KR20130088576A - Antibody specifically binding to afp, hybridoma cell producing the same, and kit for diagnosing liver cancer using the same - Google Patents

Abstract

PURPOSE: A kit for diagnosing liver cancer using an antibody which specifically binds to alpha feto protein (AFP) is provided to diagnose liver cancer using non-invasive biological samples such as blood, plasma, serum, and lymph with high specificity and sensitivity. CONSTITUTION: A monoclonal antibody specifically binds to AFP which is produced by a hybridoma cell (deposit number KCTC 12101BP or KCTC 12102BP). The hybridoma cell produces the monoclonal antibody. A kit for diagnosing liver cancer contains an antibody which specifically binds to AFP. The monoclonal antibody is used as a capture or a detector in the kit.

Description

Antibodies specifically binding to AFP, hybridoma cell producing the same, and kit for diagnosing liver cancer using the same}

The present invention provides an antibody specifically binding to AFP, a hybridoma cell line producing the antibody, a kit for diagnosing liver cancer comprising the antibody, and an antigen-antibody binding reaction using the antibody to the AFP in a sample of a liver cancer patient. It relates to a method of detection.

Despite the remarkable advances in modern medicine, cancer incidences and deaths continue to increase, resulting in large losses in individual households, national and social expenditures for their diagnosis and treatment. The development of preventive, diagnostic and therapeutic techniques for this is an international challenge. Early cancer screening is a way to dramatically reduce cancer burden by minimizing cancer occurrence and cancer death through comprehensive cancer management. The development of Alpha Feto Protein (AFP) monoclonal antibodies for the screening and monitoring of liver cancer is necessary to develop a method for rapidly detecting cancer in a large number of people in the concept of screening and monitoring cancer. It is required. Among cancers, liver cancer is the second largest in Korea and the world's most incidence, and the recurrence rate is very high. Currently, in domestic general hospitals, liver cancer is a high-tech equipment developed by Ryoshu in the United States using MODULAR ANALYTICS E170 equipment. Doing a diagnostic test. However, for the localization of diagnostic reagents as well as the localization of the equipment, development of matchable monoclonal antibodies capable of detecting AFP (Alpha Feto Protein) cancer markers is necessary.

Alpha Feto Protein (AFP) is a glycoprotein with a molecular weight of 70 kD containing 4% carbohydrate and is a tumor marker of non-seminon. Alpha Feto Protein (AFP) is structurally homologous to albumin and is produced in birthing interphase and egg yolk, peaking at 13-14 weeks of gestation, and begins to decline after 12 months of age until adult value (<10 ug / L). do. Alpha Feto Protein (AFP) is also elevated in benign diseases such as hepatitis and cirrhosis. In 95% of them, AFP (Alpha Feto Protein) is less than 200 ug / L. In the non-pregnant state, AFP (Alpha Feto Protein) is more than 1,000ug / L can be said to be cancer. The cut-off value of AFP for detecting very small and resectable hepatocellular carcinoma is 10-20 ug / L. Most cancers that occur in humans secrete several types of products, some of which cause signs and symptoms in patients. These products appear in the blood or other body fluids and are called tumor markers (cancer markers).

Most tumor markers emphasize the usefulness of follow-up rather than diagnostic usefulness. However, studies investigating the purpose of screening for tumor markers showed that 54% of tumor markers were screened for screening suspicious tumors or to determine the origin of the tumors found and only 32% were used for follow-up of existing tumors. It is said. Tumor marker tests are widely used as screening tests for tumors in most health examination centers in Korea.

Unlike most tumor markers, AFP is very useful for screening and is used to select high risk groups of hepatocellular carcinoma in areas with high prevalence of hepatocellular carcinoma, such as Korea, China, Japan, Alaska and Africa. Decreasing tumor markers indicate successful treatment and vice versa, recurrence or metastasis, and tumor markers are closely related to the prognosis and size of the tumor.

Accordingly, the present inventors have developed a method for identifying an effective monoclonal antibody, and have completed the present invention by identifying monoclonal antibodies that are increased enough to diagnose liver cancer individuals using the method.

The present invention provides a novel antibody that specifically binds AFP (Alpha Feto Protein), a hybridoma cell line producing the antibody, a liver cancer diagnostic kit comprising the antibody, and an antigen-antibody binding reaction using the antibody. It is an object of the present invention to provide a method for detecting AFP in a sample of liver cancer patients.

The present invention provides monoclonal antibodies produced by hybridoma cells with an accession number of KCTC 12101BP or KCTC 12102BP.

The present invention also provides a hybridoma cell line having an accession number of KCTC 12101BP or KCTC 12102BP to produce a monoclonal antibody that specifically binds to Alpha Feto Protein (AFP).

The present invention also provides a kit for diagnosing liver cancer comprising the antibody.

In addition, the present invention provides a method for detecting AFP in a sample of liver cancer patients through the antigen-antibody binding reaction using the AFP antibody.

When the antibody specifically binding to AFP of the present invention is used as a liver cancer diagnosis kit, the diagnosis of liver cancer is diagnosed with high specificity and sensitivity only by using non-invasive biological samples such as blood, plasma, serum, and lymph fluid. can do.

In addition, in the present invention, by capturing the monoclonal antibody produced by KCTC 12101BP hybridoma cells and using the monoclonal antibody produced by KCTC 12102BP hybridoma cells as a detector, liver cancer can be easily diagnosed with high prediction. It may be useful for developing a liver cancer diagnosis kit.

1 is a diagram showing the overall schematic of making AFP monoclonal antibodies.
Figure 2 is a Western blot by monoclonal antibodies produced by AFP antibodies, KCTC 12101BP (h-AFP-mAb-2C5h-JYC) and KCTC 12102BP (h-AFP-mAb-4B1h-JYC), which are sold commercially. The results are shown.
Figure 3 shows the Western blot results of purified AFP monoclonal antibody in a plurality of mice.
Figure 4 shows the correlation between the monoclonal antibody produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) or KCTC 12102BP (h-AFP-mAb-4B1h-JYC) and AFP antigen by Direct ELISA. The figure shown.
Figure 5 is a capture of the monoclonal antibodies produced by KCTC 12102BP (h-AFP-mAb-4B1h-JYC), monoclonal produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) Figure shows Sandwich ELISA results using raw antibodies as detectors.
Figure 6 captures the monoclonal antibody produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC), and the monoclonal antibody produced by KCTC 12102BP (h-AFP-mAb-4B1h-JYC). Sandwich ELISA results used as a detector.
7 is a diagram showing a standard curve according to AFP antigen concentration.

Another object of the present invention is to provide a monoclonal antibody produced by hybridoma cells having an accession number of KCTC 12101BP or KCTC 12102BP.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, such as hybridomas, recombinant and phage display techniques, or methods using combinations thereof. For example, monoclonal antibodies can be prepared using hybridoma techniques, such as those known in the art, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd). ed. 1988); Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981), which are incorporated herein by reference. The term "monoclonal antibody" herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody derived from a single clone, such as any eukaryotic, prokaryotic or phage clone, but not a method of production thereof.

Methods of producing and screening specific antibodies using hybridoma techniques are routine and well known in the art. By way of non-limiting example, mice can be immunized with a target antigen or cells expressing such antigen. When an immune response is detected, for example, when an antibody specific for an antigen is detected in mouse serum, the mouse spleen is recovered to separate splenocytes. The splenocytes are then fused with any suitable myeloma cells (eg, P3U1, P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2 / 0-Ag14, P3X63-Ag8-653, etc.) by known techniques. . Hybridomas were screened and by limiting dilution

Clones. The hybridoma clones are then assessed for their ability to secrete antibodies capable of binding antigen by methods known in the art. In general, ascites containing high levels of antibodies can be made by inoculating a mouse abdominal cavity with a positive hybridoma clone. In a preferred embodiment of the present invention, monoclonal antibodies specific for AFP generated from deposited hybridoma cells were identified by ELISA and Western blot.

The present invention provides a hybridoma cell line having an accession number of KCTC 12101BP or KCTC 12102BP to produce a monoclonal antibody that specifically binds to Alpha Feto Protein (AFP).

The term "hybridoma cell" of the present invention refers to a cell made by artificial fusion of two different cell types. Hybridoma cell lines are produced by fusing two or more allogeneic or heterologous cells using a substance that causes cell fusion, such as polyethylene glycol, or a specific species of virus.By forming hybridomas, different cells have different functions. Can be incorporated into cells. Such hybridoma cells can generally be prepared by fusing cancer cells which proliferate in vitro and large cells having any specific differentiation trait extracted from a living body. Representative hybridomas are lymphocyte hybridomas, hybridoma cells fused with myeloma cells and B cells, T cells and T cells that produce tumor cells, hybridoma cells, and lymphokines (bioactive substances). The tumor cells are hybridoma cells and the like, and it is apparent that the hybridoma cell line of the present invention can be fused and cultured under optimum conditions for effectively producing the autoimmune antibodies of the present invention according to the choice of those skilled in the art. In a preferred embodiment of the present invention, after obtaining B cells from the spleen of a mouse identified as liver cancer, culturing them with mouse myeloma cells (Sp2 / 0 cells), and then culturing B cell high with hepatocyte cancer-reactive antibodies Bridoma was selected and named as h-AFP-mAb-2C5h-JYC and h-AFP-mAb-4B1h-JYC, which was deposited on December 12, 2011 to the Korea Institute of Bioscience and Biotechnology. 12101BP and KCTC 12102BP.

It is still another object of the present invention to provide a monoclonal antibody having the same epitope specificity as a monoclonal antibody produced by hybridoma cells having an accession number of KCTC 12101BP or KCTC 12102BP. The portion of the antigen that is specifically recognized and bound by the antibody is referred to as an “epitope”. A similar antigenic antibody response can be seen if it has the same epitope specificity as monoclonal antibodies produced by hybridoma cells with an accession number of KCTC 12101BP or KCTC 12102BP.

Another object of the present invention to provide a kit for diagnosing liver cancer comprising an antibody that specifically binds to AFP.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether the liver cancer. When AFP of the present invention is used as a diagnostic marker for liver cancer, it is possible to determine whether liver cancer has occurred in a subject by measuring the expression level of AFP of the present invention in a subject's sample.

In the present invention, the term "diagnosis marker", "marker for diagnosis" or "diagnosis marker" is a substance which can diagnose liver cancer cells by distinguishing them from normal cells, and in cells having liver cancer compared to normal cells. Polypeptides or nucleic acids (eg, mRNA, etc.) that show an increase or decrease, organic biomolecules such as lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For purposes of the present invention, the hepatic cancer diagnostic markers of the present invention are capable of expressing specifically high levels of whole blood, blood, serum or plasma in individuals with liver cancer as compared to whole blood, blood, serum or plasma in individuals with normal liver. It is a visible anti-AFP antibody.

In the present invention, the term "liver cancer" generally refers to cancer originating from hepatocytes. Hepatocellular carcinoma (HCC) is a metastatic liver cancer that develops in the liver from primary liver cancer occurring in the liver and cancer occurring in other tissues. Most of the causes are unclear, but liver cirrhosis is often present, and hepatocellular carcinoma is found to occur in patients with cirrhosis, chronic active hepatitis B, or hepatitis B carriers. When using the autoantibody marker of the present invention, it can be diagnosed with a high level of sensitivity and specificity for the development of liver cancer from the individual.

In the present invention, an antigen protein that specifically binds to an autoimmune antibody is a molecule that can be used for detection of a marker by confirming the expression level of AFP, a marker of which expression is increased in whole blood, serum and plasma, lymph and intercellular fluid of an individual. it means. Antigen-antibody assays include Western blot, Enzyme Linked ImmunosorbentAssay (ELISA), radioimmunoassay (otA: badioimmunoassay), radioioimmunodiffusion, Ouchterlony immunodiffusion, and rockets Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip, but are not limited thereto. Through the above analysis methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in the suspected liver cancer can be compared, and thus diagnosis of liver cancer in liver cancer patients can be made. Will be.

An antigen (or polypeptide that specifically binds to an antibody) that specifically binds to an anti-AFP antibody of the present invention includes all proteins that can specifically bind to the antibody, and is limited to a specific protein or polypeptide. It doesn't work. Preferably, the antigen may include all fragments or variants thereof as long as the antigen can be recognized by the anti-AFP antibody. These antigens may consist of at least seven amino acids.

And preferably 9 amino acids, more preferably 12 or more amino acids. The antigen may also be an epitope sequence that can be recognized by the autoimmune antibody markers of the invention. The epitope sequence is not limited to the size and type of the sequence as long as it is a sequence recognizable by the antibody of the present invention.

In the liver cancer diagnosis kit, when the monoclonal antibody produced by KCTC 12101BP hybridoma cells and the monoclonal antibody produced by KCTC 12102BP hybridoma cells are used together, the cancer is diagnosed more accurately than when one antibody is used. can do. In other words, using monoclonal antibodies generated from KCTC 12101BP and KCTC 12102BP hybridoma cells, one of the two monoclonal antibodies reacts with an AFP and causes an antigen-antibody reaction to catch the other antibody. Working together, the two monoclonal antibodies work together to produce an antigenic antibody response that allows for accurate diagnosis of cancer.

The liver cancer diagnosis kit is preferably a liver cancer diagnosis kit using a monoclonal antibody produced by KCTC 12102BP hybridoma cells as a detector by capturing a monoclonal antibody produced by KCTC 12101BP hybridoma cells.

In a preferred embodiment of the present invention, a 4B1 monoclonal antibody as a capture and a 2C5 monoclonal antibody as a detector were reacted with AFP protein by Sandwich ELISA.

Still another object of the present invention is to provide a method for detecting AFP in a sample of liver cancer patient through an antigen-antibody binding reaction using an antibody that specifically binds to AFP. That is, (a) measuring the expression level of AFP from a sample of a subject suspected of liver cancer, (b) comparing the expression level of the AFP protein with the expression level of AFP in a normal control sample, and (c) the It provides a method comprising the step of confirming that the expression level of AFP of step (a) is expressed higher than the AFP expression level of the normal control sample.

Methods for measuring the expression level of the antibody are Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and It is preferably any of the protein chip methods.

As used herein, the term "sample" includes, but is not limited to, samples such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, cerebrospinal fluid, and hepatic fluid, which differ in the expression level of AFP, a liver cancer marker. Do not.

The method of detecting the AFP of the present invention may be performed by measuring the expression level of AFP from a sample of a suspected liver cancer, and comparing it with the AFP expression level from a sample of a normal subject. If a higher level of AFP is detected than, the subject can be determined as a liver cancer patient.

In the detection or diagnostic method of the present invention, one or more selected from the above methods may be used, and liver cancer may be diagnosed by performing two steps, a primary test and a confirmation test, as necessary. As described above, when the liver cancer is to be diagnosed by detecting the autoimmune antibody of the present invention by the two steps, allogenic sera screening can be used as the primary test, and as a confirmation test for distinguishing the positive and false positive reactions. Western analysis can be used.

The detection or diagnostic method of the present invention can accurately detect the AFP and diagnose the onset of liver cancer because the results are highly consistent with each result regardless of which method is used for a sample obtained from the same individual. Can be.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

<Examples>

1. Monoclonal Antibody Formation

The overall monoclonal antibody preparation is shown in FIG. 1.

(1) Immunization stage

150 μl of AFP antigen (30 μg / μl) was mixed well with 150 μl of complete Freund's adjuvant (Sigma) in the same volume to make an emulsion, and injected into the abdominal cavity of 6-week-old female BALB / C mice. Two weeks after the first immunization step, 150 μl of the antigen and 150 μl of the same amount of incomplete Freund's adjuvant (Sigma) were prepared and intraperitoneally injected. Immunization was performed up to tertiary at 2 week intervals. One week after the last immunization, 10-30 μg / μl of antigen was injected into the tail vein three days before fusion.

(2) Fusion

Myeloma cells (SP2 / 0 cells) were prepared at a density of 2.5-5 × 10 ⁴ / ml and prepared by diluting to 1/3 before 24 hours before fusion. The reagents in Table 1 were used for fusion.

Composition of reagents used for cell fusion SF-DMEM1 DMEM + 1X AA SF-DMEM2 DMEM + 2X AA RBC LB Red blood cell lysis buffer (sigma) HT 50X HT (sigma) 1vial + SF-DMEM1 10ml HAT 50X HAT (sigma) 1vial _ SF-DMEM 10ml HT media HT 50X 1ml + FBS 10ml + SF-DMEM1 30ml HAT media HAT 50X 4ml + FBS 40ml + SF-DMEM1 105ml + Blood 1 ~ 1.5ml + Thymus Cell suspension 50ml PEG PEG (sigma)

For cell fusion, the following experiment was performed.

Blood was drawn from the heart of immunized mice. Blood was stored at 4 ° C. overnight, the syrup was separated after centrifugation, divided into appropriate portions, stored at −80 ° C. , and the spleens of immunized mice were extracted. Splenocytes were eluted after washing with SF-DMEM2. The mass was allowed to settle to settle and the supernatant was taken and centrifuged for 5 minutes at 1500 rpm. Supernatants of centrifuged spleen cells were removed and filled with SF-DMEM2. Spleen cells and SP2 / 0 cells were washed by centrifugation. Supernatants of SP2 / 0 cells were removed and filled with SF-DMEM2. LB to 1ml RBCs were put in and filled with SF-DMEM2 after treatment. Spleen cells and SP2 / 0 cells were centrifuged. Centrifuged spleen cells and SP2 / 0 cells were supernatant removed and filled with 10 ml of SF-DMEM2. Spleen cells and SP2 / 0 cells were diluted 100-fold and counted to determine concentration. Spleen cells and SP2 / 0 cells were determined at a ratio of 10: 1. Spleen cells and SP2 / 0 cells of the determined concentrations were put together in a tube and centrifuged. After removing the supernatant of the centrifuged cells were placed on the alcohol cotton and dried for 30 seconds ~ 1 minutes and then slowly added PEG. The reaction was carried out as it is for 1 minute and centrifuged with SF-DMEM2. After centrifugation, the supernatant was removed and 50 ml of HT medium was charged. Then, the lid was opened and incubated for 3 hours at 37 ℃, 5% CO ² incubator. Three rats were anesthetized, and a heparinized syringe was used for heart blood collection and thymic tissue extraction. Thymic cells were eluted after washing with SF-DMEM2. The cultured cells were centrifuged at 500 rpm for 10 minutes. The supernatant of the centrifuged fusion cells was removed and mixed slowly using HAT medium. Aliquots into 96-well plates were incubated in a 37 ° C., 5% CO ² incubator.

Change medium at 8-9 days after fusion and grow well from 96 to 24 wells

Incubated in cDMEM2. After changing the medium, the supernatant of the color-changed well was harvested and filled with cDMEM2 on 5-7 days, and then ELISA was performed. After confirmation by ELISA, wells were selected, transferred to 24 wells, and cultured again.

The ELISA treated the blocking solution after coating the antigen, and the primary and secondary antibodies were identified at 450 nm wavelength after the treatment in turn.

(3) monoclonal antibody selection and ascites production

The following experiments were performed to select monoclonal antibodies.

After confirming the cell concentration of 24 wells, the cells were diluted in 15 ml of the culture medium so as to be 0.5 cells / well in a 96 well plate. The diluted cells were dispensed at 150 μl per well and examined under a microscope to check the wells containing one cell. The supernatant of the wells in which the cells were grown to some extent was harvested, and the cells were transferred to 24 wells after ELISA and cultured. The supernatant of well grown in 24 wells was floated and confirmed by ELSIA, Western Blot. Cells to create ascites were selected and the rest frozen. The identified cells were transferred to 25T / C flasks and cultured.

In order to purify the antibody after ascites production, the following experiment was performed. Several 7-8 week old females of Balb / C mice were prepared. Two mice intraperitoneally injected with 500 μl of pristane. After 7-10 days, cells grown in 75T / C culture flasks were collected and centrifuged. The supernatant was removed and 500 μl of PBS was injected into the mouse abdominal cavity. After 5-7 days after confirming that the abdomen of the mouse was swollen ascites using a syringe. Ascites was one day at 4 ℃ and centrifuged the next day to separate only the supernatant and stored at -20 ℃. After filling the appropriate amount of Protein G Fast Flow Bed stored in 20% ethanol in the column (20%), 20% ethanol was flowed down and washed with 5 Bed Volum Binding Buffer. The ascites was diluted in PBS with an appropriate amount and loaded into the Protein G Column. After binding to 3 Bed Volum Binding Buffer and diluted with 3 Bed Volum Elution Buffer into 0.5 ml fractions. Each fraction was neutralized with 35ul Neutrazing Buffer. After standing overnight at refrigeration temperature with 70% ethanol and further treated in 20% ethanol and refrigerated until the next use.

Reagent Compositions Used for Purifying Monoclonal Antibodies Buffer Composition Binding Buffer 20 mM sodium phosphate, pH 7.0 Elution Buffer 0.1M glycine buffer, pH 3.0-2.5 Neutralizing Buffer 1M Tris-HCl, pH 9.0 Storaging Solution 20% ethanol

2. Experimental results

(1) Confirmation of antigen antibody response of monoclonal antibody

In the process of making hybridoma cell lines, two high-performance cell lines were isolated with monoclonal antibodies against AFP. The isolated cell line corresponds to a hybridoma cell line with an accession number of KCTC 12101BP (h-AFP-mAb-2C5h-JYC) or KCTC 12102BP (h-AFP-mAb-4B1h-JYC). Using monoclonal antibodies produced in the cell line was compared with the actual commercially used antibodies. Commercially available antibodies (Mouse monoclonal (AFP-01) to alpha 1 Fetpprotein, Abcam co.) And KCTC 12101BP (h-AFP-mAb-2C5h-JYC) or KCTC 12102BP (h-AFP-mAb-4B1h-JYC) Antibodies from in hybridoma cell lines were treated with AFP antigens and patient samples to obtain results, as shown in FIG. 2.

Commercially available antibodies (Mouse monoclonal (AFP-01) to alpha 1 Fetpprotein, Abcam co.) Do not have antigen-antibody reactions with AFP antigen in liver cancer patient samples, but KCTC 12101BP (h-AFP-mAb Monoclonal antibodies produced from hybridoma cell lines, which are either -2C5h-JYC) or KCTC 12102BP (h-AFP-mAb-4B1h-JYC), can clearly identify antigen-antibody responses in AFP antigen and liver cancer patient samples. have.

It can be seen in FIG. 3 that the monoclonal antibodies generated in the ascites also induce an antigen-antibody reaction with the AFP antigen. In Figure 3, the first line, C, is a commercially available antibody as a control, and 1 to 4 are monoclonal antibodies taken from ascites of mice injected with KCTC 12101BP (h-AFP-mAb-2C5h-JYC) cells, 5 to 8 correspond to monoclonal antibodies taken from ascites of mice injected with KCTC 12102BP (h-AFP-mAb-4B1h-JYC) cells.

(2) Correlation with AFP Antigen and Monoclonal Antibodies Produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) or KCTC 12102BP (h-AFP-mAb-4B1h-JYC) by Direct ELISA

Direct ELISA was used to confirm the correlation between the AFP antigen and the monoclonal antibody. After antigen coating from 0 to 100 ng / 100 ul, ELISA was performed by treating monoclonal antibodies from ascites. As can be seen in Figure 4, it can be seen that the correlation of the OD value rises in a concentration-dependent manner up to 100ng for the AFP antigen.

(3) Sandwich ELISA using monoclonal antibodies produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) and KCTC 12102BP (h-AFP-mAb-4B1h-JYC)

Sandwich ELISA using monoclonal antibodies produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) and KCTC 12102BP (h-AFP-mAb-4B1h-JYC) was performed. Sandwich ELISA was performed using one of two types of monoclonal antibodies as the detector for the antigen and the other as the capture.

 Sandwich ELISA results are shown in FIGS. 5 and 6.

Monoclonal antibodies generated by KCTC 12102BP (h-AFP-mAb-4B1h-JYC) as captured, as shown in Figure 5, monoclonal produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) Raw antibodies were used as detectors. As a result, it was observed that the OD value increased in a concentration-dependent manner, and by capturing the monoclonal antibody produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC), KCTC 12102BP (h-AFP-mAb-4B1h- It can be seen that the affinity is greater than the result of FIG. 6 using the monoclonal antibody produced by JYC) as a detector.

In addition, as shown in Figure 6 monoclonal antibody produced by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) was captured, generated by KCTC 12102BP (h-AFP-mAb-4B1h-JYC) As a result of using the monoclonal antibody as a detector, it can be seen that in comparison with the results of FIG. 5 in terms of affinity.

Therefore, by executing the Sandwich ELISA using the two monoclonal antibodies, it is possible to determine whether the liver cancer more accurately than the conventional liver cancer test method, AFP (Alpha) using the MODULAR ANALYTICS E170 equipment, which is currently a universal liver cancer test method Feto Protein has been found to be able to diagnose liver cancer accurately and at a lower cost than that of Feto Protein.

(4) Sandwich ELISA for patient samples

As shown in FIG. 5, monoclonal antibodies generated by KCTC 12102BP (h-AFP-mAb-4B1h-JYC) by capture were generated by KCTC 12101BP (h-AFP-mAb-2C5h-JYC) by detector. Use of monoclonal antibodies showed the highest affinity for the AFP antigen. Therefore, AFP quantitative experiment was performed by performing a Sandwich ELISA on the patient sample to confirm whether the same result was obtained for the actual patient sample. Blood samples correspond to blood samples of patients and normal people provided by the Department of Diagnostic Laboratory Medicine, Kyungpook National University Hospital. Monoclonal antibodies generated by KCTC 12102BP (h-AFP-mAb-4B1h-JYC) were captured by coating overnight at 4 ° C. with 100 ng / 100 ul and then blocked with 100 ul of blocking buffer (1% BSA / DPBS). The patient's crude serum was added 25ul and the detector monoclonal antibody 2C5h-HRP 1: 1000, 100ul was treated at room temperature for 2 hours. 100ul of substrate solution was added for 20 minutes, and 50ul of stop solution was added to stop the color development. Thereafter, the OD value was measured at a wavelength of 450 nm.

Experimental results are shown in Figure 7 and Table 3.

As shown in Figure 7, the standard curve according to the antigen concentration was derived y = 0.019x + 0.1355. This can be obtained by measuring relative concentrations of AFP in actual patient samples. The results are shown in Table 3.

Table 3 shows the result of confirming the amount of AFP through the OD value of samples of normal people and liver cancer patients. A total of 25 experiment groups and control groups were obtained, and the results showed sensitivity 44% and specificity 80%. Through these results, the two monoclonal antibodies can be used together to make a liver cancer diagnostic kit with high specificity and sensitivity to liver cancer, and monoclonal antibodies themselves can be used as biomarkers with high affinity. It can be confirmed.

Korea Biotechnology Research Institute KCTC12101BP 20111212 Korea Biotechnology Research Institute KCTC12102BP 20111212

Claims (9)

A monoclonal antibody that specifically binds to AFP produced by hybridoma cells with an accession number of KCTC 12101BP or KCTC 12102BP. The monoclonal antibody according to claim 1, which has the same epitope specificity as said monoclonal antibody. A hybridoma cell line with an accession number of KCTC 12101BP or KCTC 12102BP that produces a monoclonal antibody that specifically binds to Alpha Feto Protein (AFP). The diagnostic kit for liver cancer comprising an antibody that specifically binds to the AFP of claim 1. The method of claim 4, wherein the kit captures a monoclonal antibody produced by KCTC 12101BP hybridoma cells and uses the monoclonal antibody produced by KCTC 12102BP hybridoma cells as a detector. Kit. A method for detecting AFP in a sample of a liver cancer patient through an antigen-antibody binding reaction using an antibody that specifically binds to AFP. 7. The method of claim 6,
(a) measuring the expression level of AFP from a sample of a subject suspected of having liver cancer;
(b) comparing the expression level of AFP with the expression level of AFP in a normal control sample; And
(C) the method comprising the step of confirming that the expression level of the AFP of step (a) is expressed higher than the AFP expression level of the normal control sample. The method of claim 7, wherein the expression level of AFP is measured by Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation. A method characterized by using any one selected from the group consisting of analytical methods, FACS and protein chip methods. The method of claim 6, wherein the sample is one or more selected from the group consisting of whole blood, serum, blood, plasma, saliva, urine, lymph, cerebrospinal fluid and intercellular fluid.

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