KR100275865B1 - IgM MONOCLONAL ANTIBODY AGAINST X-PROTEIN OF HEPATITIS B VIRUS AND COMPOSITION FOR THE DIAGNOSIS OF HEPATIC-DISEASE COMPRISING SAME - Google Patents

Abstract

PURPOSE: Provided is IgM monoclonal antibody which shows specificity to X protein of hepatitis B virus and is useful in detecting cancer cells, and measuring the level of antigens and antibodies in blood. Also, hepatitis diagnosis composition that contains the monoclonal antibody is provided. CONSTITUTION: IgM monoclonal antibody is obtained by the next steps of: i) inserting SP2/0 X-02M (KCTC 0346BP) into a rat and obtaining spleen cells after 4 weeks; ii) mixing mouse spleen cells with myeloma cells and centrifuging the cells to obtain cell lysates; iii) facilitating the growth of hybridoma in HT medium; iv) selecting hybridoma cells which produces IgM monoclonal antibody through ELISA (Enzyme Linked Immunosorbent Assay); v) separating and purifying anti-X monoclonal antibody IgM; and vi) measuring the absorbency of anti-X monoclonal antibody IgM.

Description

IGM Monoclonal Antibody to Hepatitis Virus X Protein and Composition for Diagnosing Liver Diseases Comprising the Same

The present invention relates to a monoclonal antibody (monoclonal antibody) that specifically reacts with the X protein of Hepatitis B virus (hereinafter referred to as 'HBV'), and a kit for diagnosing liver disease, including the same. Preferably, the IgM monoclonal antibody that specifically reacts with the X protein, a regulatory protein that is believed to be directly involved in the mechanism of transferring HBV-induced hepatitis to liver cancer, among the four proteins constituting the HBV virus, and liver comprising the same A composition for clinically diagnosing a disease.

HBV is a DNA virus with a coating belonging to the Hepadnavirus family. Serologically, HBV has eight subtypes, ayw, ayw2, ayw3, and ayr, depending on the surface antigen (HBsAg) of the virus. , adw, adw2, adw4 and adr. Among them, the adr, adw, and ayw subtypes are distributed worldwide, and especially the adr subtypes are mainly distributed in East Asia including Korea (Tiollais et al., Nature, 317, 489-495 (1985)). About 300 million people around the world are infected, and it is known that infection causes hepatitis or cirrhosis, and chronic hepatitis leads to liver cancer (Tiollais and Buenda, Scientific American, 246, 48 (1991)). Hu et al., PNAS, 87, 7140 (1990); Wei et al., J. Med. Virol., 45, 82 (1995).

The genome of HBV has four genes: polymerase (P), surface antigen (S; surface protein; pre-S1, pre-S2, S), and central antigen gene (C; coat protein; pre). -C) and genes encoding the X protein, of which the P, S and C genes express structural proteins, and the X gene expresses regulatory proteins.

In particular, the X protein is a protein with a molecular weight of about 17 kDa consisting of about 154 amino acids (Cheong et al., EMBOJ., 14, 143 (1995)), whose biological function and role are not yet fully understood. It has been reported so far that the X protein acts as a transactivator to increase the action of various promoters of viruses and cells (Korean Patent Application No. 96-11602; Zahm et al., Oncogene, 3, 619 (1988); Twu and Schloemer, J. Viol., 61, 3448 (1987); Spandau and Lee, J. Virol, 62, 427 (1988); and Seto et al., Nature, 344, 72 (1990) ). In other words, they do not bind directly to DNA and indirectly react with other transcription factors or signaling factors to promote expression of the gene of interest (Takada et al., Virology, 216, 80-89 (1996)).

Indeed, HBV's X protein transforms rodent cells (Rakotomahnina et al., Oncogene 9, 2613-2621 (1994)), and induces tumors (HCC) in transgenic mice transformed with X protein. (Kim et al., Nature, 351, 317-320 (1991)), it is reported that the X protein of HBV is expressed in about 90 to 100% of liver cancer cells (Ueda et al., Nature Genetics, 9 , 41-47 (1995). From this, it can be inferred that X protein is directly involved in the mechanism of transition from hepatitis caused by HBV to liver cancer.

Cytogenes identified by the function of the X protein include thymidine kinase, c-myc, c-jun, β-interferon, EGF receptor, IGF-II gene, and the like. Among these genes, c-myc or c-jun genes are well-known protocogenes, and EGF or IGF-II genes have been reported to be associated with liver cancer. Therefore, it can be seen that various proteins activated by the X protein are directly related to the mechanism of liver cancer.

Therefore, in order to determine the cause of liver cancer caused by hepatitis virus, and to prevent or delay the occurrence of liver cancer in hepatitis patients, cancer cells using the expression system of genes specifically expressed in cancerous cells of hepatitis virus genes. It is important to establish basic techniques for developing immunotherapy, cancer gene therapy, antibody diagnosis, etc. using specific T lymphocytes. Specifically, by producing the X protein and using a monoclonal antibody against it, it is possible to accurately measure the level of the X protein in the blood will be able to reliably diagnose hepatitis and liver cancer, such as diseases caused by hepatitis virus.

Therefore, the present inventors have continued to develop a method for diagnosing liver disease caused by HBV infection. As a result, the inventors of the present invention have a single ability to specifically bind to the X protein of HBV, which is directly related to the mechanism of liver cancer. By developing a cloned antibody IgM and using it to develop a liver disease diagnostic composition that can clinically diagnose human liver disease has led to the completion of the present invention.

An object of the present invention is to provide a monoclonal antibody that can specifically recognize the X protein of hepatitis B virus (HBV) and a diagnostic composition for clinically diagnosing human liver disease using the same.

1 is a fractional view of the monoclonal antibody IgM of the present invention which specifically reacts with the X protein of HBV by purification using affinity chromatography.

Figure 2 shows the results of 10% SDS-polyacrylamide electrophoresis of the anti-X monoclonal antibody IgM isolated and purified in the present invention.

3 is a graph showing the response activity of the recombinant HBV to the X antigen according to the anti-X IgM concentration isolated in the present invention.

Figure 4 is a graph comparing the epitope recognition response to the X antigen of recombinant HBV according to each dilution concentration of anti-X IgG and anti-X IgM purified in the present invention.

In accordance with the above object, the present invention provides a composition for diagnosing liver disease comprising an IgM monoclonal antibody that specifically binds to the X protein of HBV, a hybridoma cell line producing the same, and the IgM monoclonal antibody as an active ingredient.

Hereinafter, the present invention will be described in more detail.

First, the X protein immunogen of HBV is prepared by Korean Patent Application No. 96-20423 filed by the inventors. After injection into mice, spleen cells of mice were obtained and fused with myeloma cells of mice to identify hybridoma cell populations that specifically reacted only with the X protein antigen (ELISA; Enzyme). After screening with Linked Immunosorbent Assay, anti-X monoclonal antibody IgM can be isolated and purified from the obtained hybridoma cell line. The hybridoma cell line SP2 / 0X-02M, which produces the anti-X monoclonal antibody IgM, was deposited on July 8, 1997 as the accession no. KCTC 0346BP to the Gene Bank of Korea Institute of Science and Technology.

The present invention also provides a composition for diagnosing liver disease comprising an anti-X monoclonal antibody IgM as an active ingredient.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Production and Separation of Monoclonal Antibody IgM Against X Protein Antigen of HBV

(Step 1) Cell Fusion

Four weeks after injection of HBV's X protein immunogen (see Korean Patent Application No. 96-20423, the applicant's prior application) 1: 1 with Freund's incomplete adjuvant into the abdominal cavity of mice, Was collected. 10 ⁸ splenocytes obtained from the mouse and 10 ⁷ myeloma cells (SP2 / 0) of the mouse were collected in a 50 ml test tube, followed by centrifugation to obtain a cell precipitate. 1 ml of 50% polyethylene glycol (molecular weight 1,450, manufactured by Sigma Co., Ltd.) was added thereto, and reacted for 1 minute. Then, 1 ml of RPMI 1640 medium was added thereto, followed by 1 minute of dilution, and 20 ml of medium was continuously diluted. Subsequently, the supernatant was removed by centrifugation, and the cells were transferred to a separation medium (HAT medium), incubated for about 10 days, and then changed to HT medium when the hybridoma cell population started to grow clearly, thereby promoting growth.

(Step 2) Selection of Hybridoma Cells Producing Monoclonal Antibody IgM

Screening by ELISA (Enzyme Linked Immunosorbent Assay) using X protein antigen to select a hybridoma cell group that specifically reacts only with X protein antigen from the hybridoma cell group prepared in step (step 1) screening). That is, 50 μl (2 μg / ml) of X antigen per well was added to a 96 well microtiter plate to attach to the surface of the plate and the unreacted antigen was washed. The culture medium of the hybridoma cells obtained in (Step 1) was added to 50 μl per well and reacted for 1 hour, followed by washing with PBS-Tween 20 (PBS) solution sufficiently to remove the unreacted culture. To this, goat anti-mouse IgG-HRP was added and reacted at room temperature for 1 hour, and then sufficiently washed with PBST solution, followed by reaction with a substrate solution of peroxidase (OPD). The degree of reaction was measured by absorbance at 492 nm with an ELISA reader to select hybridoma cell lines that secrete antibodies having a specific high binding capacity only to the X antigen.

(Step 3) Isolation and Purification of Anti-X Monoclonal Antibody IgM

After incubating the hybridoma cell line that specifically reacts with the X protein obtained in (step 2) and injecting a total of 2 x 10 ⁶ cells into the abdominal cavity of mice, ascites were collected and PBS (pH) was collected. 7.3) Dialysis in buffer. 5 ml of goat anti-mouse IgM agarose beads were charged to the column, and then 15 ml of the solution obtained was passed through the column at a rate of 1 ml / min. After washing the column sufficiently with phosphate buffer, fractions of 0.1M glycine-0.15M NaCl (pH 2.7) solution were collected and dialyzed with phosphate buffer to separate and purify the anti-X monoclonal antibody.

1 is a fractional view of the monoclonal antibody IgM of the present invention which specifically reacts with the X protein of HBV by purification using affinity chromatography.

Figure 2 shows the results of 10% SDS-polyacrylamide electrophoresis of the purified anti-X monoclonal antibody IgM in the present invention, column M represents the standard molecular weight protein, columns 1 to 3 Concentrations of 5 μg, 10 μg and 15 μg, respectively.

(Step 4) Determination of titer of anti-X monoclonal antibody IgM

50 μl of X antigen (2 μg / ml) was added to the wells of a 96 well microtiter plate and reacted at 4 ° C. for 18 hours. All wells were washed with PBST and then 50 μl / well of anti-X monoclonal antibody IgM diluted in PBS by concentration. After reacting for 1 hour at room temperature and washing with PBST, anti-X IgG-HRP was diluted 2000-fold in PBS, 50 μl of each well was added for 1 hour, and then washed three times with PBST. Subsequently, the reaction was performed by adding OPD, and the degree of reaction was measured at 492 nm using an ELISA reader.

3 is a graph showing the response activity of the recombinant HBV to the X antigen according to the anti-X IgM concentration isolated in the present invention, as shown here, the optimal absorbance when diluted to 1000 to 2000 times, especially 2000 times Indicated.

Example 2: Comparison of Epitope Cognitive Responses to X Antigen of Monoclonal Antibody IgM and IgG

50 μl of X antigen (2 μg / ml) was added to the wells of two 96-well microtiter plates and reacted at 4 ° C. for 18 hours. All wells were washed with PBST and then 50 μl / well of anti-X monoclonal antibody IgM and IgG (see our patent application No. 96-20423) were diluted in PBS at different concentrations. After reacting for 1 hour at room temperature and washing with PBST, anti-X IgG-HRP was diluted 2000-fold in PBS, 50 μl of each well was added, and further reacted for 1 hour, followed by washing 3 times with PBST. Subsequently, the reaction was performed by adding OPD, and the degree of reaction was measured at 492 nm using an ELISA reader.

Figure 4 is a graph showing the epitope cognitive response to the X antigen of recombinant HBV according to each dilution concentration of the anti-X IgG and anti-X IgM purified in the present invention, as shown here anti-X-IgG It can be seen that the anti-X IgM of the present invention binds to different epitopes of the X antigen.

Example 3: Antibody Screening by Enzyme Immunoassay

In order to detect the presence of an antibody against hepatitis B virus X protein in human serum, the microprotein of HBV X protein (Applicant's previous Korean Patent Application No. 96-20423) at a concentration of 2 µg / ml was detected. After attaching to the plate, the serum samples were diluted 1:50 and reacted, and then the anti-human IgG-HRP conjugate was diluted 1: 2000. A sample of a normal person and a sample of chronic hepatitis, cirrhosis and liver cancer patients are shown in Table 1 below.

Clinical diagnosis Number of patients Number of antibody positive patients against X antigen Serum markers Normal people 12 0 4 HBsAg-8 HBsAg- / anti-HBs + Acute hepatitis 2 0 2 HBsAg + Chronic hepatitis 15 0 15 HBsAg + Cirrhosis 11 7 3 HBsAg + 1 HBsAg− / anti-HBs + Liver cancer 19 12 12 HBsAg + 6 HBsAg− / anti-HBs +

As described above, in normal, acute hepatitis, and chronic hepatitis patients, HBV did not show antibodies to X antigen. In cirrhosis patients, 7 out of 11 responded and 12 out of 19 liver cancer responded. . The high levels of 63.6% and 63.1%, respectively, indicate that HBV X protein expression is frequently or high in the chronic hepatitis patients with liver cirrhosis or liver cancer, leading to antibody production.

Example 4: Antigen Search by Enzyme Immunoassay

Anti-X monoclonal antibody IgM was attached to the microtiter plate for 18 hours to detect the presence of HBV X protein antigen in human serum and then washed with PBST solution sufficiently to remove unreacted culture. . Serum samples were added diluted 1: 10 and reacted for 2 hours, washed well with PBST solution, and then diluted with anti-X IgG-HRP conjugate with a secondary antibody at 1: 2000 for 2 hours. Reacted. Subsequently, the reaction was performed by adding OPD, and the degree of reaction was measured at 492 nm using an ELISA reader. A sample of a normal person and a sample of chronic hepatitis, cirrhosis and liver cancer patients are shown in Table 2 below.

Clinical diagnosis Number of patients Number of antibody positive patients against X antigen Serum markers Normal people 12 0 0 HBeAg- Acute hepatitis 2 0 0 HBeAg + Chronic hepatitis 4 0 2 HBeAg + 1 HBeAg- / anti-HBe + Cirrhosis 13 3 3 HBeAg + 4 HBeAg- / anti-HBe + Liver cancer 12 4 2 HBeAg + 4 HBeAg- / anti-HBe +

In Table 2, serum HBV X antigen was not detected in normal subjects and patients with acute and chronic hepatitis as well as antibody measurements, but only in liver cirrhosis patients and liver cancer patients. Unlike the results of the antibody search, 3 out of 13 patients had liver cirrhosis (23%), and 4 out of 12 liver cancer patients (33.3%). This is believed to be due to the low antigenicity of the HBV X antigen, unlike the antibody. However, the production of antibodies against HBV X antigen is mainly found in cirrhosis patients and liver cancer patients, indicating that the detection of HBV X antigen and antibodies is correlated with the morbidity of liver cirrhosis and liver cancer in chronic hepatitis.

As described above, expressing cancer cells can be searched using the anti-X monoclonal antibody IgM of the present invention, and in the long term, the degree of antigen and antibody in blood, liver disease caused by hepatitis virus, hepatitis and liver cancer And clinical diagnostics.

Claims (3)

IgM monoclonal antibody that specifically binds to the X protein of HBV produced by the hybridoma cell line SP2 / 0 X-02M (KCTC 0346BP). Hybridoma cell line SP2 / 0 X-02M (KCTC 0346BP), which produces an IgM monoclonal antibody that specifically binds to the X protein of HBV. The liver disease diagnostic composition comprising the IgM monoclonal antibody of claim 1 as an active ingredient.

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