JP4753366B2 - % CDT quantification method - Google Patents

Description

  The present invention relates to a method for quantifying the ratio of CDT to total transferrin (% CDT) for carrying out a simple immunoassay method capable of identifying an alcoholic patient.

  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.

  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 disialoganglioside transferrin carbohydrate (sugar) deficient transferrin (abbreviation: CDT) is called, as a method for measuring the CDT, immunological tests (W O9 6/26444: Patent Document 1 And Heil et al, Anaesthesist, 43 , 447-453, (1994): Non-Patent Document 6, etc.) and high-performance liquid chromatography (WO95 / 04932: Patent Document 2) are used.

In the immunological test method, a CDT fraction is separated with an anionic ion exchange resin and then measured by an immunoturbidimetric assay, or a competitive latex agglutination method using a CDT-specific antibody (specifically, Open 2004-051633: Patent Document 3). In order to quantify the ratio (% CDT) of carbohydrate-deficient transferrin (CDT) to total transferrin by the immunological test method, the% CDT is calculated from the CDT concentration obtained by the test method and the total transferrin concentration. Yes. In order to quantify% CDT by the high performance liquid chromatography method,% CDT is calculated from the CDT concentration obtained by the method and the total transferrin concentration.
WO96 / 26444 WO95 / 04932 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 Anaesthesist, 43, 447-453, (1994)

  In quantification of the ratio of CDT to total transferrin (ie,% CDT) by the conventional immunological test method or the high performance liquid chromatography method, the measurement of total transferrin is performed in addition to the measurement of CDT. The process of calculating% CDT has been performed.

  Therefore, an object of the present invention is to provide a% CDT quantification method capable of directly measuring% CDT without the need to measure total transferrin.

  The method for quantifying% CDT of the present invention that solves the above-mentioned problems uses substance A that reacts nonspecifically with transferrin isoform and substance B that reacts specifically with carbohydrate-deficient transferrin (CDT), By selecting non-specifically the transferrin isoform in the body fluid with the substance A and specifically selecting CDT with the substance B, the amount of total transferrin in the body fluid can be measured without measuring the total amount of transferrin. This is a method for quantifying the ratio of CDT (% CDT).

  In the method for quantifying% CDT of the present invention, “substance A that reacts nonspecifically with transferrin isoform” is used in the sense that it does not react specifically with a specific isoform of transferrin. ing.

  The substance A that reacts nonspecifically with the transferrin isoform includes an anti-transferrin antibody. The substance B that specifically reacts with CDT includes CDT-specific antibodies.

  In the method for quantifying% CDT according to the present invention, the transferrin in the body fluid is selected nonspecifically by the substance A that reacts nonspecifically with the transferrin isoform, and the transferrin is used in an excess amount. Thus, the% CDT can be directly measured without being affected by the variation of transferrin in each body fluid and without having to measure the total transferrin.

  A method applicable to the method for quantifying% CDT of the present invention is a heterogeneous measurement method. For example, when labeling substance B that specifically reacts with CDT (that is, a CDT-specific antibody). Examples include radioisotope-labeled immunoassay, enzyme-labeled immunoassay, and fluorescence-labeled immunoassay. Alternatively, it is possible to measure directly by mass change or the like without labeling the CDT specific antibody. The method for quantifying% CDT of the present invention may be carried out in any reaction format as long as it is a heterogeneous system. These quantification methods may be used or automated.

  In the method for quantifying% CDT of the present invention, one of the reagents used in radioisotope-labeled immunoassay, enzyme-labeled immunoassay, and fluorescent-labeled immunoassay is the detection or quantification of CDT in body fluids. In order to make it possible, it is necessary to label, but for these labeling substances, known substances may be used, 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 method for quantifying% CDT of the present invention,% CDT can be quantified by forming a sandwich complex by the reaction of the substance A, transferrin isoform and substance B.

  A preferred embodiment for carrying out the method for quantifying% CDT of the present invention is that the substance A is bound to a solid phase. In the case of immobilizing the substance A, 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 preferred method for quantifying% CDT in the measurement method is (1) measurement depending on the amount of CDT in the complex of various isoforms formed by the reaction of the substance A, transferrin isoform, and substance B. (2) Complexes of various isoforms by reacting substance A and substance B with transferrin isoforms in body fluids in advance. (3) a value measured depending on the amount of CDT in the formed complex, (4) a value measured depending on the amount of CDT obtained in the step, and This is a method of calculating% CDT from a calibration curve prepared in advance.

  The value obtained as a function of the amount of CDT obtained by the method for quantifying% CDT of the present invention is directly proportional to% CDT. Examples of values measured depending on the amount of CDT include absorbance, fluorescence intensity, emission intensity, and radioactivity.

The substance A suitable for use in the method for quantifying% CDT of the present invention preferably includes an antibody against transferrin. A particularly preferred antibody is an antibody produced from the hybridoma NCDT076 deposited with the Patent Organism Depositary (IPOD) ( accession date: March 14, 2006, accession number: FERM P-20842 ).

Substance B suitable for use in the immunoassay method of the present invention includes CDT-specific antibodies, and any substance that selectively reacts with the CDT fraction against transferrin isoforms other than CDT may be used. A particularly preferred CDT-specific antibody is an antibody produced from the hybridoma NCDT503 ( accession date: March 14, 2006, accession number: FERM P-20844 ) deposited at the Patent Organism Depositary (IPOD).

  The antibody against transferrin and the CDT-specific antibody may be a polyclonal antibody or a monoclonal antibody, and may be an antiserum or a purified fraction thereof, or a known isotype or subclass. Furthermore, it may be an antibody fragment that can bind to an antigen as necessary.

  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 CDT quantification method of the present invention, since it is not necessary to measure total transferrin and% CDT can be directly measured, it becomes possible to identify an alcoholic patient with a simple technique with high accuracy. .

  According to the CDT quantification method of the present invention, when transferrin in a body fluid is selected nonspecifically, the transferrin is in an excess amount relative to a substance that reacts nonspecifically with the transferrin isoform. This makes it possible to directly measure% CDT without being affected by the transferrin variation in each body fluid and without having to measure total transferrin.

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

[Example 1] Preparation of antibody-producing hybridoma A CDT fraction (antigen solution 0.5 mg / ml) contained in a mixed state of natural disialotransferrin and asialotransferrin was prepared as an immunizing antigen. 500 μl of this antigen solution was mixed with 500 μl of adjuvant (TiterMax Gold: trade name, TiterMax Co.) and emulsified, and immunized subcutaneously to BALB / c mice at 5 weeks of age. Similar booster immunizations were repeated 3 times every 2 weeks. Meanwhile, blood was collected during immunization, and the antibody activity in the 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. Spleen cells were fused by reacting with mouse myeloma cells (P3 × 63Ag8.653) in the presence of polyethylene glycol 4000 (Merck) for 2 minutes. 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.

  Confirmation of the antibody-producing hybridoma was performed by sandwich ELISA. 50 μl of each anti-mouse IgG 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, blocked with 0.5% BSA-PBS, and used as a screening plate.

  50 μl of the 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 was reacted at room temperature for 1 hour. After washing in the same manner, 50 μl of an alkaline phosphatase-labeled anti-transferrin polyclonal antibody was added and reacted at room temperature for 1 hour. . After washing again, the alkaline phosphatase activity was developed 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 and all transferrin isoforms A hybridoma (NCDT076) having antibody activity against was selected.

The two selected hybridomas were cloned twice by limiting dilution to obtain established strains. The resulting hybridomas NCDT503 and NCDT076 were deposited at the Patent Organism Depository Center (IPOD), respectively (deposit date: March 14, 2006, deposit numbers: FERM P-20844, FERM P-20842 ).

[Example 2] Preparation of antibodies The two monoclonal antibody-producing hybridomas established in Example 1 (hybridomas NCDT076 and NCDT503) were each grown in the peritoneal cavity of mice for mass production of monoclonal antibodies. Two types of monoclonal antibodies were obtained from each mouse ascites and purified by column chromatography using a protein G column (trade name, manufactured by GE).

Example 3 Determination of Antibody Subclass The two antibody subclasses obtained in Example 2 were determined using Isostrip (trade name, manufactured by Roche). The subclass of each antibody is shown in Table 1 below.

[Example 4] Confirmation of antibody specificity using sandwich ELISA The two antibodies prepared in Example 2 were confirmed for specificity to transferrin isoform using sandwich ELISA as follows. It was.

  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 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 the antibody was confirmed by comparing the strength of the reaction with transferrin isoform. The results of the reactivity of the selected hybridomas to the transferrin isoform are shown in Table 2 below.

[Example 5] Confirmation of antibody specificity using Western blot The two monoclonal antibodies prepared in Example 2 (monoclonal antibody produced by hybridoma NCDT076 and monoclonal antibody produced by hybridoma NCDT503) were subjected to western blotting. Using, confirmation of specificity for transferrin isoform was performed as follows. For comparison, confirmation was similarly performed using an anti-transferrin polyclonal antibody (manufactured by Shiba Goat Co., Ltd.) that reacts with all transferrin isoforms.

  Add the CDT fraction and transferrin other than CDT to the gel for isoelectric focusing (Novex IEF gel pH3-7: trade name, manufactured by Invitrogen) after dilution with a dedicated sample buffer so that each lane contains 5 μg. Then, after electrophoresis in a dedicated electrophoresis buffer, it was transferred to a PVDF membrane with a dedicated buffer. After the transfer, the PVDF membrane was blocked with Block Ace (trade name, manufactured by Dainippon Pharmaceutical Co., Ltd.), and then reacted with the antibody prepared in Example 2 prepared at 10 μg / ml with PBS at room temperature for 1 hour. Furthermore, after washing 3 times with PBS, it was reacted with alkaline phosphatase-labeled anti-mouse IgG antibody for 1 hour at room temperature. After washing again, color was developed with BCIP and NBT reagents.

  A photograph showing the development of a Western blot showing the specificity of these antibodies is shown in FIG.

  According to FIG. 1, it can be seen that the monoclonal antibody produced by hybridoma NCDT076 reacts nonspecifically with the transferrin isoform, and the monoclonal antibody produced by hybridoma NCDT503 is a CDT-specific antibody.

[Example 6] Preparation of solid-phase plate An antibody (NCDT076) showing antibody activity to all transferrin isoforms obtained in Example 2 was immobilized on a sandwich ELISA plate as follows.

  100 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.

[Example 7] Preparation of alkaline phosphatase-labeled anti-CDT antibody ALP-la belling kit-NH2 (trade name, manufactured by Dojindo) against monoclonal antibody (NCDT503) having antibody activity against the CDT fraction obtained in Example 2 above. ) Was used for alkaline phosphatase labeling.

[Example 8] Setting of specimen dilution rate On the solid-phased plate prepared in Example 6 above, diluted samples (10, 20, 40, 80, 160, 320, 640, 1280, iron-saturated patient sera of alcohol poisoning) were prepared. 2560, 5120, 10240, and 20480-fold dilutions), and 100 μl of each was added to the wells and allowed to react for 1 hour at room temperature. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 100 μl of the alkaline phosphatase-labeled anti-CDT antibody prepared in Example 7 was added and reacted at room temperature for 1 hour. After washing again, the alkaline phosphatase activity of the sandwich immune complex bound to the solid-phased plate was developed by the Kind-King method, the absorbance at 490 nm was measured with a microplate reader, and the saturation region of the sample dilution rate was examined. . The results are shown in FIG. 2 in a graph with the specimen dilution rate on the horizontal axis and the absorbance on the vertical axis.

  As shown in the graph of FIG. 2, it can be seen that the sample dilution ratio of 100 times or less is the saturation region.

  Furthermore, the change in the measured value of the absorbance in the case of four types of specimen dilution ratios of 25 times, 50 times, 100 times, and 200 times using specimens with% CDT of 2.5% to 6.5% is the same as described above. The results confirmed by the method are shown in FIG. 3 as a graph with% CDT on the horizontal axis and absorbance on the vertical axis. As shown in the graph of FIG. 3, no change in the absorbance measurement value was observed between the specimen dilution factor of 25 to 200 times regardless of% CDT. Thereby, the specimen dilution rate was set to 50 times in the following examples.

[Example 9] Setting of dilution ratio of labeled antibody Iron-saturated solid plate prepared in Example 6 and 50-fold diluted healthy serum and alcohol-poisoned patient serum were added to 100 μl wells and reacted at room temperature for 1 hour. I let you. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 100 μl of a diluted solution of alkaline phosphatase-labeled anti-CDT antibody (2-fold dilution series from 500-fold) was added and 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 results are shown in FIG. 4 as a graph with the labeled antibody dilution ratio on the horizontal axis and the absorbance on the vertical axis. As shown in the graph of FIG. 4, the range in which the difference between the measured values of the healthy subject serum and the alcoholic patient serum is remarkably recognized, the labeled antibody dilution ratio is within 10,000 times, preferably within 5000 times, more preferably 1000 times. In the following examples, a dilution ratio of labeled antibody of 500 times was selected.

[Example 10] Preparation of% CDT standard solution and absorbance (OD)
By combining a sample having a high% CDT and a sample having a low% CDT, eight kinds of standard solutions having various values that gradually increase the% CDT from 0 to 12.7% were artificially prepared. The obtained eight kinds of standard solutions of% CDT were added to 100 μl wells at a 50-fold dilution to the solid-phased plate prepared in Example 6 and reacted at room temperature for 1 hour. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 100 μl of alkaline phosphatase-labeled anti-CDT antibody diluted at the set magnification of Example 5 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 measurement range was confirmed. The results are shown in FIG. 5 in a graph with% CDT on the horizontal axis and absorbance on the vertical axis. As shown in the graph of FIG. 5, since% CDT and absorbance are linearly proportional from the low value (0%) to the high value (12%) of% CDT, the graph of FIG. It can be seen that it can be used as a calibration curve in the range of 12%.

[Example 11] Measurement of specimen Healthy individuals and alcoholic patients in sandwich ELISA using specimen dilution ratio (50 times) and labeled antibody dilution ratio (500 times) that can be set as shown in Examples 8 and 9 above Absorbance of 40 samples in total was measured as follows.

  After diluting 40 samples of iron-saturated serum 50 times, 100 μl was added to the wells of the solid-phased plate and reacted at room temperature for 1 hour. Subsequently, after washing with a washing solution (0.05% Tween-PBS), 100 μl of alkaline phosphatase-labeled anti-CDT antibody diluted at the set magnification (500 times) of Example 5 was added and 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. Using the calibration curve (FIG. 5) prepared in Example 10,% CDT was calculated from the absorbance of each sample obtained in the above step.

  In addition, using a reagent made by Bio-rad as a control test, the% CDT of each specimen obtained in the above step and the% CDT of a reagent made by Bio-rad are regression by the least square method. Analysis was performed to obtain a correlation coefficient and a regression equation. The correlation diagram obtained is shown in the graph of FIG.

Regression equation y = 1.148x−0.938 Equation (1)
Correlation coefficient R = 0.799
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 in the sample is measured. % CDT is calculated. As shown in FIG. 6, the measured value of% CDT of the present invention measured by sandwich ELISA was found to have a very strong positive correlation with the measured value of% CDT by a method using a reagent manufactured by Bio-Rad.

  The method for quantifying% CDT according to the present invention uses non-specific selection for all transferrin isoforms in body fluid, and further uses carbohydrate-deficient transferrin (CDT) therein with an antibody having high selectivity. Since the measurement is performed, the obtained measurement value is directly proportional to% CDT. Therefore, the% CDT obtained by the method of the present invention is useful as a test method for identifying alcoholic patients who are highly sensitive, specific, quick and inexpensive.

2 is a photograph showing a Western blot showing the specificity of two types of monoclonal antibodies prepared in Example 2. FIG. It is a graph which shows the relationship between the light absorbency which shows the alkaline phosphatase activity of the sandwich immune complex couple | bonded with the solid-phased plate, and a sample dilution rate. It is a graph which shows the relationship between% CDT and a light absorbency in case a sample dilution rate is four types. It is a graph which shows the relationship between a labeled antibody dilution rate and a light absorbency about healthy subject serum and alcoholic patient sera. It is a graph which shows the relationship between% CDT using 8 types of standard solutions of% CDT, and a light absorbency. It is a graph which shows the correlation of% CDT of sandwich ELISA and control test (biorad)% CDT about a total of 40 serum of healthy persons and alcoholic patients.

Claims (9)

Using substance A that reacts non-specifically with transferrin isoform and substance B that reacts specifically with carbohydrate-deficient transferrin (CDT),
By selecting non-specifically the transferrin isoform in the body fluid with the substance A and specifically selecting CDT with the substance B, the amount of total transferrin in the body fluid can be measured without measuring the total amount of transferrin. A method for quantifying the percentage of CDT (% CDT) comprising :
The method for quantifying the CDT ratio (% CDT), wherein the substance B is a CDT-specific antibody produced by the hybridoma NCDT503 (FERM P-20844).   The method for quantifying% CDT according to claim 1, which is carried out under a condition that an excess amount of transferrin isoform in a body fluid is excessive with respect to the amount of the substance A.   The method for quantifying% CDT according to claim 1 or 2, wherein a sandwich complex is formed by the reaction of the substance A, a transferrin isoform, and the substance B.   The method for quantifying% CDT according to claim 1, wherein the substance B is labeled.   The method for quantifying% CDT according to any one of claims 1 to 4, wherein the substance A is bound to a solid phase. (1) A calibration curve of% CDT and a value measured depending on the amount of CDT in a complex of various isoforms formed by the reaction of the substance A, transferrin isoform and substance B Create it in advance,
(2) A complex of various isoforms is formed by reacting the substance A and the substance B with the transferrin isoform in the body fluid,
(3) measuring a value measured depending on the amount of CDT in the formed complex;
(4) The% CDT according to any one of claims 1 to 5, wherein% CDT is calculated from a value measured depending on the amount of CDT obtained in the step and the calibration curve prepared in advance. How to quantify.   The method of quantifying% CDT according to claim 1, wherein the value measured depending on the amount of CDT is absorbance. The method for quantifying% CDT according to any one of claims 1 to 7, wherein the substance A that reacts nonspecifically with the transferrin isoform is an anti-transferrin antibody. The method for quantifying% CDT according to claim 1-8, wherein the substance A is a monoclonal antibody produced by a hybridoma NCDT076 (FERM P-20842).