JP2003277398A - Antibody against transferrin-including immune complex, method of producing the same, hybridoma and immunity measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody that can identify a patient with alcoholism and a patient with hepatitis, a method of producing the antibody, a hybridoma for producing the antibody and a simple immunoassay using the antibody. <P>SOLUTION: The antibody to be provided selectively reacts with an immune complex and the immune complex is composed of transferrin and immunoglobulin. The content of the immune complex in the body fluid is high in a patient with alcoholism but low in a patient with non-alcoholism and the difference can be measured with the antibody. Since the immune complex in the body fluid is determined with the antibody against the immune complex composed of transferring and immunoglobulin, the patients with alcoholism or hepatitis can be simply identified with high exactness. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody capable of identifying an alcoholic patient and / or a liver disease patient,
A method for producing the antibody, a hybridoma producing the antibody,
The present invention relates to an immunoassay method using the antibody.

[0002]

BACKGROUND OF THE INVENTION Alcohol poisoning not only causes great damage to alcoholics and their individuals, but also results in lost productivity and enormous annual health care costs associated with alcohol poisoning. Therefore, early recognition and treatment of alcoholism is the best and cost effective for individuals and society. To identify individuals at risk of comorbid drinking in various populations under these circumstances,
What is needed is a method that is sensitive, specific, yet rapid and inexpensive. Many tests have been developed in the last 30 years to detect severe drinkers.

For example, γ-glutamyl transpeptidase (γ-GTP), mean corpuscular
volume) (MCV), aspartate aminotransaminase (AST) or alanine aminotransaminase (ALT), α-lipoprotein and ferritin have been used as biochemical markers for alcohol abuse for many years, , The diagnostic sensitivity and specificity of these tests was unsatisfactory. Also, while the assessment of the level of alcohol intake is very important for knowing its effect during treatment, the reports of alcohol consumption by alcoholics themselves were unreliable. In view of such a situation, a simple clinical test method that is correlated with long-term excessive alcohol consumption has been desired.

Stibler et al., Acta Med Scan, 206 , 275-
281, (1979), 60 g of transferrin isoform with high isoelectric point daily for over 1 week.
An increase was observed in 81% of those who took the above ethanol, and it was reported that the isoform having a high isoelectric point returns to a normal level when alcohol is absent for 10 days or more.

Serum transferrin is a glycoprotein having a molecular weight of 79.5 kD and consisting of a single polypeptide chain having two N-linked polysaccharide chains. These polysaccharide chains are branched and have a terminal sialic acid residue,
There are six transferrin isoforms, depending on different levels of sialylation. Wong and Regoeczi are Int.
In J. Peptide Res. 9, 241-248, (1977), human transferrin is heterogeneous in nature and states that different isoforms exist with different levels of sialylation. In fact, there are six such isoforms, pentacialo, tetrasialo, trisialo, disialo, monosialo and asialotransferrin.

In normal healthy individuals, tetrasialo is predominant, whereas in the blood of alcoholics, asialo, monosialo, disialo and to some extent elevated levels of trisialo are found. (Van Eijk et al, Clin Chim Acta 132 ,
167-171, (1983), Stibler, Clin Chim, 37 , 2029-203
7, (1991) and Stibler et al, “Carbohydrate-def.
icient transferrin (CDT) in serum as a marker of h
igh alcohol consumption ", Advances in the Bioscienc
es, (Ed Nordmann et al), Pergamon, 1988, Vol.71, p
ages 353-357).

The asialo, monosialo, disialo and trisialo isoforms are sugar chain-deficient transferrin (abbreviation:
It is called CDT). CDT is alcohol consumption,
In particular, it has been found to be an effective marker for detecting and monitoring chronic alcohol consumption. CDT
, Unlike conventional tests (eg γ-GTP or MCV), can be used for screening for high alcohol intake in patients with liver disease, so the test method for CDT is WO-96 / 26444 and Heil et al. al
, Anaesthetist, 43 , 447-453, (1994) and the like. In their approach, a diluted serum sample is passed through an anion 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 this eluate was determined by the immunoturbidimetric method (immu
noturbidimetric assay). In addition, a method for quantifying CDT by high performance liquid chromatography (WO95 / 04932) is also known.

[0008]

However, all of the above-mentioned conventional examination methods cannot be directly applied to many automatic analyzers for clinical examination commonly used in clinical examination sites,
The inspection method is also based on a relatively complicated procedure such as application of an ion exchange resin or high performance liquid chromatography.

On the other hand, Makhlouf et al. Attempted to produce a monoclonal antibody against CDT in order to directly measure only CDT. For the purpose of obtaining a monoclonal antibody that specifically recognizes CDT, a synthetic peptide of 6 amino acids before and after the 413th and 611st asparagine to which a sugar chain is bound is used as an immunogen to prepare an antibody. It was considered to apply the antibody to an immunoassay for CDT measurement (WO93 / 06133). On the other hand, E
R. Trimble et al. (Biochem Soc Trans, 26 (1), S48 (1998)
In addition, since the monoclonal antibody prepared by the method of Makhlouf et al. Using a synthetic peptide as an immunogen did not react with CDT, its epitope is hidden in the transferrin molecule and cannot be applied to detection.

In view of the above situation, the present inventors, under a completely new idea, an antibody for identifying an alcoholic patient or a liver disease patient, a method for producing the antibody, a hybridoma, and a simple method using the antibody. It is intended to provide a simple immunoassay method.

[0011]

The present inventors have for the first time found that there are many immune complexes consisting of transferrin and immunoglobulin in patients with alcoholic liver disease or alcoholics. No such report has been found before. The present inventors have paid attention to this point and completed the present invention.
The present inventors thought that an alcoholic patient could be identified by measuring the amount of an immune complex consisting of transferrin and immunoglobulin. Therefore, the inventors used immobilized anti-transferrin antibody and enzyme-labeled anti-immunoglobulin G (IgG) antibody. The amount of immune complex was measured by sandwich immunoassay. The results, as expected, were that not only were hepatitis virus, e.g. B
The present inventors have confirmed that even in patients with liver disease due to type C and type C, a large amount of the immune complex consisting of transferrin and immunoglobulin is measured with a significant difference from that of a healthy individual.

That is, the antibody of the present invention is (1) an antibody that selectively reacts with an immune complex, and (2) the immune complex is an immune complex composed of transferrin and immunoglobulin, and (3) ) An antibody characterized in that the amount of the immune complex in a body fluid is measured by a significant difference that it is high in alcoholics and low in non-alcoholics. In the present specification, the "significant difference" means that the risk rate (P) is 0.05 (5%) or less, preferably 0.01 (1%).
It indicates that there is a difference with the following probability.

The immune complex consisting of transferrin and immunoglobulin is not limited to the above alcoholics,
A large amount is produced in body fluids of patients with alcoholic liver disease, liver disease due to hepatitis virus, etc. with a significant difference from healthy solids. The antibody of the present invention is an antibody that selectively reacts also with an immune complex composed of transferrin and immunoglobulin produced in a body fluid of a patient with alcoholic liver disease, a patient with liver disease due to hepatitis virus or the like. Therefore, by measuring the immune complex using the antibody of the present invention, it is possible to identify patients with alcoholic diseases, alcoholics, and patients with liver diseases due to hepatitis virus and the like.

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

The method for producing an antibody of the present invention is (1) an immune complex consisting of transferrin and immunoglobulin, wherein the amount of the immune complex in body fluid is high in alcoholic patients and low in non-alcoholic patients. Is immunized into the animal by immunizing the immune complex, which is measured with a significant difference, to produce an antibody that selectively reacts with the immune complex in the body of the animal, and (2) the antibody is produced from the animal. Characterized by collecting.

The hybridoma of the present invention is a hybridoma in which the antibody having the above-mentioned characteristics is a monoclonal antibody and has the ability to produce the monoclonal antibody.

The first immunoassay method of the present invention is characterized in that the above-mentioned antibody of the present invention is brought into contact with a body fluid, and an immune complex selectively reacting with the antibody is detected or quantified. . That is, the antibody used in the above-described immunoassay method of the present invention includes the following aspects 1) to 4). 1) An antibody that selectively reacts with an immune complex, wherein the immune complex is an immune complex consisting of transferrin and immunoglobulin, and the amount of the immune complex in body fluid is high in alcoholics. An antibody characterized by being significantly less measured in non-alcoholic patients. 2) An antibody in which the antibody in 1) is a monoclonal antibody; 3) An antibody in which the antibody in 1) is a polyclonal antibody; 4) Obtained by immunizing an animal with an immune complex composed of transferrin and immunoglobulin as an immunogen. Antibodies.

In the first immunoassay, the antibody against the immune complex reacts only with the immune complex consisting of transferrin and immunoglobulin, so that the immune complex can be measured with high accuracy.

The second immunoassay method of the present invention is characterized in that a body fluid is contacted with an anti-transferrin antibody and an anti-immunoglobulin antibody to detect or quantify an immune complex consisting of transferrin and immunoglobulin. .

In the second immunoassay method, an immunoassay method using a solid phase carrier is used, and transferrin antibody can be selected as an antibody to be immobilized. In this case, transferrin and immunization are used. Since not only the complex with globulin but also transferrin is captured by the immobilized antibody, the measurement sensitivity may be lower than that of the first immunoassay method.

The immunoassay method of the present invention enables identification of alcoholics and / or patients with alcoholic liver disease, and enables monitoring of alcohol consumption. The immunoassay method of the present invention is an immunoassay method that is quicker and easier to operate than the prior art assay methods for identifying alcoholics and / or patients with alcoholic liver disease or monitoring alcohol consumption. In addition, the immunoassay method of the present invention can also measure an immune complex composed of transferrin and immunoglobulin produced in a body fluid of a patient with liver disease caused by hepatitis virus or the like.

In the present invention, the transferrin constituting the immune complex includes various transferrins produced by patients with liver diseases and / or alcoholics.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION Immunoassay Method The methods applicable to the first and second immunoassay methods of the present invention include, as heterogeneous assay methods, radioisotope-labeled immunoassay, enzyme-labeled immunoassay. The measurement method, the fluorescence-labeled immunoassay method, the immunoturbidimetric method, the latex slide agglutination method, the latex immunoturbidimetric method, the sandwich method and the like can be mentioned. Examples of the homogeneous system include a fluorescence quenching method, a fluorescence enhancement method, an energy transfer method, an agglutination method and a nephelometry method. The immunoassay method of the present invention may be performed in any reaction format,
It may be either homogeneous or heterogeneous. These immunoassays may be manual or automated.

In the reagents used in the immunoturbidimetric method, the latex slide agglutination method, and the latex immunoturbidimetric method,
To enable the detection or quantification of immune complexes consisting of transferrin and immunoglobulins in body fluids, the turbidity based on aggregation formed depending on the antigen-antibody reaction is visually or, for example, in the case of immunoturbidimetry. 340 nm, in the latex immunoturbidimetric method, it is desirable to measure spectroscopically using a wavelength near 540 nm.

Labeling substance One of the reagents used in the above-mentioned immunoassay method is a radioisotope-labeled immunoassay method, an enzyme-labeled immunoassay method, and a fluorescence-labeled immunoassay method. In order to enable detection or quantification of the complex, it is necessary to label, but known labeling 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) Fluorophore: fluorescein isocyanate, rhodamine, Texas red, etc. 3) Radioisotope element: I ¹²⁵ ,
I ^131, etc. 4) Bioluminescent substance: luciferin, aequorin, etc. 5) Chemiluminescent substance: Isoluminin, acridinium ester, oxalate ester, etc. It can be carried out.

Solid phase carrier 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 and nitrocellulose. Examples of the shape include microtiter plates, vials, latex beads, dip strips and the like.

Body fluid The body fluid which is the target of the immunoassay method of the present invention may be serum, plasma, saliva or other body fluid, but serum is preferred.

First immunoassay method 1) Acquisition of immunogen and antibody Consists of at least one isoform transferrin and immunoglobulin purified or partially purified from body fluids of patients with alcoholic liver disease and / or alcoholics The immune complex can be used as an immunogen to immunize an animal to obtain an antibody that selectively reacts with the immune complex composed of transferrin and immunoglobulin.

For example, the immunogen has been purified or partially purified from the sera of patients with alcoholic liver disease and / or alcoholics by affinity chromatography using anti-transferrin antibodies that react with all transferrin isoforms. However, more preferably, by gel chromatography, a fraction having a molecular weight higher than that of transferrin alone, that is, an immunocomplex fraction composed of transferrin and immunoglobulin may be used as an immunogen.

2) Polyclonal antibody, hybridoma, and monoclonal antibody Polyclonal antibody is obtained by immunizing an animal with the above immunogen. A known method can be applied to a method for immunizing an animal, and an immunization protocol required for the method can be easily determined by those skilled in the art. For example, the antibody of the present invention is prepared by combining an immunogen mixed with an adjuvant with a suitable host animal (rabbit,
Goats, horses or other mammals). Injection of immunogen is continued until a suitable titer of antiserum is obtained. After collecting the antiserum, it is preferable that the antiserum is further purified, if necessary, using a known technique such as affinity chromatography or ion exchange chromatography.

Further, the antibody of the present invention is obtained from a fused cell (hybridoma) obtained by fusing a cell obtained from an immunized animal (eg, rat, hamster, mouse or other mammal) with an immortalized cell. Obtainable. For example, a mouse can be used. In this case, after immunizing the immunogen by a known technique, the target hybridoma is obtained by the method of Köhler and Milstein (Nature 256, 495, 1975) using spleen cells and mouse myeloma cells collected from the immunized mouse. Is possible. The selection of hybridomas producing the desired antibody is carried out by using an immunocomplex composed of transferrin and immunoglobulin used as an immunogen,
An enzyme-linked immunosorbent assay in which transferrin and immunoglobulin are immobilized is used. Select one that does not react with transferrin and immunoglobulin but strongly reacts only with the immune complex. Finally, cloning is performed by the limiting dilution method to obtain the target hybridoma. The monoclonal antibody can be prepared from the obtained hybridoma in a culture system or mouse abdominal cavity which is a known technique, and can be purified by a known technique such as affinity chromatography or ion exchange chromatography as in the case of the polyclonal antibody.

Antibodies suitable for use in the immunoassay of the present invention may be polyclonal or monoclonal antibodies, antisera or purified fractions thereof, or known isotypes or subclasses. Further, it may be an antibody fragment capable of binding to an antigen if necessary. That is, the only condition for the antibody is the immune complex, which is measured by the significant difference that it is predominant in at least alcoholics and less in non-alcoholics,
That is, it can selectively react with an immune complex composed of transferrin and immunoglobulin.

3) Detection Method of Immune Complex As a preferable detection method, the sandwich method is recommended. In this assay method, for example, an antibody that selectively reacts with an immune complex composed of transferrin and immunoglobulin is immobilized. By reacting the body fluid with the immobilized antibody, the immune complex contained in the body fluid binds to the immobilized antibody. After washing the unreacted material, labeled antibody is added, which binds to the immune complex already bound to the immobilized antibody. This labeled antibody is preferably an antibody that reacts with all immune complexes, such as an anti-transferrin antibody. The amount of bound labeling component is proportional to the amount of immune complex of transferrin and immunoglobulin in the body fluid.

In the immunoassay method of the present invention, a polyclonal or monoclonal antibody that selectively reacts with an immune complex composed of transferrin and immunoglobulin is used as a capture antibody, that is, a solid-phase antibody for immobilizing on a solid phase. Is desirable.

Second Immunoassay Method Specifically, a solid-phased anti-transferrin antibody, an enzyme-labeled anti-immunoglobulin G (IgG) antibody, and A (IgA)
Sandwich immunoassay with a combination of antibodies or M (IgM) antibodies; or immobilized anti-immunoglobulin G (IgG) antibodies, A (IgA) antibodies or M (Ig)
M) A sandwich immunoassay using a combination of an antibody and an enzyme-labeled anti-transferrin antibody can detect or quantify an immune complex composed of transferrin and immunoglobulin contained in a body fluid.

In the second immunoassay method of the present invention, transferrin or immunoglobulin is also captured by the immobilized antibody, so that the second immunoassay method is
The measurement accuracy drops slightly.

[0038]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to embodiments and the accompanying drawings.

Example 1 Comparison of Amount of Immune Complex Containing Transferrin and Immunoglobulin in Serum of Healthy Subjects and Patients with Alcoholic Liver Disease Present in panel serum of 96 healthy subjects and 92 subjects with alcoholic liver disease The amount of immune complex to be measured was measured by sandwich enzyme immunoassay (ELISA) using two kinds of antibodies. That is, 100 μl of each anti-human transferrin polyclonal antibody prepared at 10 μg / ml with 10 mM phosphate buffer (pH 7.2) (abbreviation: PBS) was added to a 96-well microplate, and the mixture was reacted at 4 ° C. overnight. After that, it was washed once with PBS and PBS containing 0.5% bovine serum albumin (BSA).
Blocking was performed with (B-PBS) to obtain a screening plate. 100 μl of a 50-fold diluted solution of each serum with PBS was added to the wells and reacted at room temperature for 1 hour. After washing with PBS, 100 μl of an alkaline phosphatase-labeled anti-human IgG antibody (manufactured by Sigma) was added and reacted at room temperature for 1 hour. After washing, the alkaline phosphatase activity was developed by the Kind-King method, and the absorbance (OD) at 490 nm was measured with a microplate reader to detect the immune complex.

The results are shown in the graph of FIG. As shown in FIG. 1, it was revealed that the amount of the immune complex composed of transferrin and immunoglobulin was significantly high in the patients with alcoholic liver disease.

[Example 2] Preparation of immunogen 20 ml of pooled serum derived from an alcoholic patient was bound with an anti-transferrin polyclonal antibody.
The fraction was passed through a Sepharose 4B (Pharmacia) column to capture the total transferrin fraction in serum presumably containing an immune complex consisting of transferrin and immunoglobulin. 10 column volumes of PBS
After washing the column with, the column was eluted with an acid (0.1 M glycine-hydrochloric acid buffer pH 2.3). After dialysis against PBS overnight, the acid-dissociated immune complex was reconstituted and the gel filtration column (ULTROGEL AcA34: trade name, BIOS
The fraction of the immune complex consisting of transferrin and immunoglobulin and the fraction of transferrin were separated and purified by chromatography (EPRA). The collected fraction of the immunocomplex composed of transferrin and immunoglobulin was concentrated by ultrafiltration to obtain an immunogen.

FIG. 2 shows the elution profile when the total transferrin fraction in serum was applied to a gel filtration column (solid line OD280 nm), and the elution fraction of the immune complex consisting of transferrin and immunoglobulin (broken line OD490).
nm) is shown. The detection of the immune complex was carried out by sandwich ELI using an anti-human transferrin polyclonal antibody as the immobilized antibody and an anti-human immunoglobulin G antibody as the labeled antibody.
Since it is performed on SA, the elution fraction of the immune complex is indeed an immune complex consisting of transferrin and immunoglobulin. FIG. 2 shows that a fraction of the immune complex is detected in the higher molecular region than the major transferrin peak. This result indicates that the fraction is an immune complex consisting of transferrin and immunoglobulin.

The range indicated by the arrow in FIG. 2 represents the range of fractionation of the purified antigen (immunocomplex composed of transferrin and immunoglobulin) used in the following examples.

Example 3 Preparation of Hybridoma An antigen solution (0.5 mg / ml) was prepared using the purified antigen obtained in Example 2 above. The antigen solution 50
500 μl of Freund's complete adjuvant was mixed with 0 μl to emulsify, and 5-week-old BALB / c mice were subcutaneously immunized. As a booster, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant, and the same preparation was repeated every two weeks for three times. During that time, blood was collected at the time of immunization to measure the antibody activity in the blood against the antigen. Fourteen days after the final immunization, 500 μl of the antigen solution was intraperitoneally administered, and three days later, the spleen was extracted. Splenocytes are mouse myeloma cells (P3-NS1-Ag4-1 (NS
-1) and polyethylene glycol 4000 (manufactured by Merck) were reacted for 2 minutes to fuse the two. After fusion, the cells were suspended in a selective medium, dispensed into a 96-well culture plate, and cultured in a CO ₂ incubator at 37 ° C. Then, the hybridoma was prepared by replacing half of the culture medium once a week.

Example 4 Selection of Antibody-Producing Hybridomas Against Immunogen (Primary Screening) Confirmation of antibody-producing hybridomas was performed by ELISA in which the immune complex used as the immunogen was immobilized. That is, 10 μg / m 2 with PBS in a 96-well microplate
100 μl of antigen prepared in each well
In addition, the reaction was performed overnight at 4 ° C. Thereafter, the plate was washed once with PBS, blocked with B-PBS, and used as a plate for screening. 100 μl of a solution obtained by diluting the culture supernatant of the well in which the growth was observed with PBS by 10 times was added to the well and reacted at room temperature for 1 hour. Subsequently, after washing with PBS, 100 μl of an alkaline phosphatase-labeled anti-mouse immunoglobulin (BIOSOURCE INTERNATIONAL) solution was added, and the mixture was reacted at room temperature for 1 hour. After washing again, alkaline phosphatase activity was developed by the Kind-King method, OD at 490 nm was measured by a microplate reader, and a monoclonal antibody-producing hybridoma having an antibody activity against the immune complex was selected.

Example 5 Selection of Antibodies Reactively Reacting with Immune Complex Consisting of Transferrin and Immunoglobulin (Secondary Screening) A monoclonal antibody-producing hybridoma having an antibody activity against an immunogen is a large-scale production of monoclonal antibody. Therefore, it was proliferated in the abdominal cavity of the mouse. The thus obtained mouse ascites monoclonal antibody was purified by column chromatography using protein A agarose (manufactured by Bio-Rad). An antibody that does not react with transferrin and immunoglobulin but selectively reacts with an immune complex composed of transferrin and immunoglobulin was selected by ELISA. That is, 100 μl each of transferrin, immunoglobulin, or an immune complex solution consisting of transferrin and immunoglobulin prepared in PBS at 10 μg / ml was added to a 96-well microplate, and reacted at 4 ° C. overnight. Then, the plate was washed once with PBS and blocked with B-PBS to obtain a screening plate. 100 μl of a monoclonal antibody solution adjusted to 10 μg / ml was added to these wells and reacted at room temperature for 1 hour. After washing with PBS, 100 μl of alkaline phosphatase-labeled anti-mouse immunoglobulin antibody solution was added, and the mixture was reacted at room temperature for 1 hour. After subsequent washing, alkaline phosphatase activity was developed by the Kind-King method, and OD at 490 nm was measured by a microplate reader. The results are shown in FIG. 3 as a graph in which the vertical axis represents OD.

A monoclonal antibody-producing strain (CT861) which showed a high reaction against an immune complex composed of transferrin and immunoglobulin was used as a specific antibody (CT8) against the immune complex composed of transferrin and immunoglobulin.
61 antibody) hybridomas were selected. The monoclonal antibody producing strain (CT861) was transferred to FERM at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology.
Deposited as P-18762.

[Example 6] Confirmation that CT861 antibody recognizes immune complex composed of transferrin and immunoglobulin in serum Monoclonal antibody producing strain (C
To confirm that the CT861 antibody produced by T861) recognizes an immune complex consisting of transferrin and immunoglobulin in blood, pooled serum from a patient with alcoholic liver disease was subjected to gel filtration column (ULTROGEL AcA34: trade name: BIOSEPRA) Sandwich ELISA with CT861 antibody immobilized by chromatography
The measurement was performed at A. That is, 100 μl of each CT861 antibody prepared in PBS at 10 μg / ml was added to a 96-well microplate and reacted at 4 ° C. overnight. After that, wash once with PBS, B-PB
A plate for measurement was obtained by blocking with S. 100 μl of each fraction was added to these wells and reacted at room temperature for 1 hour. After washing with PBS, 100 μl of an alkaline phosphatase-labeled anti-human transferrin antibody solution was added, and the mixture was reacted at room temperature for 1 hour. After continuous washing,
Alkaline phosphatase activity is developed by the Kind-King method, and 490 nm is read by a microplate reader.
Was measured. The result is shown in FIG. 4 as a graph in which OD is plotted on the vertical axis.

FIG. 4 shows the elution profile (solid line OD280 nm) and CT when serum was applied to a gel filtration column.
It is the figure which showed the fraction (broken line OD490nm) which 861 reacts. The white arrow indicates the molecular weight range of transferrin, the black arrow indicates the molecular weight range of immunoglobulin, and the gray arrow indicates the molecular weight range of the immune complex composed of transferrin and immunoglobulin. As shown in Figure 4, CT86
It was shown that 1 antibody did not react in the molecular weight range of transferrin and immunoglobulin, but only in the molecular weight range of the immune complex consisting of transferrin and immunoglobulin. It can be confirmed that the CT861 antibody reacts only with the immune complex and not with transferrin and immunoglobulin.

Example 7 Clinical Test for Identification of Liver Disease Patients Using CT861 Antibody In order to detect an immune complex which is the basis for identification of liver disease patients, various liver disease patients (alcoholic liver Disease patient 50
Samples, 20 hepatitis B virus patients, and 20 hepatitis C virus patients) were used in a sandwich ELISA in which CT861 antibody was immobilized using 92 panel sera of each sample and 96 sera of healthy subjects. went.

That is, a CT861 antibody prepared at 10 μg / ml with PBS was added to a 96-well microplate.
100 μl was added to each well and reacted at 4 ° C. overnight. After that, the plate was washed once with PBS and blocked with B-PBS to prepare a plate for measurement. 100 μl of serum diluted 20-fold with PBS was added to these wells,
The reaction was carried out at room temperature for 1 hour. After washing with PBS, 100 μl of an alkaline phosphatase-labeled anti-human transferrin antibody solution was added, and the mixture was reacted at room temperature for 1 hour. After subsequent washing, the alkaline phosphatase activity was
-King method was used for color development, and OD at 490 nm was measured with a microplate reader.

The results of the analysis using these measured values are shown in FIG. Fig. 5-1 is a comparison between alcoholic liver disease patients and healthy subjects, Fig. 5-2 is a comparison between hepatitis B virus patients and healthy subjects, and Fig. 5-3 is a comparison between hepatitis C virus patients and healthy subjects. is there. As shown in Fig. 5, it was shown that CT861 antibody shows significantly higher reactivity to serum of liver disease patients than healthy people, and this measurement system can clearly distinguish liver disease patients and healthy people. It turns out that it is a different measurement system.

Example 8 Clinical Test for Identification of Liver Disease Patients Using Anti-Human Transferrin Antibody and Anti-Human Immunoglobulin Antibody In order to detect an immune complex which is the basis for identification of a liver disease patient, various tests were conducted. Liver disease patients (alcoholic liver disease patients 50
Sandwich ELIS for detection of immune complex using each panel serum of 96 samples, 20 samples of hepatitis B virus patient, 20 samples of hepatitis C virus patient) and 96 samples of healthy person serum
The measurement was performed in A as follows.

That is, 10 μM of 10 mM phosphate buffer pH 7.2 (PBS) was added to a 96-well microplate.
An anti-human transferrin polyclonal antibody solution prepared at g / ml was added to each well in an amount of 100 μl and reacted at 4 ° C. overnight. After that, it was washed once with PBS and PBS containing 0.5% bovine serum albumin (BSA)
Blocking was performed with (B-PBS) to obtain a screening plate. 100 μl of a 50-fold diluted solution of each serum with PBS was added to the wells and reacted at room temperature for 1 hour. After washing with PBS, 100 μl of an alkaline phosphatase-labeled anti-human IgG antibody (manufactured by Sigma) was added and reacted at room temperature for 1 hour. After washing, the alkaline phosphatase activity was developed by the Kind-King method, and the absorbance (OD) at 490 nm was measured with a microplate reader to detect the immune complex. The result of analysis using these measured values is shown in FIG. FIG. 6-1 is a comparison between an alcoholic liver disease patient and a healthy person, FIG. 6-2 is a comparison between a hepatitis B virus patient and a healthy person, and FIG. 6-3 is a comparison between a hepatitis C virus patient and a healthy person. is there. According to FIG. 6, it can be seen that this measurement system is a measurement system capable of clearly distinguishing between a liver disease patient and a healthy person.

Example 9 Usefulness of CT861 Antibody as a Marker for Alcohol Poisoning Measured values of γ-GTP and sugar chain deficient transferrin (CDT) as known markers for alcohol poisoning, and C
The sandwich ELISA measurement values using the T861 antibody were used to determine 55 alcohol-intoxicated patient sera and 1 healthy individual serum.
The four samples were compared as follows.

The measurement of γ-GTP was carried out by measuring the γ-GTP activity in serum using a γ-GTP measuring reagent (GTP HA test Wako, manufactured by Wako Pure Chemical Industries, Ltd.) according to the protocol. CDT measurement is performed using a CDT measurement reagent (% C
DT TIA Standard Version: trade name, manufactured by Bio-Rad) was used to measure the ratio of CDT to total transferrin in serum (% CDT) according to the protocol. In Table 1, the measured values of sandwich ELISA using CT861 antibody and the measured values of γ-GTP are shown as positive and negative concordance rates. As shown in Table 1, γ-GTP
It can be understood that the sandwich ELISA using the CT861 antibody is always positive for the 34 specimens that are positive.

[0057]

[Table 1]

In Table 1, γ-GTP was negative and CT
Table 2 shows the results of measuring% CDT of 21 specimens showing positive results in the sandwich ELISA using the 861 antibody, in terms of positive and negative concordance rates. As shown in Table 2, all the 21 specimens of γ-GTP negative and CT861 antibody positive in Table 1 above were alcohol poisoning patient specimens showing% CDT positive.

[0059]

[Table 2]

Sandwich EL using CT861 antibody
The measured value of ISA and the measured value of% CDT are shown in Table 3 by the concordance rate of positive and negative. As shown in Table 3, it can be seen that the sandwich ELISA using the CT861 antibody always shows positive for the 37 samples with positive% CDT.

[0061]

[Table 3]

In Table 3, γ-GTP was measured for 18 specimens showing a negative% CDT measurement value and a positive sandwich ELISA using the CT861 antibody. Shown as a rate. As shown in Table 4, all 18 specimens positive for CT861 antibody
It can be seen that the sample is a γ-GTP-positive alcoholic patient.

[0063]

[Table 4]

Thus, when both or either of γ-GTP and% CDT is positive, the sandwich ELISA using the CT861 antibody shows positive, and γ-GTP
When both CT and% CDT were negative, the sandwich ELISA using the CT861 antibody gave a negative result, indicating that the CT861 antibody is useful for diagnosing alcoholics.

Using the above 69 samples, R
An OC curve was drawn. FIG. 7-1 shows R of the γ-GTP measurement system.
The OC curve is shown. Fig. 7-2 shows RO of% CDT measurement system.
The C curve is shown. FIG. 7-3 shows a ROC curve of sandwich ELISA using CT861 antibody. γ-GTP
The area under the curve in FIG. 7-1 is 0.791, and the area under the curve in FIG. 7-2 is 0.901 for% CDT, whereas the sandwich ELISA using the CT861 antibody is shown in FIG. 7-3. The area under the curve is 0.998, CT8
It can be seen that the sandwich ELISA using the 61 antibody is an excellent measurement system for diagnosing alcoholics.

[0066]

EFFECTS OF THE INVENTION According to the present invention, since an antibody against an immune complex composed of transferrin and immunoglobulin can be provided, it is possible to accurately identify an alcoholic patient or a liver disease patient by a simple method.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of comparing the amount of an immune complex consisting of transferrin and immunoglobulin present in serum between a healthy subject and an alcoholic liver disease patient by the sandwich ELISA method shown in Example 1. Is.

FIG. 2: Elution profile when the total transferrin fraction in serum was applied to a gel filtration column (solid line OD280
nm) and the elution fraction of the immune complex consisting of transferrin and immunoglobulin (dashed line OD490 nm).

FIG. 3 shows CT8 by the ELISA method shown in Example 5.
It is a figure which shows that 61 is highly specific to the immune complex which consists of transferrin and an immunoglobulin.

FIG. 4 is a diagram showing an elution profile (solid line OD280 nm) when the serum shown in Example 6 is applied to a gel filtration column and a fraction (represented by broken line OD490 nm) in which CT861 reacts.

FIG. 5 is a diagram showing that the sandwich ELISA method using CT861 shown in Example 7 is a measurement system that clearly distinguishes healthy subjects from patients with liver disease.

FIG. 6 shows that the sandwich ELISA in which the anti-human transferrin antibody shown in Example 8 is immobilized and the anti-human immunoglobulin G antibody is used as a labeled antibody is a measurement system that can clearly distinguish healthy subjects and liver disease patients. It is a figure.

FIG. 7 shows a γ-GTP measurement system (FIG. 7-1),% C.
It is a figure which shows each ROC curve of the DT measurement system (FIG. 7-2) and the sandwich ELISA using CT861 antibody (FIG. 7-3), and the sandwich ELIS using CT861.
It shows that A is an excellent measurement system for diagnosis of alcoholics.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/577 C12R 1:91 // (C12N 5/10 C12N 5/00 B C12R 1:91) (C12P 21/08 C12R 1:91) F term (reference) 4B064 AG26 AG27 CA10 CA20 CC24 DA13 4B065 AA92X AB05 AC14 BA08 CA25 CA46 4H045 AA11 CA42 CA43 DA75 DA76 EA50 FA72

Claims (10)

[Claims] 1. An antibody that selectively reacts with an immune complex, (2) the immune complex is an immune complex composed of transferrin and immunoglobulin, and (3) the immune complex. An antibody characterized in that the amount in body fluid is high in alcoholics and low in non-alcoholics with a significant difference. 2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody. 3. The antibody according to claim 1, wherein the antibody is a polyclonal antibody. 4. The antibody according to any one of claims 1 to 3, which is obtained by immunizing an animal with the immune complex as an immunogen. 5. An immunocomplex composed of (1) transferrin and immunoglobulin, wherein the amount of the immunocomplex in the body fluid is large in alcoholic patients and small in non-alcoholic patients, which is a significant difference. Immunizing an animal with the immune complex to produce an antibody that selectively reacts with the immune complex in the body of the animal, and (2) collecting the antibody from the animal. A method for producing an antibody. 6. The method for producing an antibody according to claim 5, wherein the antibody is a monoclonal antibody. 7. The method for producing an antibody according to claim 5, wherein the antibody is a polyclonal antibody. 8. A hybridoma having the ability to produce the monoclonal antibody of claim 2. 9. An immunoassay characterized by contacting a body fluid with the antibody according to any one of claims 1 to 4 and detecting or quantifying an immune complex that selectively reacts with the antibody. Method. 10. An immunoassay method comprising contacting a transferrin antibody and an anti-immunoglobulin antibody with a body fluid to detect or quantify an immune complex composed of transferrin and immunoglobulin.