JP2007254428A - Antibody against natural unmodified cdt, method for producing antibody, hybridoma and immunoassay method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody enabling the identification of alcohol dependent patients, a method for producing the antibody, a hybridoma producing the antibody and a simple immunoassay method to use the antibody. <P>SOLUTION: The antibody can selectively react with unmodified natural carbohydrate-deficient transferrin (CDT). CDT concentration can be directly determined by the use of the antibody without performing the denaturation treatment of body fluid. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an antibody capable of identifying an alcoholic patient, a method for producing the antibody, a hybridoma producing the antibody, and a simple immunoassay method using the antibody. The antibody is an antibody that selectively reacts with native carbohydrate-deficient transferrin (CDT), and the antibody can be used to directly measure the CDT concentration without the need to denature the body fluid. .

  Alcohol poisoning not only causes great damage to alcoholic patients and their stakeholders, but also results in lost productivity and huge annual health care costs associated with alcoholism. Thus, early recognition and treatment of alcohol addiction is best and cost effective for individuals and society. Under such circumstances, there is a need for a test method that is sensitive, specific, rapid and inexpensive.

  For example, clinical such as γ-glutamyl transpeptidase (γ-GTP), mean corpuscular volume (MCV), aspartate aminotransaminase (AST) or alanine aminotransaminase (ALT), α-lipoprotein and ferritin Test items have been used for many years as biochemical markers of alcohol abuse, but the diagnostic sensitivity and specificity of these tests have not been satisfactory.

Stibler et al. Found that isoforms with a high isoelectric point in transferrin increased in 81% of those who took 60 g or more of ethanol daily for more than a week and had a higher isoelectric point when they banned alcohol for more than 10 days. It has been reported that the isoform possessed returns to normal levels (Acta Med Scan, 206 , 275-281, (1979): Non-Patent Document 1).

Serum transferrin is a glycoprotein consisting of a single polypeptide chain having a molecular weight of 79.5 kD and having two N-linked polysaccharide chains. These polysaccharide chains are branched and have terminal sialic acid residues. Wong and Regoeczi state in Int. J. Peptide Res. 9 , 241-248, (1977) (Non-patent Document 2) that human transferrin naturally has various isoforms depending on the level of sialylation. In fact, there are six isoforms: pentasialo, tetrasialo, trisialo, disialo, monosialo and asialotransferrin.

It has been found that asialo, monosialo, and disialotransferrin are present at elevated levels in the blood of alcoholic patients compared to normal healthy individuals (van Eijk et al, Clin Chim Acta 132 , 167 -171, (1983): Non-patent document 3, Stibler, Clin Chem, 37 , 2029-2037, (1991): Non-patent document 4, and Stibler et al, "Carbohydrate-deficient transferrin (CDT) in serum as a marker of high alcohol consumption ", Advances in the Biosciences, (Ed Nordmann et al), Pergamon, 1988, Vol. 71, pages 353-357): Non-patent document 5).

Asialo, monosialo, and disialotransferrin are called carbohydrate (sugar chain) -deleted transferrin (abbreviation: CDT). CDT has been found to be an effective marker for detecting and monitoring alcohol consumption, particularly chronic alcohol consumption. WO96 / 26444 (patent document 1) and Heil et al, Anaesthesist, 43 , 447-453, (1994) (non-patent document 6) and the like have been reported as immunological test methods for CDT. In these techniques, a diluted serum sample is passed through an anionic ion exchange resin, allowing the normal transferrin isoform to be retained on the resin while allowing the CDT fraction to pass through. The CDT content of the eluate is measured by an immunoturbidimetric assay or the like. In addition, a method for quantifying CDT by high performance liquid chromatography is also known from WO95 / 04932 (Patent Document 2).

  All of the conventional examination methods for alcoholic patients are based on relatively complicated procedures such as application of ion exchange resin or high performance liquid chromatography.

  Meanwhile, Makhlouf et al. Attempted to produce a monoclonal antibody against CDT in order to directly measure only CDT. They made an antibody using a synthetic peptide of six amino acids around the 413th and 611th asparagine to which the sugar chain of transferrin is bound as an immunogen, and the antibody was used as an immunoassay for CDT measurement. (WO93 / 06133: Patent Document 3).

  In contrast, ER.Trimble et al. Detected that the monoclonal antibody produced using a synthetic peptide as an immunogen by the method of Makhlouf et al. Did not react with CDT, and its epitope was concealed in the transferrin molecule. It was stated that it cannot be applied (Biochem Soc Trans, 26 (1), S48 (1998): Non-Patent Document 7).

On the other hand, JP-A-2004-051633 (Patent Document 4) describes an antibody that selectively binds to CDT in an aqueous solution and does not need to bind CDT to a solid phase. The antibody is an antibody obtained by using a recombinant non-glycated transferrin obtained by a genetic recombination technique as an immunogen, or using an enzyme deglycosylated as a non-glycated transferrin as an immunogen. That is, an antibody obtained by using artificially produced CDT as an immunogen.
WO96 / 26444 WO95 / 04932 WO93 / 06133 JP 2004-051633 A Acta Med Scan, 206, 275-281, (1979) Int.J.Peptide Res. 9, 241-248, (1977) Clin Chim Acta 132, 167-171, (1983) Clin Chem, 37, 2029-2037, (1991) Advances in the Biosciences, Pergamon, 1988, Vol.71, pages 353-357 Anaesthetist, 43, 447-453, (1994) Biochem Soc Trans, 26 (1), S48 (1998)

  In order to easily detect CDT in body fluid, it is desirable that CDT in body fluid can be measured without pretreatment. For this purpose, it is necessary to obtain an antibody that recognizes the structure of natural CDT.

  On the other hand, as described above, the region in the vicinity of the sugar chain deletion site may be concealed in the CDT molecule, as is understood from the report of ER.Trimble et al. A strong three-dimensional structure is formed from the large number of disulfide bonds in the transferrin molecule, and it is assumed that it is difficult to obtain an antibody that recognizes an epitope near the sugar chain deletion site.

  Also, there is a problem that CDT artificially prepared as an immunizing antigen is greatly different from natural CDT. For example, in the case of the gene recombinant of the above-mentioned Patent Document 4, the three-dimensional structure of the protein tends to be incomplete because it contains a disulfide bond, and in the case of a deglycosylated product by an enzyme, a denaturation treatment is required during preparation. . Furthermore, the recombinant and deglycosylated products have a problem that amino acid substitution occurs.

  As a method for purifying natural CDT from a body fluid, affinity chromatography using a column bound with an anti-transferrin antibody is generally used. However, this method includes a method in which transferrin is bound to an anti-transferrin antibody in a column, and then this antigen-antibody complex is denatured under acidic conditions to elute transferrin containing CDT for purification. However, CDT obtained by this method is not preferable as an immunogen because it is highly likely to be denatured.

  In view of such problems, the present invention selectively produces natural CDT without using artificially produced CDT as an immunogen, which may generate antibodies that do not react with CDT in body fluids. An object of the present invention is to provide an antibody, a method for producing the antibody, a hybridoma, and a simple immunoassay using the antibody for identifying an alcoholic patient who binds and has a high correlation with the CDT concentration measured by an existing method. And

  Furthermore, in addition to the above-described object, the present invention constructs a native and natural CDT preparation method in which the denaturation is not performed, and obtains an anti-CDT antibody that recognizes the native and native CDT structure. The purpose is to do.

  As a result of diligent research, the present inventor constructed a native and natural CDT preparation method that does not undergo denaturation, and obtained an anti-CDT antibody that recognizes the native and native CDT structure.

  That is, the anti-CDT antibody of the present invention is an anti-CDT antibody that selectively reacts with native CDT and does not react or substantially does not react with glycosidase-digested transferrin. The anti-CDT antibody of the present invention is an antibody that recognizes a three-dimensional structure without recognizing the primary structure of native CDT.

  As used herein, the term “natural” is used in the sense that artificially produced CDTs such as synthetic peptides, gene recombinants or enzyme digests are not used as immunogens, “Non-denatured” means that natural (biologically derived) antigens can also be denatured by purification methods, etc., with pH fluctuations, heat, organic solvents, surfactants (solubilizing agents), etc. It is used in the sense that there is no denaturation. The term “under non-denaturing conditions” is used to mean a condition that maintains an unmodified state and does not denature. In the present specification, the term “natural CDT” generally means native CDT derived from a living body.

  The anti-CDT antibody of the present invention may be a monoclonal antibody or a polyclonal antibody.

  A particularly preferred monoclonal antibody is an antibody produced from hybridoma NCDT503 (FERM AP-20844) deposited with the Patent Organism Depositary (IPOD).

The anti-CDT monoclonal antibody of the present invention has no or substantially no reactivity to the following P1-P6 peptide region. P1 and P2 were prepared by the present inventor, and correspond to antibody recognition sites contained in the peptide sequence shown in Patent Document 3 (Claim 3 of WO93 / 06133). P3-P6 was prepared by the present inventor, and corresponds to the antibody recognition site contained in the peptide sequence shown in Patent Document 4 (Claim 4 of Japanese Patent Application Laid-Open No. 2004-051633). The underlined part in the peptide sequence of P1-P6 indicates the antibody recognition site of Patent Document 3 or Patent Document 4.
P1: VLAENYNKSDNCE (SEQ ID NO: 1)
P2: QHLFGSNVTDCSG (SEQ ID NO: 2)
P3: VVAR SMGGKEDLI WELL (SEQ ID NO: 3)
P4: IAKIM NGE ADAMSL (SEQ ID NO: 4)
P5: YEKYLGEEYVKAV (SEQ ID NO: 5)
P6: SK LSMGSGLNLSE PN (SEQ ID NO: 6)
The antibody of the present invention can be obtained by a method in which an animal is immunized with the native CDT antigen as an immunogen and collected from the animal.

  Preferably, the antibody of the present invention immunizes an animal with a native, native CDT antigen obtained by purifying or partially purifying the CDT antigen under non-denaturing conditions using a body fluid of an alcoholic patient. Can be obtained from the animal.

  The immunogen used in the antibody production method of the present invention can be, for example, one purified or partially purified from a body fluid such as serum from an alcoholic patient under non-denaturing conditions. Preferably, the body fluid is saturated with iron. CDT prepared by contact with a solution and then non-denaturing conditions such as ion exchange chromatography can be used as an immunogen. More preferably, the body fluid is brought into contact with an iron saturated solution, and then contaminating proteins such as albumin and globulin are removed, and then the CDT prepared by fractionation under non-denaturing conditions such as ion exchange chromatography is used as an immunogen. Can do. The albumin can be removed using a blue sepharose column, and the globulin can be removed using a Protein G column. These means for removing contaminating proteins and the means for preparing fractions are useful as means for purifying CDT under non-denaturing conditions.

  The polyclonal antibody, hybridoma, and monoclonal antibody of the present invention can be collected from the animal by immunizing the animal with the immunogen. Known methods can be applied to immunize animals, and the immunization protocol required for them can be easily determined by those skilled in the art. For example, an antibody of the invention can be made by injecting an immunogen mixed with an adjuvant into a suitable host animal (rabbit, goat, horse or other mammal). The immunogen injection is continued until an appropriate titer of antiserum is obtained. The antiserum is preferably further purified after collection using known techniques such as affinity chromatography and ion exchange chromatography as necessary.

  The antibody of the present invention can be obtained from a fused cell (hybridoma) obtained by fusing a cell obtained from an immunized animal (eg, rat, hamster, mouse or other mammal) and an immortalized cell. it can. For example, a mouse can be used. In this case, after immunizing the immunogen by a known technique, the desired hybridoma is obtained by the Kohler and Milstein method (Nature 256, 495, 1975) using spleen cells and mouse myeloma cells collected from the immunized mouse. Is possible. Selection of a hybridoma that produces the target antibody can be accomplished by enzyme-linked immunosorbent assay using CDT or transferrin other than CDT used as an immunogen, or by immobilizing an anti-mouse IgG antibody, and reacting the hybridoma culture supernatant. In addition, after reaction with CDT in a solution state or transferrin other than CDT, a sandwich ELISA that detects with an anti-transferrin antibody is used, and one that does not react with transferrin other than CDT but reacts strongly only with CDT is selected. Finally, cloning is performed by a limiting dilution method to obtain a target hybridoma. A monoclonal antibody can be prepared from the obtained hybridoma in a known culture system or mouse intraperitoneal and purified by known techniques such as affinity chromatography and ion exchange chromatography as in the case of the antiserum.

  The present invention is also an immunoassay method comprising contacting a body fluid with the antibody of the present invention described above, and detecting or quantifying CDT that selectively reacts with the antibody. For example, it is a method for detecting or quantifying CDT by contacting an anti-CDT antibody and an anti-transferrin antibody with a body fluid. In the method for detecting or quantifying CDT of the present invention, a bodily fluid may be contacted with a plurality of anti-CDT antibodies having different recognition sites, and CDT contained in a specimen may be detected or quantified.

  Anti-CDT antibodies suitable for use in the immunoassay methods of the present invention may be polyclonal or monoclonal antibodies, antisera or purified fractions thereof, or known isotypes or subclasses. Furthermore, it may be an antibody fragment that can bind to an antigen as necessary. That is, the only condition for the antibody is that it can react selectively to CDT, which is measured at a significant difference that it is at least more alcoholic and less alcoholic.

  Examples of the heterogeneous measurement method applicable to the immunoassay method of the present invention include a radioisotope-labeled immunoassay method, an enzyme-labeled immunoassay method, and a fluorescence-labeled immunoassay method. Examples of the homogeneous system include a fluorescence quenching method, a fluorescence enhancement method, an energy transfer method, an aggregation method, and a turbidimetric method. The immunoassay method of the present invention may be performed in any reaction format, and may be either homogeneous or heterogeneous. These immunoassays may be used or automated.

  In the immunoassay method, one of the reagents used for radioisotope-labeled immunoassay, enzyme-labeled immunoassay, and fluorescent-labeled immunoassay is to enable detection or quantification of CDT in body fluids. However, known substances may be used as the labeling substances, and examples thereof include the following.

1) Enzyme: alkaline phosphatase, peroxidase, glucose oxidase, β-galactosidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, etc. 2) Phosphor: fluorescein isocyanate, rhodamine, Texas red, etc. 3) Radioisotope element: I ¹²⁵ , I ^131, etc. 4) Bioluminescent materials: luciferin, aequorin, etc. 5) Chemiluminescent materials: isoluminol, acridinium ester, oxalate ester, etc. The binding of these labeled products to a desired reagent is carried out using known techniques. It can be carried out.

  In the immunoassay method, it is used for radioisotope-labeled immunoassay, enzyme-labeled immunoassay, fluorescent-labeled immunoassay, and aggregation method that requires a carrier in a homogeneous system, which is a heterogeneous measurement method. One of the reagents to be obtained requires immobilizing the antibody. The material and shape of the carrier used for immobilizing the antibody can be selected from known ones depending on the measurement application. Examples thereof include polystyrene, polyethylene, polypropylene, polyacrylamide, glass, agarose, nitrocellulose and the like. Examples of the shape include microtiter plates, vials, latex beads, dip strips, and the like.

  The body fluid to be subjected to the immunoassay method of the present invention may be serum, plasma, saliva, or other body fluid, but is preferably serum.

  According to the present invention, it is possible to provide an anti-CDT antibody that selectively reacts with native and native CDT and does not react with or substantially does not react with glycosidase-digested transferrin. Thus, it is possible to detect or quantify the body fluid containing native CDT in a simple manner with a simple technique. By using this antibody, it is not necessary to denature the body fluid, and the CDT concentration can be measured directly.

  Hereinafter, the present invention will be described in detail with reference to examples and the accompanying drawings.

[Example 1]: Preparation of native and natural CDT fractions Saturated iron solution (250 μl of 0.5 M NaHCO ₃ and 180 μl of 10 mM FeCl ₃ ) was added to 10 ml of human serum with a high CDT level, and incubated at 4 ° C. for 1 hour, followed by defatting. The supernatant of the solution to which the solution (100 g / l dextran sulfate 100 μl and 1 M CaCl ₂ 500 μl) was added was collected. Furthermore, albumin was removed using a blue sepharose column (Blue Sepharose 6 Fast Flow: trade name, manufactured by GE Healthcare Bioscience Co., Ltd.), and then a Protein G column (Hi Trap Protein G HP: trade name: GE Healthcare Bio). After removing proteins other than transferrin by removing globulin using Science Co., Ltd., an anion exchange column (MonoQ HR 5/5: trade name, manufactured by GE Healthcare Bioscience Co., Ltd.) is used. CDT fractions containing native disialotransferrin and asialotransferrin were prepared.

  FIG. 1 is a graph showing a CDT fraction elution profile by anion exchange chromatography. An arrow A in FIG. 1 indicates an absorbance curve at a wavelength of 280. Arrow B shows the absorbance curve at wavelength 460. The SDS-PAGE (NuPAGE gel: trade name, manufactured by Invitrogen Corporation) of each fraction shown in the elution profile of FIG. 1 and photographs of silver staining and western blotting for isoelectric focusing are shown in FIG. As shown in FIG. 2, it can be seen that tetrasialo, trisialo, disialo, monosialo and asialotransferrin could be prepared in an unmodified and highly pure form as five isoforms depending on the amount of transferrin sugar chain.

[Example 2]: Preparation of glycosidase-digested transferrin under denaturing conditions 20 mg of human transferrin (PENTEX HUMAN TRANSFERRIN: trade name, manufactured by MILES DIAGNOSTICS) 10 μl of 10% sodium dodecyl sulfate (abbreviation: SDS) and 10 mM phosphate buffered saline (PH 7.2) (abbreviation: PBS) 90 μl was added and denatured at 37 ° C. for 1 hour. To this solution, 10% Tween 20 (Tween is a registered trademark of ICI Chemicals & Polymers, manufactured by Wako Pure Chemical Industries, Ltd., polyoxyethylene (20) sorbitan monolaurate) 50 μl, 0.1 M EDTA 10 μl, PBS 900 μl, 10 units Of glycosidase (N-glycosidase F, recombinant: trade name, manufactured by Roche Diagnostics Co., Ltd.) was added and reacted at 37 ° C. for 24 hours. The solution after the reaction is precipitated with protein using trichloroacetic acid and acetone, and after SDS is removed, the solution is passed through a lectin (ConA) affinity chromatography column (ConA-Agarose: trade name, manufactured by Honen Corporation). Adsorbing and removing undigested transferrin with glycosidase and obtaining a glycosidase-digested transferrin crude fraction. Subsequently, glycosidase-digested transferrin whose purity was enhanced by performing chromatography on the fraction using an anion exchange column (MonoQ HR 5/5: trade name, manufactured by GE Healthcare Biosciences). Prepared.

  Further, a modified transferrin as a control was prepared in the same manner except that no glycosidase was added.

  FIG. 3 is a graph showing the elution profile of glycosidase digested transferrin in lectin (ConA) affinity chromatography. FIG. 4 shows a graph of profiles separated by chromatography using an anion exchange column (MonoQ HR 5/5: trade name, manufactured by GE Healthcare Bioscience Co., Ltd.). A photograph of SDS-PAGE and isoelectric focusing of the obtained fraction is shown in FIG. As shown in FIG. 5, it can be seen that the sugar chain of transferrin was removed by glycosidase digestion.

[Example 3]: Preparation of hybridoma The fraction containing disialotransferrin and asialotransferrin obtained in Example 1 was used as a CDT antigen. The antigen was prepared in an antigen solution having a concentration of 0.5 mg / ml. 500 μl of the antigen solution was mixed with 500 μl of adjuvant (TiterMax Gold: trade name, manufactured by TiterMax Co.) and emulsified, and immunized subcutaneously in 5-week-old BALB / c mice. Similar booster immunizations were repeated 3 times every 2 weeks. Meanwhile, blood was collected during immunization and the antibody activity in blood against the antigen was measured. Fourteen days after the final immunization, 100 μl of the antigen solution was administered intraperitoneally, and three days later, the spleen was removed. Splenocytes were fused by reacting for 2 minutes in the presence of mouse myeloma cells (P3 × 63Ag8.653) and polyethylene glycol 4000 (trade name, manufactured by Merck). After the fusion, it was suspended in a HAT selection medium, dispensed into a 96-well culture plate, and cultured in a CO ₂ incubator at 37 ° C. to prepare a hybridoma. For comparison, a hybridoma was prepared in the same manner as described above using the glycosidase-digested transferrin prepared in Example 2 as an immunizing antigen.

[Example 4]: Selection of antibody-producing hybridomas for CDT fraction For each hybridoma obtained in Example 3, the antibody-producing hybridomas of the present invention were selected by sandwich ELISA. 50 μl of anti-mouse antibody prepared to 10 μg / ml with PBS was added to a 96-well microplate, and each was allowed to react at 4 ° C. overnight. Thereafter, the plate was washed once with PBS, blocked with 0.5% BSA-PBS, and used as a screening plate. 50 μl of culture supernatant of a well in which hybridoma growth was observed was added to the well and allowed to react at room temperature for 1 hour. Subsequently, after washing with a washing solution (0.05% Tween-PBS), the CDT fraction described in Example 1 was reacted at room temperature for 1 hour. After washing in the same manner, 50 μl of alkaline phosphatase-labeled anti-transferrin polyclonal antibody was added, For 1 hour. The alkaline phosphatase-labeled anti-transferrin polyclonal antibody used was prepared by binding alkaline phosphatase (Roche) to the anti-transferrin polyclonal antibody (Shibayagi Co., Ltd.) by the periodic acid method. After washing again, the alkaline phosphatase activity was colored by the Kind-King method, the absorbance at 490 nm was measured with a microplate reader, and a monoclonal antibody-producing hybridoma NCDT503 having antibody activity against the CDT fraction was selected. Monoclonal antibody-producing hybridoma TFNG405 was selected from hybridomas obtained by using glycosidase-digested transferrin as an immunogen for comparison.

[Example 5]: Cloning of antibody-producing hybridoma against CDT fraction Each hybridoma selected in Example 4 was cloned twice by limiting dilution to obtain established strains. The obtained hybridoma NCDT503 was deposited at the Patent Organism Depositary (IPOD) (Reception Date: March 14, 2006, Reception Number: FERM AP-20844). For comparison, a hybridoma TFNG405 prepared by using glycosidase digested transferrin as an immunizing antigen and selected in Example 4 was similarly obtained.

[Example 6]: Preparation of antibody For each established strain obtained in Example 5, a monoclonal antibody-producing hybridoma having antibody activity against the CDT fraction was allowed to grow in the mouse abdominal cavity for mass production of monoclonal antibodies. It was. The thus obtained monoclonal antibody in each mouse ascites was purified by column chromatography using a protein G column (manufactured by GE).

[Example 7]: Determination of antibody subclass The subclass of each monoclonal anti-CDT antibody obtained in Example 6 was determined using Isostrip (registered trademark, manufactured by Roche).

  The subclass of each antibody is shown in Table 1 below.

[Example 8]: Confirmation of antibody specificity using sandwich ELISA Each monoclonal antibody (hybridoma NCDT503-producing antibody and hybridoma TFNG405-producing antibody) prepared in Example 6 above was used in Example 1 using a sandwich ELISA. Specificity for the prepared transferrin isoform was confirmed. 50 μl of each antibody prepared to 10 μg / ml with PBS was added to a 96-well microplate and allowed to react at 4 ° C. overnight. Thereafter, the plate was washed once with PBS and blocked with 0.5% BSA-PBS. Thereafter, 50 μl of transferrin isoform prepared in Example 1 was added to the well and reacted at room temperature for 1 hour. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 50 μl of alkaline phosphatase-labeled anti-transferrin polyclonal antibody (the same as that prepared in Example 4 above) was added and reacted at room temperature for 1 hour. After washing again, the alkaline phosphatase activity was colored by the Kind-King method, the absorbance at 490 nm was measured with a microplate reader, and the specificity of each antibody was confirmed by comparing the strength of the reaction to transferrin isoform. The results are shown in Table 2 below.

  As shown in Table 2, the monoclonal antibody of the present invention (hybridoma NCDT503-producing antibody) specifically reacted with natural CDT fractions (asialotransferrin and disialotransferrin) in solution, but transferrin other than CDT. Tetrasialotransferrin did not react with glycosidase digested transferrin, an artificially produced CDT. On the other hand, a hybridoma TFNG405-producing antibody for comparison obtained by immunizing N-glycosidase F-digested transferrin was artificially prepared with natural CDT fractions (asialotransferrin and disialotransferrin) as shown in Table 2. It reacted with both CDT (glycosidase digested transferrin).

[Example 9]: Confirmation of antibody specificity using Western blot The transferrin isoform prepared in Example 1 above using the Western blot for the monoclonal antibody (hybridoma NCDT503-producing antibody) prepared in Example 6 above. The specificity for was confirmed. After diluting the gel for isoelectric focusing (Novex IEF gel: trade name, manufactured by Invitrogen Corporation, pH 3-7) with CDT fraction and transferrin other than CDT to 5 μg per lane with a dedicated sample buffer Then, after electrophoresis in a dedicated electrophoresis buffer, it was transferred to a PVDF membrane with a dedicated buffer. The PVDF membrane after transfer was blocked with Block Ace (trade name, manufactured by Dainippon Pharmaceutical Co., Ltd.), and then reacted with an antibody prepared to 10 μg / ml with PBS at room temperature for 1 hour. Further, after washing 3 times with PBS, it was reacted with an alkaline phosphatase-labeled anti-mouse IgG antibody (Goat F (ab ') Anti-Mouse Ig's, Alkaline Phosphatase Conjugate AMI4405: trade name, manufactured by BIOSOURCE) at room temperature for 1 hour. After washing again, color was developed with BCIP NBT reagent (trade name, manufactured by Nippon Bio-Rad Laboratories, Inc.). The results are shown in FIG. 6 as a photograph showing the specificity of the antibody (strength of reaction to transferrin isoform) by Western blotting.

  As shown in FIG. 6, it can be seen that the monoclonal antibody of the present invention (hybridoma NCDT503-producing antibody) specifically reacted with natural CDT fractions (asialotransferrin and disialotransferrin) in Western blot.

[Example 10]: Correlation test using a sandwich ELISA combined with an anti-transferrin polyclonal antibody Using the monoclonal antibody (hybridoma NCDT503-producing antibody) prepared in Example 6 above, the sandwich ELISA method was used for healthy subjects and alcoholic patients. A total of 32 samples were measured. As a control test, measurement was performed using a reagent manufactured by Bio-Rad. The control test is a method for measuring% CDT in transferrin using a column. The amount of total transferrin before separation on the column and the amount of CDT after separation are measured, and the CDT concentration and% in the sample are measured. CDT (ratio of CDT to total transferrin) was calculated.

  For the measurement by the sandwich ELISA, 50 μl of each antibody prepared to 10 μg / ml with PBS was added to a 96-well microplate and allowed to react at 4 ° C. overnight. Thereafter, the plate was washed once with PBS and blocked with 0.5% BSA-PBS. Thereafter, 50 μl of each serum from a healthy person and an alcoholic patient was added to the well and allowed to react at room temperature for 1 hour. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 50 μl of alkaline phosphatase-labeled anti-transferrin polyclonal antibody (the same as that prepared in Example 4 above) was added and reacted at room temperature for 1 hour. After washing again, the alkaline phosphatase activity was colored by the Kind-King method, the absorbance at 490 nm was measured with a microplate reader, and the correlation with the measured value of the control test was confirmed. The result is shown in FIG.

  As shown in FIG. 7, the antibody of the present invention obtained using the native and native CDT fraction as an immunogen showed a good correlation with the control test CDT concentration. On the other hand, the monoclonal antibody (hybridoma TFNG405 producing antibody) obtained using the glycosidase-digested transferrin used for comparison as an immunogen did not show a good correlation with the control test CDT concentration. Therefore, since the absorbance of the hybridoma NCDT503-producing antibody of the present invention correlates with the CDT concentration in the specimen, the CDT concentration in the specimen can be quantified by measuring the absorbance value.

[Example 11]: Confirmation of antigen recognition site of antibody The reactivity of the monoclonal antibody (hybridoma NCDT503-producing antibody) prepared in Example 6 was examined as follows. Peptides of the following six amino acid sequences shown in P1-P6 were bound to an ELISA plate, and then 50 μl of each antibody prepared to 10 μg / ml was added to each well and reacted at room temperature for 1 hour. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 50 μl of alkaline phosphatase-labeled anti-mouse IgG antibody (Goat F (ab ') Anti-Mouse Ig's, Alkaline Phosphatase Conjugate AMI4405: trade name, manufactured by BIOSOURCE) In addition, the mixture was reacted at room temperature for 1 hour. After washing again, the alkaline phosphatase activity was colored by the Kind-King method, and the absorbance at 490 nm was measured with a microplate reader.

The underlined part in the peptide sequence of P1-P6 indicates the antibody recognition site of Patent Document 3 or Patent Document 4. The results are shown in Table 3 below.
P1 VLANYNKSDNCE (SEQ ID NO: 1)
P2 QHLFGSNVTDCSG (SEQ ID NO: 2)
P3 VVAR SMGGKEDLI WELL (SEQ ID NO: 3)
P4 IAKIM NGE ADAMSL (SEQ ID NO: 4)
P5 YEKYLGEEYVKAV (SEQ ID NO: 5)
P6 SK LSMGSGLNLSE PN (SEQ ID NO: 6)

  According to Table 3, it can be seen that the monoclonal antibody (hybridoma NCDT503-producing antibody) does not recognize P1-P6 (a peptide containing the recognition site of the antibody of Patent Document 3 or Patent Document 4).

  Next, in order to examine the antigen recognition site of the antibody prepared in Example 6, the reactivity to the primary structure of the antigen protein was first confirmed as follows. That is, the CDT antigen was covalently bound to an ELISA plate (immobilizer amino: trade name, manufactured by NUNK), and then the antibody prepared to 10 μg / ml was added to a well subjected to denaturation with 4M urea and reduction with 0.1M DTT. 50 μl was added per well and allowed to react at room temperature for 1 hour. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 50 μl of alkaline phosphatase-labeled anti-transferrin polyclonal antibody (the same as that prepared in Example 4 above) was added and reacted at room temperature for 1 hour. After washing again, the color of alkaline phosphatase activity was developed by the Kind-King method, and the absorbance at 490 nm was measured with a microplate reader to confirm whether the primary structure of the protein was recognized. The results are shown in Table 4 below.

  As shown in Table 4, the antibody of the present invention (hybridoma NCDT503-producing antibody) has a primary structure obtained by denaturing and reducing the CDT antigen recognized by the antibody (hybridoma TFNG405-producing antibody) obtained by immunizing glycosidase-digested transferrin. It turns out that it does not react. It can also be seen that the antibody of the present invention does not recognize the primary structure of the CDT antigen, but recognizes the three-dimensional structure.

  The anti-CDT antibody of the present invention can be used in a method for directly measuring CDT concentration without denaturing a body fluid.

  The anti-CDT antibody of the present invention can be used in a simple immunoassay for identifying an alcoholic patient who selectively binds to natural CDT and has a high correlation with the CDT concentration measured by an existing method.

It is a graph which shows the CDT fraction elution profile by anion exchange column chromatography. The photographs of SDS-PAGE and isoelectric focusing of each fraction shown in the elution profile of FIG. 1 are shown. It is a graph which shows the elution profile of N-glycosidase F digestion transferrin in a lectin (ConA) affinity chromatography. It is a graph which shows the profile isolate | separated by the anion exchange chromatography after a lectin (ConA) affinity chromatography. Fig. 5 is a photograph showing SDS-PAGE and isoelectric focusing of the fraction obtained in Fig. 4. It is a photograph which shows the specificity (strength of reaction with respect to transferrin isoform) of the antibody by Western blotting. It is a graph which shows the correlation test by sandwich ELISA combined with the anti- transferrin polyclonal antibody.

Claims (14)

  An anti-CDT antibody that reacts selectively with native CDT antigen and does not react or substantially does not react with glycosidase digested transferrin.   The anti-CDT antibody according to claim 1, wherein the antibody recognizes a three-dimensional structure.   The anti-CDT antibody according to claim 1 or 2, which is obtained by immunizing an animal using a native and natural CDT antigen as an immunogen.   Claims obtained by immunizing an animal with the native and native CDT antigen as an immunogen obtained by purifying or partially purifying the CDT antigen under non-denaturing conditions using the body fluid of an alcoholic patient. 3. The anti-CDT antibody according to 1 or 2.   The anti-CDT antibody according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.   6. The anti-CDT antibody according to claim 5, wherein the monoclonal antibody is produced from hybridoma NCDT503 (FERM AP-20844). The anti-CDT antibody according to claim 5 or 6, wherein the monoclonal antibody has no reactivity or substantially no reactivity to the next P1-P6 peptide region.
P1: VLAENYNKSDNCE
P2: QHLFGSNVTDCSG
P3: VVARSMGGKEDLIWELL
P4: IAKIMNGEADAMSL
P5: YEKYLGEEYVKAV
P6: SKLSMGSGLNLSEPN   The anti-CDT antibody according to any one of claims 1 to 4, wherein the antibody is a polyclonal antibody.   A hybridoma NCDT503 (FERM AP-20844) capable of producing a monoclonal antibody that selectively reacts native CDT and does not react or substantially does not react with glycosidase digested transferrin.   A method for preparing a CDT antigen, which comprises using a body fluid of an alcoholic patient and purifying or partially purifying the CDT antigen under non-denaturing conditions.   A CDT antigen characterized by contacting a body fluid of an alcoholic patient with an iron saturated solution, then removing contaminating proteins under non-denaturing conditions, and then fractionating under non-denaturing conditions to obtain a CDT antigen. Preparation method. An N-glycosidase-digested transferrin reacts selectively with the CDT antigen in the animal body by immunizing the animal with the CDT antigen obtained by the CDT antigen preparation method according to claim 10 or 11 as an immunogen. Producing an anti-CDT antibody that does not react or substantially does not react with
A method for producing an anti-CDT antibody, which comprises collecting the anti-CDT antibody from the animal.   An immunoassay method comprising contacting the body fluid with the anti-CDT antibody according to any one of claims 1 to 8, and detecting or quantifying CDT that selectively reacts with the antibody. The anti-CDT antibody according to any one of claims 1 to 8 is brought into contact with a body fluid, and the absorbance of the reaction solution obtained by the reaction is measured to detect or quantify CDT selectively reacting with the antibody. An immunoassay method characterized by comprising: