molecular substances with Monoclonal Antibodies", p. 11-14. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6161234A (en) * 1999-05-10 2000-12-19 Samina Produktions - Und Handels Gmbh Lying surface with lamellar grid
EP1779926A3 (fr) * 2005-10-24 2008-06-25 Universität für Weiterbildung Krems Système pour la purification extracorporelle du sang ou de plasma

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