JPS61241666A - Method for measuring quantity of anti-human liver specific antigen antibody in serum by using monoclonal antibody - Google Patents

Abstract

PURPOSE:To make possible the measurement of the quantity of the anti-humal liver specific antigen antibody in the serum of a person to be examined by an immunochemical method using the monoclonal antibody for the antigen for the human liver bacteria. CONSTITUTION:The monoclonal antibody for the antigen for the human liver bacteria is immobilized and the serum of the person to be examined and the anti-serum for the monoclonal antibody thereof are added thereto so that the immobilized monoclonal antibody and the anti-serum are conjugated. The anti-serum immune globulin labeled by enzyme, radio isotope or phosphor is added thereto and is conjugated therewith. The quantity of the conjugated anti-serum immune globulin is immunochemically measured, by which the measurement of the quantity of the anti-human liver specific antigen antibody in the serum of the person to be examined is made possible.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to the production of antibodies against hepatocyte antigens in serum, so-called autoantibodies, using novel monoclonal antibodies that specifically act against human hepatocyte antigens. Concerning methods for measuring quantities.

The present invention also relates to a liver disease diagnostic method using the above-mentioned novel monoclonal antibody.

[Prior art] Conventionally, liver function was diagnosed using methods that fixed the activity of enzymes present in the liver, such as At-T (alanine aminotransferase) and AST (aspartate aminotransferase). Although these methods allow us to obtain a variety of information about liver function and liver diseases, they are not necessarily specific to liver function, and for accurate measurement and diagnosis, there is a need for a means to obtain other information. .

[Background of the Invention] The present inventors first described a novel monoclonal antibody that specifically acts against human hepatocyte antigens and its I! proposed the J construction method (patent application 1986-2 filed on February 15, 1985)
No. 6532).

This new monoclonal antibody that specifically acts against human hepatocyte antibodies was obtained by fusion of mouse myeloma cells with mouse poetry cells obtained by immunizing mice with human hepatocyte antigens. Fused cells capable of producing monoclonal antibodies against human hepatocyte antigens are cultured in a culture medium or transplanted intraperitoneally into mice to produce the monoclonal antibodies in the culture medium or, alternatively, in ascites fluid. This was obtained by

By the way, it is known that human serum contains so-called autoantibodies, which are antibodies that react with various antigens derived from human hepatocyte tissue. In the process of researching the behavior of the above-mentioned novel monoclonal antibody that specifically acts against human hepatocyte antigens, we measured the level of autoantibodies in serum using this monoclonal antibody, and in this way. They discovered that the amount of autoantibodies measured by the method was closely related to the presence or absence of liver disease.

In other words, when the amount of autoantibodies is measured using this monoclonal antibody, it is found that a large amount of autoantibodies is present in the serum of subjects with liver disease compared to that of normal subjects, as detailed below. It was discovered that the presence or absence of liver disease can be detected by monitoring the amount of autoantibodies. Therefore, the measurement of autoantibodies in serum according to the present invention has introduced new means and possibilities for the diagnosis of liver diseases.

[Specific Contents of the Invention] The present invention provides a method for measuring anti-human liver-specific antigen antibodies in the serum of a subject by an immunochemical method using a monoclonal antibody against human hepatocyte antigens. It is.

More specifically, the present invention involves immobilizing a monoclonal antibody against a human hepatocyte antigen, adding an antiserum against the monoclonal antibody in the serum of a subject, and combining the immobilized monoclonal antibody with the antiserum. Then, antiserum immunoglobulin labeled with an enzyme, radioisotope, fluorophore, etc. is added to the antiserum bound to the immobilized monoclonal antibody, and the amount of the bound antiserum immunoglobulin is determined immunochemically. The present invention provides a method for measuring the amount of anti-human liver-specific antigen antibodies in the serum of a subject.

In order to prepare this monoclonal antibody, the present inventors produced a monoclonal antibody against human hepatocyte antigen by fusing mouse splenocytes obtained by immunizing a mouse with human hepatocyte antigen and mouse myeloma cells. The mouse myeloma cells used in this case include various strains known in the art, such as ST2-NS1 strain, 83 strain, P3U1 strain, MPC-1 strain.
Examples include 1 strain, SP2 strain, and X63 strain. These various known mouse myeloma cell lines are used as fusion cells for producing monoclonal antibodies used in the method of the present invention.
As one of these many myeloma cell lines, the following example uses mouse myeloma cell line ST2-NS1.

The fused cells obtained using this mouse myeloma cell line ST2-NS1 were named fused cell line H-2 by the present inventors. The monoclonal antibody produced by this fused cell strain 1''-2 is also referred to as H-2 monoclonal antibody of the present invention.

The monoclonal antibody obtained in this way is 10G2
This is a monoclonal antibody specific to human hepatocyte antigen, which is an immunoglobulin belonging to 6.

The present inventors also found that the amount of autoantibodies against liver tissue-derived antigens produced in the human liver was significantly different by -8= between subjects with liver disease and normal subjects, as detailed below. However, it has become possible to determine the presence or absence of liver disease by assaying the amount of autoantibodies described above using the monoclonal antibody according to the present invention.

This monoclonal antibody can therefore be used for pathological diagnosis of various human liver diseases.

In this way, the measuring method of the present invention has no correlation with the test results of ALT, which is one of the conventional liver function tests, and thus makes it possible to measure liver function from a completely different perspective.

[Detailed Description of the Present Invention] The monoclonal antibody specific for human hepatocyte antigen used in the method of the present invention is produced as follows.

(1) Preparation of human hepatocyte antigen Human liver group I! (normal needle) is extracted with an aqueous sucrose solution, the extract is subjected to ultracentrifugation, and the supernatant is subjected to gel filtration. Gel filtration is preferably Sephalex G-10
0 (manufactured by Pharmacia, USA) column using Tris
Elute with a buffer consisting of -HcL Na C1° EDTA, and transfer the first peak fraction of the eluate to Repharose (USA, USA).
6B column (manufactured by Pharmacia) and eluted with the above buffer.

The antigenic substance is obtained from the first peak fraction of the eluate.

(2) Preparation of immunized animal cells A mouse is immunized with human hepatocyte antigen, and splenocytes are collected from the animal. Immunization is carried out by methods known per se.

(3) Creation of fused cells The immunized splenocytes described above and mouse myeloma cells are fused according to a conventional method. The fused cells are selectively cultured in hyboxanthine-aminoprilan-thymidine (11AT) medium. A group of antibody-producing cells is selected by radioimmunoassay (RIA) and cloned to obtain a fused cell [''-2 strain that produces monoclonal antibodies specific to human hepatocyte antigens.

(4) Preparation of monoclonal antibodies Monoclonal antibodies are produced and accumulated in the culture solution or ascites by culturing the fused cell 1"-2 strain in a medium or transplanting it into the peritoneal cavity of a mouse to cause ascites cancer. Alternatively, monoclonal antibodies are collected from ascites using a conventional method.

The fused cell line H-2 thus obtained has the following characteristics.

(Origin) These are fused cells of mouse myeloma cells (ST2-NS1) and mouse spleen cells.

(form) It shows a morphology similar to that of mouse myeloma cells.

(function) Constantly produces immunoglobulin IgG2b.

(Proliferative property) Shows the same proliferative property as myeloma cells. for example,
Proliferates approximately 10 times in 12 hours.

(Storability) It can be stored for a long time at temperatures below -80°C.

(Optimal growth conditions) I) l-17.2, temperature 37°C (medium) 1 in RPMI l640 (Gibco, USA)
Contains 0% beef tallow face serum.

The above-mentioned fused cell strain H-2 was applied for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 12, 1985 (Notice of Rejection of Deposit Acceptance, No. 192, No. 192).

Next, a method for measuring the amount of anti-human liver-specific antigen antibodies, that is, autoantibodies against liver tissue-derived antigens produced in the human liver, in the serum to be tested using the monoclonal antibodies obtained as described above will be described.

The measurement principle used in the present invention is as follows. That is, a monoclonal antibody against the antigen corresponding to the autoantibody is immobilized, and an antiserum (anti-idiotype antiserum) against the monoclonal antibody and a sample body fluid (blood or bone fluid) are prepared.
The autoantibody in the sample body fluid is quantified by contacting the monoclonal antibody and the immobilized monoclonal antibody and subjecting them to a competitive reaction, and then determining the amount of antiserum that has bound to the immobilized monoclonal antibody. .

Conventional methods for quantifying autoantibodies include, for example, Ophthalony-13-
・H- method and indirect fluorescence method using tissue sections, which each require tissue-extracted antigens and tissue sections, and it has often been difficult to obtain these tissue-extracted antigens and tissue sections. On the other hand, the water droplet method uses monoclonal antibodies and antiserum that can be stably supplied, and there are no problems in obtaining reagent raw materials compared to the conventional method.

This method enables the detection and quantification of autoantibodies in a wide range of ways, and is not used only for the quantification of autoantibodies against human liver-specific antigens.

Measurement of autoantibodies in serum using this monoclonal antibody is performed by immunochemical assay. For example, this monoclonal antibody is made into the same phase, a fixed amount of the monoclonal antibody with a known concentration is added thereto, then an antiserum obtained by immunizing an animal, such as a rabbit, with this monoclonal antibody is added, and then the above-mentioned solid phase is added. The monoclonal antibody and the added monoclonal antibody are combined with each other, washed and immobilized, and the bound antiserum is removed and the antiserum labeled with an enzyme, radioisotope, fluorophore, etc. Immunoglobulin is added and bound to the antiserum bound to the immobilized monoclonal antibody, and the amount of bound antiserum immunoglobulin is measured. For example, when the EL-recognizing enzyme is peroxidase, the antiserum immunoglobulin is colored with O-phenyldiamine or hydrogen peroxide, and measured by its absorbance at 490 tuR.

In addition to these measurement methods, methods using reactions such as latex agglutination reactions are also available. In this way, a known amount of the added monoclonal antibody and the immobilized monoclonal antibody competitively react with the antiserum, so the antiserum is distributed to both, but it is not immobilized by washing. Only the antiserum bound to the monoclonal antibody will remain. The amount of antiserum bound to the immobilized monoclonal antibody is thus determined depending on the amount of monoclonal antibody added. Therefore, if the amount of this bound antiserum is measured by binding, for example, enzyme-labeled antiserum immunoglobulin, the amount of antiserum bound to the immobilized monoclonal antibody corresponding to a known amount of added monoclonal antibody can be measured. The hanging will be revealed. This operation is repeated by varying the amount of the seven clonal antibodies added, and a test line as shown in FIG. 1 is obtained. Next, the serum to be tested whose traces of autoantibodies are unknown is added to the immobilized monoclonal antibody, further antiserum is added to this, and the amount of antiserum bound to the immobilized monoclonal antibody is measured in the same manner as the labeled antiserum. It is measured using serum immunoglobulin and applied to the previously obtained calibration curve to determine the amount of autoantibodies in the serum to be tested.

The monoclonal antibody described above is usually immobilized in a plastic well (plyastic well).
If there is a part of this plastic well where the monoclonal antibody is not immobilized, the monoclonal antibody to be added next or the autoantibody in the serum will be further immobilized, causing a measurement error. For protection, protection with proteins such as fetal bovine serum is carried out.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Preparation of fused cells (1) Human normal liver tissue was homogenized with 2.5% sucrose, ultracentrifuged at 100,000 g, and the supernatant obtained after 1 hour was purified using Sephadex G- 100. Elution was carried out using 0.IM Tris-HCl (
pl-18,0), 0.2M NaCu, 0.1ME
DTA buffer was used. The obtained fraction of the first peak was applied to a Sepharose 6B column, and the first peak eluted with the same buffer was collected and used as the antigen substance used below.

For details on the purification of antigenic substances, please refer to the method of Me Farlane et al.
Purification and Character
erization 0fhLllan 1iver-
specific 1lolIlbrane 1ipo
protein (1-8P)), “Clinical Experimental Immunology J (Clinical
EXperillletnta11+1111un01
0(IV) 27.381-390 (1977)).

(2) 1 g of 160I dissolved in 70 India complete adjuvant is injected subcutaneously and half intraperitoneally into 11 Balb/c mice (5 weeks old, male). One month later, subcutaneous administration was performed in the same manner, and another month later, the same injection was performed. Four days after the final administration, the spleen was removed, and the splenocytes were cultured in serum-free culture medium (RPMI 1640 (manufactured by Gibb 1) 7.5mM).
HFPE5. buffered with pl-17,2).

(3) Myeloma for A Mouse myeloma cells (SP2/N5I) were subcultured in RPM1 1640 medium containing 10% tallow serum. This myeloma cell (SP2/N5I
) was cultured at 23.7°C in a carbon dioxide incubator with an air flow containing 5% carbon dioxide. It was found that these cells were selectively inhibited in hyboxanthin-aminobuterine-thymidine medium w1.

(4) Prepare a RPMI suspension of 11 M momme 11 mouse myeloma cells (SP2/N5I) (2X 107 cells), mix with the splenocyte suspension (7X 107 cells), and centrifuge at 400X9 for 5 minutes. The supernatant was removed and a mixed precipitate containing both cells was collected. Add 50% (weight/volume) polyethylene glycol solution 1#Il to the precipitation! was added at 37° C. for 1 minute with gentle stirring to fuse both cells. Thereafter, the reaction was stopped by gradually adding 15 d of RPMr 1640 medium without fetal bovine serum, and the reaction was stopped at RPHT 1640.
The cells were washed ~3 times (400×g, 5 minutes) to obtain fused cells.

The above fused cells were added to RPMI supplemented with 15% fetal bovine serum.
1640 medium to obtain a suspension containing 2 x 10a cells/-, and place 0.1 d of each into a 96-well tissue culture plate using a carbon dioxide incubator in an air stream of 95% air and 15% carbon dioxide. After culturing at 37°C for 24 hours, hyboxanthine-aminopterin-thymidine (+-IAT) selection medium o and iId were added and cultured. Step 1 - Continue culturing while replacing the IAT medium.
After day 0, the culture medium was replaced with 1-IT medium several times.
On the 14th day, two supernatants of each culture solution were taken, and antibody-producing cell groups were selected by radioimmunoassay (RIA).

Selection was performed using a dilution method, and cells were grown so that one fused cell was contained in each well. Cloning was performed by diluting the obtained clone several times and repeating the culture.

In this way, a fused cell line H-2 was obtained which produced a monoclonal antibody specific to human hepatocyte antigens.

The above operations were performed by Köhler and Milstein (1975).
[Kohler Ge and Milstein Tsu ■-1 (K
6hler, Gand Milstein, C0 “Nature) I 256 495 (1
This was carried out according to the conventional method established by 975).

Example 2 Preparation of Monoclonal Antibody According to Row A - 11 Jl Balb/c mice (6-8 weeks old, female) were treated with pristene (mineral oil derived from shark) at 0.5#+1 per mouse! One week after the intraperitoneal administration, approximately 10 6 to 10 7 antibody-producing cells of the H-2 strain were transplanted into the peritoneal cavity of the mouse, and 1 to 2 weeks later, the accumulated ascites was collected. Ascites was fractionated by ammonium sulfate fractionation, and precipitates from 0 to 50% fractions were collected, dissolved in 0.1M phosphate buffer (pH 8.0), and dialyzed. The supernatant of the dialysate is r
) Protein A-Sephas CL-/l B column (USA,
V, O, 21 acetic acid and 0 adsorbed on (manufactured by Pharmacia)
．． Elution was performed using a mixture of 14M NaCl. After elution, the mixture was neutralized with Tris powder and thoroughly dialyzed against 0.1M phosphate buffer (pi-17,4) to obtain 1''-2 monoclonal antibody.

Example 3 Measurement of anti-human liver-specific antigen antibodies in serum (1) A suspension was prepared with 550 μg of I+-2 monoclonal antibody and an equivalent amount of complete 70% indoor adjuvant, It was injected subcutaneously into the front paws and back of domestic rabbits. After 3 weeks, equal amounts of 14
-2 monoclonal antibody was injected intravenously. Furthermore 1.
23 [1 hour later, each drug was administered intravenously in the same manner, and on the third day after the final administration, whole blood was collected from the carotid artery to obtain serum. The obtained serum was treated with normal mouse IO to obtain an antiserum (anti-idiotype antiserum).

(2) LJ21 We used H-2 monoclonal antibodies to detect autoantibodies similar to H-2 monoclonal antibodies, that is, anti-human liver-specific antigen antibodies, in patients with liver disease and to understand the relationship between them and pathological conditions. CompeNtive E L
The amount of autoantibodies was measured by the ISA method.

H-2 monoclonal antibody was diluted to 2.5 μg/Id with phosphate buffer (pH 7.4), added at 501 tM to a 96-well tissue culture plate, incubated at 37°C for 1 hour, and diluted with phosphate buffer (pH 7.4). (Wash three times with pl(γ, 4). Phosphoric acid 11 buffer (pH 7,4
) Add 200 μm to each well, incubate for 1 hour at 31° C., and wash three times with phosphate buffer (pH 7,4). 0.2% bovine serum albumin 0.05%'l we
H-2 with phosphate buffer (pH 7,4) containing en 20
Dilute the monoclonal antibody to 500 ng/
In addition to each well at each concentration of me, an additional 20,000
Add 50μρ of anti-H-2 antiserum diluted twice,
Incubate for 1 hour at ℃.

After washing three times, 50 μρ of peroxidase-labeled anti-inflammatory immunoglobulin diluted 20,000 times was added and incubated at 37° C. for 1 hour. After washing three times, O-phenyldiamine and hydrogen peroxide were added according to a conventional ELISA method, and absorbance between 490 and 490 was measured.

The results obtained using the H-2 monoclonal antibody to obtain the standard curve are shown in FIG. A linear relationship was observed between the natural logarithm of the concentration and the absorbance in the concentration range of 4 nMd to 650 (trig/aal') of the 1''-2 monoclonal antibody.

Next, for application to patients with liver disease, patient serum was diluted 100 times using the method described above.

The antibody in the subject's serum was calculated as 81 traces corresponding to H-2 monoclonal antibody. As a result, as shown in Table 1, 323±104 nO/#li! of 11 healthy subjects! In contrast, in 14 cases of chronic active hepatitis, the number of cases was 533 ± 154 no.
/1trf! %685 (:301n) in 7 cases of liver cirrhosis
(1/#11!, all significantly (p<0.001
, Wilcoxon Rank-Sum Test). Based on these results, it is possible to measure the concentration of antibodies, that is, anti-human liver-specific antigen antibodies, in the serum of subjects who have similar autoantibodies, using antiserum against 1-1-2 monoclonal antibody. It was discovered that

Table 1 Anti-human liver-specific antigen antibodies detected in serum of patients with liver disease 1

[Brief explanation of the drawing]

The attached drawing shows a calibration line prepared using H-2 monoclonal antibody and its antiserum.

Claims (1)

[Scope of Claims] 1) A method for measuring the amount of anti-human liver-specific antigen antibodies in a test serum by an immunochemical method using a monoclonal antibody against a human hepatocyte antigen. 2) Immobilize a monoclonal antibody against a human hepatocyte antigen, add the serum to be tested and an antiserum against this monoclonal antibody, and combine the above immobilized monoclonal antibody with the antiserum to bind to the immobilized monoclonal antibody. 2. The method according to claim 1, which comprises further adding and binding an enzyme-labeled antiserum immunoglobulin to the obtained antiserum, and measuring the amount of the bound antiserum immunoglobulin. 3) Monoclonal antibodies are fused mouse splenocytes obtained by immunizing mice with human hepatocyte antigens and mouse myeloma cells, and have the ability to produce monoclonal antibodies against the human hepatocyte antigens. The monoclonal antibody can be obtained by culturing the cells in a culture medium or transplanting them into the peritoneal cavity of a mouse to cause ascites cancer, producing the monoclonal antibody in the culture solution or ascites, and collecting the monoclonal antibody from the culture solution or ascites. A method according to claims 1 and 2, wherein the method is performed by: 4) The monoclonal antibody was used to target mouse myeloma cells ST
The method according to claim 3, which is produced from the fused cell H-2 strain obtained using the 2-NS1 strain. 5) The method according to claim 2, wherein the monoclonal antibody is immobilized on a plastic plate. 6) Claim 2, wherein the antiserum is obtained by administering a monoclonal antibody to a domestic rabbit, and the enzyme-labeled antiserum immunoglobulin is an enzyme-labeled anti-rabbit immunoglobulin. the method of. 7) The enzyme used for labeling is peroxidase, and the amount of bound antiserum immunoglobulin is measured by coloring due to the reaction between O-phenyldiamine and hydrogen peroxide. Method described. 8) A method for diagnosing liver disease by measuring the amount of anti-human liver-specific antigen antibodies in the serum to be tested by an immunochemical method using a monoclonal antibody against human hepatocyte antigens. 9) Immobilize a monoclonal antibody against human hepatocyte antigen, add the test serum and antiserum against this monoclonal antibody, and combine the above immobilized monoclonal antibody and antiserum to bind to the immobilized monoclonal antibody. 9. The method for diagnosing liver disease according to claim 8, which comprises further adding and binding an enzyme-labeled antiserum immunoglobulin to the obtained antiserum, and measuring the amount of the bound antiserum immunoglobulin. 10) Monoclonal antibodies are cell fusions of mouse splenocytes obtained by immunizing mice with human hepatocyte antigens and mouse myeloma cells, and have the ability to produce monoclonal antibodies against the human hepatocyte antigens. The monoclonal antibody can be obtained by culturing the cells in a culture medium or transplanting them into the peritoneal cavity of a mouse to cause ascites cancer, producing the monoclonal antibody in the culture solution or ascites, and collecting the monoclonal antibody from the culture solution or ascites. The method for diagnosing a liver disease according to claims 8 and 9, which is a method for diagnosing a liver disease. 11) The monoclonal antibody was used to target mouse myeloma cells S
The method for diagnosing liver disease according to claim 10, which is produced from the fused cell H-2 strain obtained using the T2-NS1 strain. 12) The liver disease diagnostic method according to claim 9, wherein the monoclonal antibody is immobilized on a plastic plate. 13) Claim 9, wherein the antiserum is obtained by administering a monoclonal antibody to a domestic rabbit, and the enzyme-labeled antiserum immunoglobulin is an enzyme-labeled anti-rabbit immunoglobulin. How to diagnose liver disease. 14) The enzyme used for labeling is peroxidase, and the amount of bound antiserum immunoglobulin is measured by coloring due to the reaction between O-phenyldiamine and hydrogen peroxide. The liver disease diagnosis method described.