WO1987003376A1 - Monoclonal antibody and process for its preparation - Google Patents

Abstract

Monoclonal antibody for bilirubin obtained from hybridoma cells prepared by cell fusion of antibody-yielding cells obtained by immunizing animal with bilirubin and tumor cells capable of growing in a culture medium, and a process for preparing the antibody.

Description

 TECHNICAL FIELD The present invention relates to a monoclonal antibody and a method for producing the same, and more particularly, to a monoclonal antibody having bilirubin specificity and a method for producing the same.

 Heme proteins, such as hemoglobin, which have lost their function in the living body, have their porphyrin ring opened and converted to biliverdin, because their iron is recovered, and are reduced in the next step. Become a bilirubin

Bilirubin contained in biological samples can be roughly classified into fat-soluble bilirubin (diazo reaction indirect reaction), bilirubin in water-soluble bilirubin (direct reaction type), bilirubin salt or ester, etc. Gaparu (Yamaoka et al .: Proc. Japan Acad., 27, 715-721, 1951) ₀ Furthermore, the presence of isomers in free bilirubin has been confirmed It has been found that about 97% of bilirubin is the 1X isomer of free bilirubin, and a small amount is the 1X and 1Xδ isomers. Also, various derivatives have been reported in the ester form of bilirubin. confirmed til ₃ conventionally, bilirubin, to produce a § zone dyes Jiazo reaction is measured I chemically analyzed Oru and Iu indirect quantitative analysis Nyo its § zone dye Tita. However, since bilirubin and its conjugates are unstable substances and change during the azo dye formation reaction and during the extraction separation operation, such chemical methods determine the composition and structure of bilirubin and the like. to it is possible that I _(also in this partial忻法, material to produce a § zone dyes other than bilirubin Ya, also I-vivo yellow substance Nyo such mosquito σ Chin When the constant鼂値varies significantlyぃ ぅ Significant disadvantages c In addition, the conventional method of Biliyi only allows direct measurement of bilirubin and total bilirubin values, and also requires the chemical decomposition of the original bilirubin to measure. It is necessary to know the exact structure of the original bilirubin. Therefore, this method can only be used to know the degree of jaundice, and not to understand the growth, disease state, etc. of the dandelion. There is a fatal drawback. There has been a strong demand for a direct separation method in which the free bilirubin-bilirubin conjugate can be separated and quantified without decomposing the original structure.

 As a method of directly praying bilirubin, a separation method using high performance liquid chromatography (HPLC) has been reported (Komoda, Yamazaki et al .: Proc. Japan Acad., 55B. 89-93, 1979), In this method, bilirubin and its conjugate can be separated and fixed with high detection sensitivity without decomposing the bilirubin and its conjugate. However, this method requires about one hour and a long time for sample separation, it is not always guaranteed that the separated amount j is a uniform material, and expensive equipment. It is far from practical because of the need for training and the need to train skilled technicians.

Therefore, there has been a strong demand for the development of a direct analytical method that can easily separate and determine a free bilirubin-bilirubin conjugate without decomposing the original structure and in a short time. Therefore, the present invention has high specificity and detection sensitivity for free bilirubin, bilirubin conjugate, and the like, and enables a large number of samples to be analyzed in a short time without chemically decomposing bilirubin and the like. It is an object of the present invention to provide a monoclonal antibody which can be quantitatively determined by a method and a method for producing the same.

 In this specification, the term “bilirubin” is a generic term for free bilirubin, its conjugate, and the like, unless otherwise specified. '

 The monoclonal antibody according to the present invention can be obtained from a hybridoma cell obtained by fusing cells producing an antibody specific for bilirubin with a tumor cell having the ability to divide and proliferate in a culture system.

 In order to produce a body against bilirubin according to the present invention, animals such as mice and rats are first immunized with bilirubin as an antigen. The antigen used in the present invention is bilirubin including various isomers and derivatives as described above. When immunizing animals such as mice using this bilirubin as an antigen, each bilirubin may be used as a single substance or as a mixture. When bilirubin is used as a mixture, a hybridoma having an antibody having specificity for each bilirubin can be selected when screening the produced antibodies. In order to obtain bilirubin as a single substance, it can be obtained by chemical synthesis, or it can be separated from biological samples, for example, by high performance liquid chromatography, and then separated from each fraction. Each bilirubin can be unified.

 A method for immunizing animals such as mice using bilirubin as an antigen can be performed according to a conventional method. The amount of antigen to be used and the number of immunizations / interval vary depending on the animal's immune response ability and the degree of antigen purification. If the amount of antigen is too small, immunization will not be sufficient and it will be necessary to repeat the administration of the antigen.However, even in the normal case, it is preferable to perform booster immunization in addition to the initial immunization.ぃ. Immunization is usually carried out using an adjuvant, and as the adjuvant, Freund's adjuvant, alum or the like can be used.

The antibody-producing cells are prepared from an organ having the cells several days after the final immunization, but it is usually preferable to prepare the cells 3 to 3 days after the final immunization. It is preferable that the antibody-producing cells thus prepared are fused with tumor cells as quickly as possible. However, even if the cells are left for several hours, a high percentage of hybridomas can be formed. In order to increase the fusion efficiency, various methods such as rupture of erythrocytes in cells before fusion can be used. As the tumor cells to be fused with the antibody-producing cells, myeloma cell lines such as P33X63 · Ag8 (X63), P3 · NS-1 / 1 · Ag4.1 · (NS-1) are the most preferable. As myeloma cell lines, those derived from BALB / c mice are common, and those obtained from mice of other strains or fused with cells obtained from rats, etc. Can form hybridomas at a high rate. Can be. However, it is desirable that the animal species from which the myeloma cell line was derived and the donor animal species of the antibody-producing cells match. For cell fusion, it is preferable to use cells in the logarithmic culture phase.

 In the present invention, NS-1 medium is generally used as a medium used for culturing hybridomas. It is preferable to use RPMI as a basal medium of this medium, and glutamin, pyruvic acid and the like can be added. In addition, fetal calf serum (FCS) can be added to the NS-1 medium.

 Cell fusion can be performed using a cell fusion reagent. As a cell fusion reagent, polyethylene glycol (PEG) is usually used. As the polyethylene glycol to be used, various average molecular weights can be used, and generally those having an average molecular weight of 1,000, 1500, 2000, 4000 and 6000 are used. The concentration of polyethylene glycol is usually used in the range of 30% -50%. The method of cell fusion is not limited at all. For example, a PEG solution may be added to the antibody-producing cells and myeloma cells and shaken or shaken. Mix cells and PEG solution and centrifuge at low speed. The mixing ratio of the antibody-producing cells and the tumor cells is not particularly specified, but is preferably about 1: 1 to 20: 1 (antibody-producing cells: tumor cells).

 Hybridomas obtained by cell fusion as described above were separated, for example, from hypoxanthine-amino-aminopriterin-thymidine, in order to separate them from cells that had not been fused. It can be obtained by culturing in a medium (HAT medium) and selecting hybridomas.

 Next, the antibody activity contained in the culture supernatant of the hybridoma obtained in this way is examined, and only the clone having the desired pile activity is cloned. This cloning can be performed according to a conventional method. The target antibody activity is re-examined from the obtained culture supernatant, and only antibody-positive cells are further cultured. It is preferable to perform cloning more than once in this area.

 A large amount of the clone having the desired antibody activity obtained in this manner is cultured, and the desired monoclonal antibody is injected into the peritoneal cavity of an animal such as a mouse or the like having a tissue-compatible antigen. Antibodies can be obtained. Such a monoclonal antibody is extremely useful because it can be cryopreserved and a uniform sample can always be obtained.

The monoclonal antibody obtained in this manner acts with high specificity for specific bilirubin. Therefore, the use of this monoclonal antibody makes it possible to perform a direct quantitative analysis of bilirubin in a very short time and efficiently, which was impossible with the conventional separation method. Furthermore, the use of this monoclonal antibody chemically degrades bilirubin. Since it can be quantified without the need, the etiology and pathological condition of jaundice can be accurately analyzed. For example, not only immediately after birth but also within the mother, various congenital metabolic abnormalities can be diagnosed, and appropriate treatment strategies can be determined, which is extremely useful in diagnosis. You. Furthermore, in the diagnosis of adults, it is possible to diagnose hematological diseases, liver diseases, and malignant tumors. .

Example 1

 (Antigen preparation method and immunization method)

 The bilirubin α-isomer and serum albumin were dissolved in 0.1 ml of 0.02 M phosphate buffer (ρΗ7.8) to give 100 g of each 10 / ig. This solution was gently stirred at 4 ° C for about 30 minutes under light shielding. The same amount of Freund's complete adjuvant was added to the antigen thus obtained.

 0.1 ml of this solution was intraperitoneally injected into a 4-week-old female mouse (BalbZc). The same operation was repeated three times at weekly intervals for booster immunization, and the following operation was performed on the third day after the final immunization. (Cell fusion)

The spleen of the immunized mouse was excised as described above, cut into small pieces, and passed through a method to obtain a suspension. To this, red blood cells were lysed by adding 0.83% NH ₊ C1 / Tris buffer (pH T.2), and then washed three times with RPMI (37 ° C) to prepare spleen cells. On the other hand, X63. 6.5.3 was used as myeloma cells, and washed similarly to spleen cells to prepare myeloma cells.

 The spleen cells and myeloma cells obtained in this manner are mixed at a ratio of 10: 1 and contacted with 45% polyethylene glycol 6000 at 37 ° C for 8 minutes, and at 500 G for 3 minutes on the way. Cell fusion was performed by centrifugation.

 The resulting mixture was suspended in 20% fetal calf serum (FCS) RPMI and dispensed into a 96-well microplate. The HAT medium was exchanged every three days, and screening described later was performed only on the wells that had formed colonies two weeks later.

The antibody-positive gel was transferred to a 24-well plate using HT medium, and then transferred to a culture bottle to determine the number of cells. The cells were suspended in 20% neonatal serum (NCS) -RPI and adjusted to a cell count of 5 × 10 ⁶ to 1 × 10 ⁷ cells / tube. The same amount of 20% dimethyl sulfoxide and 60% RPM 1 was added to the tube, dispensed into tubes, frozen at 80 ° C for 1 day, and stored in liquid nitrogen.

One part was 5 x 10 weeks before injection of 0.5 ml of 2,4,10,14-tetramethylpentadecane intraperitoneally 1 to 2 weeks ago to increase antibody titer. Inject ⁶ to 1 x 10 ⁷ cells (ibridooma) in 0.5 ml or less of phosphate buffered saline (PBS) and intraperitoneally. — Δ-entered. Ascites had accumulated within 1-2 weeks after this treatment.

(Screening of antibodies)

After dissolving 5 mg of bilirubin (Br) in 0.5 ml of 0.05% NaOH, 10 ml of 0.05M-tris / HCI buffer (pH 6.8) containing NaCl is added, and the solution obtained in each gel of polystyrene plate is added. Aliquots of 100 β 1 were added and left at room temperature for 2 hours or at 4 ° C overnight. Next, after washing with PBS (pH 7.2) for 3 minutes, the culture supernatant of the hybridoma was added, and the cells were cultured at 37 ° C for 30 minutes. After washing in the same manner with PBS ¾, Usagi was coupled Ureaze anti Mausu F (a ') ₂ Imuno globulin 0.25 © shea serum Arubumi emissions (BSA) -? 100〃 1 added which was diluted 100-fold with PBS Then, the cells were recultured at 37 ° C for 30 minutes. After the completion of the culture, the cells were washed three times with PBS and three times with distilled water, and added with a substrate substrate solution (CSL), and left at 37 ° C for 30 minutes. Those that turned purple were considered positive.

 Furthermore, the reactivity of BSA and the BSA-Br complex with ascites obtained by the method described above was examined: First, BSA was brought to a concentration of 1% with 0.05M carbonate buffer (pH 9.6). The mixture was dissolved in water and left on a polystyrene plate at 4 ° C overnight. In the next step, 5 mg of bilirubin dissolved in 10 ml of 0.06 M phosphate buffer (PH 7.8) (previously dissolved in 0.5 ml of 0.05N NaOH was dispensed in 100 〃 portions and left at 4 C for 30 minutes. — Br compression socks were generated, while BSA-only wells containing bilirubin were incubated with buffer only.

 The gel prepared in this manner was treated in the same manner as the antibody screening procedure described above, except that ascites was diluted 100 times with 0.25% BSA-PBS. The control that we did was awesome! The total protein content of commercially available egret serum containing the gG antibody was prepared by the Lowry method to be almost the same as that in ascites fluid. In addition, the same anti-mouse IgG immunoglobulin was used as the second antibody.

 A monoclonal antibody against bilirubin obtained by this method, which does not react with BSA but reacts only with BSA-Br complex sox, was used.

 As a result, of the 1055 clones, 14 (1.3%) were strongly positive and 6 (0.6%) were positive

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 In addition, the results of ascites-based assays revealed that four clones produced monoclonal antibodies against bilirubin.

 Next, the figure shows a graph of the results of high-speed liquid chromatography σ-matography of bilirubin isomers and bilirubin conjugates in bile of iK ordinary people. In this drawing, DG is a diglucuronide conjugate, MG is a monoglucuronide conjugate, 'IX is a free bilibin IXα isomer, IX; 3 is a free bilirubin IXS isomer, C is Controls are shown respectively.

Claims

The scope of the claims

1. Monoclonal antibody against bilirubin obtained from hybridoma cells obtained by fusing antibody-producing cells obtained by immunizing an animal with bilirubin and tumor cells capable of dividing and growing in a culture system. Null antibody.

 2. The monoclonal antibody according to claim 1, wherein the bilirubin is free bilirubin or a bilirubin conjugate.

 3. The monoclonal antibody according to claim 2, wherein the bilirubin conjugate is a monoglucuronic acid conjugate or a diglucuronic acid conjugate.

 4. The monoclonal antibody according to claim 2, wherein the animal to be immunized is a mouse or a rat.

 5. The monoclonal antibody according to claim 1, wherein the antibody-producing cell is a mouse spleen cell.

6. Monoclonal antibody _{c according} to claim 1, wherein the ffiift cells are myeloma cells. 7. Divide and propagate in a culture system with antibody-producing cells obtained by immunizing animals with bilirubin. A method for producing a monoclonal antibody, comprising obtaining a monoclonal antibody against bilirubin from hybridoma cells by fusing with a tumor cell having the ability.