KR20100016784A - A rapid diagnostic kit for clonorchis sinensis comprising the clonorchis sinensis-specific antigen and a monoclonal antibody for production of the clonorchis sinensis-specific antigen - Google Patents

Abstract

PURPOSE: A kit for quickly diagnosing clonochiasis-specific antigen is provided to conveniently use in one step method and enable poin-of-care testing(POCT) without expensive equipment. CONSTITUTION: A kit for diagnosing clonochiasis contains clonochiasis-specific antigen which is isolated from monoclonal antibody. The monoclonal antibody which is specific to the clonochiasis antigen is an monoclonal antibody Cs1. The diagnosis kit is a diagnosis kit of strip form using immunochomatography. A method for producing the diagnosis kit comprises: a step of purifying antigen from clonorchis sinenesis; a step of preparing fusion cells which produce monoclonal antibody to the antigen; a step of selecting monoclonal antibody having high reactivity to clonorchis sinensis antigen; a step of isolating antigen which is specific to the selected monoclonal antigen; and a step of producing diagnosis kit using the specific antigen.

Description

A rapid diagnostic kit for Clonorchis sinensis comprising the Clonorchis sinensis-specific antigen and a monoclonal antibody for production of the Clonorchis sinensis-specific antigen}

The present invention cloned a fusion cell line obtained by fusing immunized rat splenocytes and myeloma cells using a member of the hepatic insecticide that reacts strongly with the antibody in the blood of a hepatic nematode to produce a monoclonal antibody and purify it to have good reactivity. The present invention relates to a quick diagnosis kit for hepatitis and to a method for diagnosing hepatitis using the same. The present invention also relates to monoclonal antibodies used in the above diagnostic kits and fusion cells producing them.

Gandistoma is a disease caused by parasites called hepatococcus parasites in the bile ducts or extrahepatic bile ducts and gallbladder in human liver. Hepatitis can cause inflammation of the bile ducts or block the bile ducts, causing symptoms such as lethargy, abdominal pain, indigestion and jaundice.

Hepatitis B is a foodborne parasite that infects the human body through freshwater ash. Liverworms live in the human liver, bile ducts outside the liver, and in the gallbladder, irritate bile ducts or gallbladder and cause severe inflammation due to secondary bacterial invasion, resulting in cholangitis or cholecystitis. In addition, when a large number of hepatic worms are infected with intrahepatic bile ducts, they may block the biliary tract and cause biliary obstruction. When the liver worms block the biliary tract, bile stagnates, and in this state, the dead caries or eggs are nuclei, and gallstones are said to be good. Most importantly, these biliary obstructions and chronic inflammation can lead to bile duct cancer.

Praziquantel is used for the treatment of hepatitis infection. The drug has excellent insecticidal effects against hepatic as well as lung insects. However, there are side effects that can cause abdominal pain by pressing the bile ducts by repeated doses, and there is a risk of re-infection even after complete treatment with Fraziquantel rather than causing resistance to the body.

Hepatitis B infection is not a major problem in its own treatment, but complications are caused if the infection is left unattended. Since various complications such as cholangiocarcinoma, bile duct stones, and purulent cholangitis are more serious diseases than hepatitis infection itself, it may be very important to diagnose the infection early and to accelerate the treatment before it develops into a serious disease.

According to the data conducted by the Ministry of Health and Welfare and the Korea Health Care Association, a nationwide fecal nematode survey, the rotary worm infection has almost disappeared recently. Insects have become a major cause of parasitic infections, especially since they cause clinically serious infections and side effects. The current diagnostic methods of hepatic pneumoniae infection include intra-fecal egg test, ultrasonography and endoscopic retrograde cholangiography for imaging, skin test for intradermal reaction, and parasite antibody test through blood test.

Fecal egg test is to find the eggs of hepatic insects in feces. This test is the most general method because it can not only determine the most accurate infection, but also estimate the extent of infection by measuring the number of eggs in the feces.

The C.S skin test can detect whether a liver worm has entered the body, but has the disadvantage of being positive even if all of the worms die after treatment. It is not possible to accurately diagnose the current infection. Mostly used early in the diagnosis process, 80-90% of those infected with hepato-pneumoniae are positive, but more than 50% of patients continue to respond positively for 5 to 20 years after treatment.

Ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), and endoscopic retrograde cholangiopancreatography can be used to identify hepatic insufficiency due to irregular enlargement of the bile ducts in the liver. It is a way to recognize bile ducts, liver inflammation, bile duct stones and bile duct cancer.

The serological method is a method of tentatively diagnosing a hepatic repellent infection by detecting antibodies in the serum when the eggs are not spawned or the eggs do not produce eggs. Serological tests for parasite-specific antibodies include complement binding, immunofluorescence, immunoelectrophoresis, immune agar diffusion, and enzyme immunoassay.

As described above, there are various diagnostic methods, but one of the important problems in insecticidal and worm infections is that there are no diagnostic methods with high sensitivity and specificity in the laboratory. Detecting eggs or cysts directly in human specimens or tissues is the most obvious method, but it is problematic to use low sensitivity and sometimes too invasive methods.

In addition, clinical symptoms and radiologic examinations may be helpful for diagnosis, but they are not a way to reliably differentiate from other diseases or confirm the causative parasites. Hepatitis and lung respiratory tract tests, which are commonly used as screening tests, are known to have high sensitivity but low specificity and do not distinguish between vertigo and past infections (Chung et al., 1995; Sandun et al., 1959; Walton and State). , 1959; Kim Dong-chan et al., 1969; Jeong Ho-yeon et al., 1989), cross-reactions caused by the use of articles made by grinding the entire body are not suitable for diagnostic tests.

To compensate for this, complementary binding, immunofluorescence, immunoelectrophoresis, immunoagar diffusion, and enzyme-immunoassays have been developed to serologically test parasite-specific antibodies since the 1950s.However, ELISA methods have been developed in terms of sensitivity and specificity and convenience. Because of its superiority, the ELISA method has been used in the clinical laboratory for diagnosis of filamentous, manic phenomena, serological epidemiology, monitoring of therapeutic effects, and healing decisions. However, as with all the above methods, the ELISA method requires expensive equipment and specialists, so it is almost impossible to test in rural areas or island areas where Gandistoma patients frequently occur.

In view of the above, the present inventors have made a monoclonal antibody specific for hepatitis and isolated and purified hepatitis specific antigen from the crude extract using immuno-affinity chromatography. Then, by using these antigens as a raw material, a diagnostic kit for hepatitis can be prepared, and the diagnosis kit can be used to diagnose non-specific reactions with other parasites. Therefore, rapid immunochromatography has been developed. The present invention has been completed by confirming that hepatitis can be efficiently diagnosed in the field without the help of manpower and expensive equipment.

An object of the present invention is superior to sensitivity and specificity than the egg test and intradermal reaction test, which is a liver dystomosis diagnostic method, and is a simpler and more economical diagnostic kit than enzyme immunoassay. It is intended to provide a diagnostic kit. The diagnostic kit containing the hepatobacterium specific antigen of the present invention overcomes the low sensitivity and specificity raised as a problem in the conventional diagnostic kit and shows high agreement with the results of enzyme immunoassay, so that it can be easily used for the diagnosis of Gandistoma. Even more effective.

Another object of the present invention to provide a method for diagnosing liver dystomia using the diagnostic kit.

Another object of the present invention is to provide a novel monoclonal antibody specific for hepatitis antigens for producing the hepatitis specific antigen.

Still another object of the present invention is to provide a fusion cell for producing the hepatotoxic specific monoclonal antibody.

In one aspect, the present invention provides a hepatomegaly diagnostic kit comprising hepatomegaly antigens produced using hepatomegaly antigen specific monoclonal antibodies. Diagnostic kit according to the present invention is superior to the specificity and sensitivity compared to the existing diagnostic kit, it is a quick, easy diagnostic kit that has the advantage that can be tested at high speed in the field without the use of high technology, personnel, and equipment.

As used herein, the term "diagnosis" refers to identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine the onset of hepatic nematode.

As used herein, the term “hepatic nematode” is also referred to as hepatostomoma, and is referred to as Clonorchis sinensis ) refers to a disease or condition caused by an infection of a parasitic worm.

As used herein, the term "hepatitis-specific antigen" refers to an antigen isolated and purified from a novel hepatitis-specific monoclonal antibody developed by the inventor. The hepatobacilli specific monoclonal antibody is preferably a monoclonal antibody Cs1 specific for a hepatomegaly antigen, produced by fusion cells of Accession No. KCTC 11182BP.

As a specific example, the hepatitis specific antigen was prepared by the following method. The present inventors prepared hepatitis antigens from hepatitis filaments isolated from the host and reacted with the sera of a number of hepatomegaly (Gandistoma) patients to find hepatitis antigens that react strongly with hepatitis specific antibodies (FIG. 1). Hepatitis F. antigens that react strongly with hepatosiosis specific antibodies were extracted and injected into mice using immunogens to produce fusion cells that secrete one type of monoclonal antibody that specifically reacts with hepatotoxic antigens. Monoclonal antibodies produced in the fusion cells were purified by affinity chromatography using protein G-Sepharose filler. The purified monoclonal antibody was reacted with CNBr-activated Sepharose to prepare a monoclonal antibody-Sepharose filler. It was packed into a column and used to purify hepatitis specific antigen.

Purification of hepatitis-specific antigens was performed by first pulverizing tissues by adding phosphate buffered saline (PBS) and then immunoaffinity chromatography using a monoclonal antibody-Sepharose-filled column. Hepatitis B specific antigens were purified and used in the diagnostic kit of the present invention.

Thus, in one aspect, the present invention provides a method of making a diagnostic kit using the hepatobacterium specific antigen.

In a preferred embodiment, the method for preparing the hepatodiaphragm diagnostic kit comprises the steps of: (a) purifying the antigen from the hepatic decay; (b) preparing a fusion cell producing a monoclonal antibody against the antigen of step (a) and producing a monoclonal antibody therefrom; (c) selecting monoclonal antibodies highly reactive to hepatotoxic antigens in the monoclonal antibody of step (b); (d) separating and purifying the specific antigen for the monoclonal antibody of step (c); And (e) preparing a diagnostic kit using the specific antigen of step (d).

Specifically, highly reactive hepatomegaly members are selected from the sera of patients with hepatic nematodes and immunized mice using them. Then, using the splenocytes and myeloma cells of the immunized mice to produce a fusion cell line from which monoclonal antibodies are produced. The monoclonal antibodies thus produced are purified, and among them, those having high reactivity to hepatotoxic antigens are selected and used for the production of kit raw materials to produce a hepatotoxicosis diagnostic kit.

Assay systems for use in the kits of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography and radiation split immunoassay devices, flow-through devices, and the like. . Preferably, a diagnostic kit in the form of a strip or a device using immunochromatography may be used.

The immunochromatographic assay (ICA) is also called a rapid test because of its speed and simplicity. The principle is that hepatomegaly antibodies in the serum of a sample are bound to colloidal gold particles. It reacts with the tracer antigen and then develops by binding to protein A, which is fixed on the inner surface of the micropore, during the migration through the micropore of the nitrocellulose membrane by capillary action. By forming a band, positive and negative are visually discriminated. Immunochromatography diagnostic methods are now widely used for the testing of hormones, antigens, antibodies, pharmaceutical components, etc. because of the simple procedure and rapid results.

In the diagnostic kits of the present invention, the antigen-antibody complex is detected by chromosomal particle binding, which may include, for example, colloidal gold particles or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. ). In one preferred embodiment, the present invention uses gold colloids as color body particles.

In a preferred embodiment, the diagnostic kit of the present invention comprises protein G or anti-human immunoglobulin in addition to protein A as a capture agent that binds to the antigen-antibody complex. can do. In a preferred embodiment, the present invention uses Protein A as a capture.

In a specific embodiment, the liver dystomoma diagnostic kit using the immunochromatography method of the present invention is a glass fiber in which a nitrocellulose membrane and an antigen-gold condensate in which two invisible lines are drawn on the surface are stored in a dry state. It consists of two main parts of the pad. Two different antibodies are immobilized on an invisible line on the nitrocellulose membrane. Specifically, the test line (T) at the lower side is Protein A (Protein A), and the control line (C) at the upper side is streptoavidin (Streptoavidin) is fixed. The fiberglass pad contains the antigen-gold condensate, which conjugates hepatitis B-specific antigen and Biotin-BSA to the gold particles, and the biotin-bovine albumin-gold condensate is absorbed by the fiberglass pad and dried. It is. When the sample is added to the sample drop of the kit, the antigen-gold condensate stored in the glass fiber pad is hydrated and binds to the antibody in the sample and simultaneously moves through the micropores in the nitrocellulose membrane by capillary action. do. Subsequently, protein A (Protein A), which is immobilized on the invisible line, reacts with the antibody bound to the antigen-gold condensate, resulting in a visible red line due to the red color of the antigen-gold condensate. In addition, streptoavidin, which is immobilized on the upper line, can react with biotin-serum albumin-gold condensate even without antibodies to hepatotoxicity, so it must be present in each test, which confirms whether the test was performed properly. Can be. In other words, if there is a hepatitis antibody, the test line and the control line of the kit appear at the same time, and if there is no hepatitis antibody, the test line does not appear and only the control line appears.

In another aspect, the present invention provides a method of diagnosing liver dystomosis using the diagnostic kit.

In the method for diagnosing Gandistoma of the present invention, the collected blood (serum or plasma) is allowed to be absorbed by contacting the sample dropping unit of the above Diagnostic Kit, and then left. Determine if positive.

As used herein, the term “biological sample” refers to serum, plasma, and the like of a mammal including a human being suspected or tested for hepatomegaly, and may be referred to herein as a “sample” or “sample” for convenience. The biological sample may be used diluted or undiluted before contacting the sample dropping portion of the diagnostic kit of the present invention.

In the case where the biological sample has a hepatitis antibody, as the hepatitis specific antigen in the diagnostic kit of the present invention binds to the antibody and passes the test line, the complex in which the hepatitis specific antigen and the antibody in the biological sample are bound to the capture body of the test line The color change occurs at the inspection line above the membrane for visual identification as it engages. However, if there is no hepatotic antibody in the biological sample, it will not bind with the hepatotoxic antigen of the present invention and no change will occur in the test line on the membrane. This makes it possible to easily and quickly diagnose hepatic respiratory tract by using the diagnostic kit of the present invention.

As another aspect, the present invention relates to a composition for diagnosing hepatomegaly comprising a hepatitis specific antigen isolated from a monoclonal antibody specific for hepatitis.

The hepatobacterial specific antigen is an antigen isolated and purified from a monoclonal antibody specific for hepatotoxicity, and the monoclonal antibody is specifically a monoclonal antibody Cs1 produced by a fusion cell of Accession No. KCTC 11182BP.

Hepatitis specific antigens contained in the composition of the present invention are antigens isolated and purified from monoclonal antibodies selected from fusion cells secreting anti-hepatitis specific monoclonal antibodies that specifically react to the hepatitis antigens prepared by the present inventors. The composition for diagnosing hepatic pneumonia of the present invention is significantly higher in sensitivity and specificity for hepatic pneumonia than other conventional compositions (see Example 7).

In another aspect, the present invention provides novel monoclonal antibodies specific for hepatotoxic antigens used in the production of the raw material of the diagnostic kit.

As used herein, the term 'monoclonal antibody' refers to a highly specific antibody directed against a single antigenic site as it is known in the art. Typically, unlike polyclonal antibodies that include different antibodies directed against different epitopes, monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding and also have another advantage of not being contaminated by other immunoglobulins because they are synthesized by fusion culture. Such monoclonal antibodies are known in the art by hybridoma methods (see Kohler and Mislstein (1976) European Journal of Immunology 6: 511-519) or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991, etc.).

In a specific embodiment, the monoclonal antibody of the present invention immunizes a mouse with an immunogen of a member of a hepatotoxic insect, which strongly reacts with an antibody in the blood of a liver dystomosis (or hepatomegaly) patient, and then splenocytes thereof into myeloma cells (myeloma). ) Is an antibody produced by fusion cells (hybridoma) to generate fusion cells, and select fusion cells that specifically bind to hepatotoxic antigens.

Specifically, as illustrated in the flowchart of FIG. 2, proteins were isolated through electrophoresis and immunoblot was performed by reacting with hepatostomosis-positive blood to identify antigens that are highly reactive with hepatotoxicosis antibodies. These were extracted from gels and used as immunogens.

Using the prepared antigen as an immunogen, the mice were injected and selectively fused to one type of fusion cells secreting anti-hypertoxin monoclonal antibodies that specifically react with hepatotoxic antigen. More specifically, in order to select fusion cells that secrete anti-hepatitis-specific monoclonal antibodies that specifically respond to hepatitis antigens, ELISA plates prepared by binding the hepatitis antigens extracted by electrophoresis were prepared. The reactivity of was screened. Plates bound to hepatobacterium antigens were selected for fusion cells secreting monoclonal antibodies to react, and the monoclonal antibodies secreted therefrom were named Cs1 and used in the preparation of the diagnostic kit of the present invention. Each of the selected fusion cell lines was injected into the mouse abdominal cavity, and after a certain period, ascites was collected and monoclonal antibodies were isolated therefrom.

Fusion cells producing the monoclonal antibody Cs1 that selectively recognizes the hepatotoxic antigen of the present invention were deposited on August 30, 2007 to KCTC (Korean Collection for Type Cultures), an international depository institution located at 52, Eeun-dong, Yuseong-gu, Daejeon. Was deposited.

In another embodiment, the present invention provides a fusion cell of Accession No. KCTC 11182BP, which produces the monoclonal antibody Cs1.

The present invention purifies the hepatitis specific antigen from hepatitis and clones a fusion cell line obtained by fusion of splenocytes and myeloma cells of immunized mice using the same to produce monoclonal antibodies and to purify them to select highly reactive monoclonal antibodies. By using the immunochromatography method can provide a quick diagnosis kit of hepatitis, which is designed to include hepatitis specific antigen obtained by purifying the hepatitis, in particular, the antigen, using the hepatitis diagnosis kit is more specificity and sensitivity than the existing diagnostic kit, It is very easy to use, one-step, and can provide a diagnostic method that can be quickly point-of-care testing (POCT) without the need for expensive equipment.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  1: reactive with hepatotoxicosis specific antibodies Hepatitis  Antigen purification

1) Identification of Hepatotoxic Antigens Reactive with Hepatosiosis Specific Antibodies

This test was performed using hepatotoxicosis antibody positive sera provided in the Department of Parasitology, Wonkwang University School of Medicine (Iksan, Korea). The blood used in this study has been prepared over the years as blood identified as a patient with hepatic pneumonia through an egg test and an intradermal reaction test. These blood were used for immunoblot.

Hepatitis was obtained from patients and patients from the Department of Parasitology, Wonkwang University School of Medicine (Iksan, Korea) and the Department of Parasitology, Hanyang University School of Medicine (Seoul, Korea). After washing the liver worms three times with physiological saline, pulverizing with a tissue grinder (tissue grinder; PolyTron, Kinematica AG, Switzerland) for 5 minutes using 5 ml of physiological saline per 10 mg of carcass, and 20 minutes at 10,000 rpm. The supernatant was taken by centrifugation. The supernatant was placed in a 10% acrylamide gel and subjected to electrophoresis. Protein antigens in the electrophoretic gels were transferred to nitrocellulose membranes, and then reacted with the blood of patients with hepatic pneumonia at 37 ° C. for 1 hour. It was washed with physiological saline containing 0.1% Tween 20 and then reacted with an anti-human IgG-HRP condensate. Thereafter, color development was carried out using a membrane for membrane (Membrane 1 component TMB, BioFX, USA) (Fig. 1).

2) Purification of Hepatitis B Specific Antigen

Purification of hepatitis B antigens isolated by electrophoresis was performed using electro-elution. SDS-PAGE was performed as shown in FIG. 1A, and then antigens having a molecular weight of 7 kDa, which are strongly reacted with hepatic pneumonia, were identified. Thereafter, as shown in FIG. 1B, a large amount of hepatitis antigens were subjected to electrophoresis under the same conditions to cut and collect a gel having a molecular weight of 7 kDa, followed by an electroelute (ElectroEluter Model 422; Bio-Rad, USA). Was electro-elution using. The extracted hepatitis antigen solution was dialyzed with physiological saline containing 0.1% SDS and 0.1% Tween 20.

Example  2 : Hepatitis  Against antigen Monoclonal  Antibody Construction

1) Immunization of Antigens

Six to eight weeks old female mice (BALB / C, Daehan Biolink Co., Ltd.) were prepared by making a suspension containing the same volume of complete Freund's adjuvant, each containing 100 µg of hepatitis antigen. Intraperitoneal). Two weeks later, incomplete Freund's adjuvant was used to inject once more with the same technique, and again two weeks later with the same technique. Two days after the last immunization, blood was collected from the tail, serum was obtained, diluted in phosphate buffer (1/1000), and the titer of the antibody was determined by enzyme linked immunosorbant assay (ELISA). If titers were low, they were immunized again after 2 weeks.

2) Fusion of Splenocytes and Myeloma Cells

After the spleen was removed from the immunized mice, the spleen was crushed by a tissue grinder, and the cell suspension was placed in a container and centrifuged to recover the cells. Myeloma cells were recovered from the cell culture flasks and suspended in RPMI1640 to calculate the cell number. Splenocytes were also counted. 1 x 10 ⁷ myeloma cells and 1 x 10 ⁸ splenocytes were transferred to a 50 ml container, and then, after adding an appropriate amount of RPMI1640, the cells were recovered by centrifugation at 200 x g for 5 minutes. The recovered cells were suspended and slowly shaken, and 1 ml of PEG (50% polyethylene glycol) solution was added for 1 minute. After 5 minutes 1 ml of RPMI1640 was added over 30 seconds and again 3 ml of RPMI1640 were added over 30 seconds. Again, 17 ml of RPMI1640 was added over 1 minute, and then 20 ml of RPMI1640 was added and reacted for 5 minutes. After centrifugation at 200 x g for 5 minutes, the medium was removed. After careful suspension of RPMI1640 containing 50 ml of 1% HAT (hypoxathine-aminopterin-thymidine), 100 μl / well of the fused cells were placed in a 96-well cell culture vessel containing feeder cells. After 37 ° C., 5% CO ₂ Cells were cultured in an incubator.

3) Antibody production using cloning and ascites of fusion cell line

After about 10 days, when it is confirmed that the fusion cells grow and form colonies, the medium is taken from a 96-well cell culture container, and the wells which produce antibodies against hepatitis F antigen purified by Eliza method are identified, and the cells of the wells are recovered. The cells were transferred to a 24-well cell culture vessel and cultured, and then cloning was continued until a stable monoclonal cell line was obtained. After cloning was completed, transferred to a T flask (T flask), incubated in large quantities, frozen and stored in liquid nitrogen.

0.5 ml of incomplete Freund's adjuvant was injected into the abdominal cavity of 6-8 week old rats (BALB / C). On day 1, fused cells were suspended and counted in phosphate buffer, and 1.5 × 10 ⁶ cells per mouse were suspended in 0.5 ml phosphate buffer and injected intraperitoneally. After 1-2 weeks, ascites were properly charged, the asctic fluid was collected using a syringe and then stored frozen.

4) Purification of Monoclonal Antibodies

Ammonium sulfate was added to the collected ascites at a concentration of 10% and mixed for 30 minutes. And the supernatant was taken after centrifugation for 30 minutes at 15,000rpm. Again, ammonium sulfate was added to the supernatant at a concentration of 50%, and left at 4 ° C for 30 minutes. After centrifugation at 15,000 rpm for 30 minutes, the supernatant was discarded and the precipitate was suspended in 20 mM phosphate buffer (pH 7.0). This suspension was dialysis in 20 mM phosphate buffer for at least 18 hours. After dialysis, the dialysis solution was injected into a Protein G-coupled column equilibrated with 20 mM phosphate buffer solution (pH 7.0), and once all were injected, the non-adsorbed substances were removed using the phosphate buffer solution. The antibody bound to the column was eluted with a 100 mM glycine solution (pH 2.8) solution. At this time, 1M Tris (pH 9.0) solution was added to 1/10 volume of the eluate to correct the pH. The eluted antibody solution was concentrated and dialyzed in 150mM phosphate buffer solution, and after dialysis, the amount of antibody was confirmed by Bradford assay and stored frozen until use.

The production process of the monoclonal antibody according to the present invention is shown in FIG.

5) Selection of monoclonal antibody and calculation of dissociation constant (K _d )

Purified monoclonal antibodies were measured using the Eliza method to select antibodies highly reactive with hepatotoxic antigens. Hepatitis B antigens extracted from the gels were coated on a 96 well ELISA plate at a concentration of 2 μg / ml. Each purified monoclonal antibody was diluted in stages to react with the hepatitis antigen-coated plate, and after the reaction, the unbound antibody was removed by washing. Goat anti-mouse immunoglobulin G (IgG) coupled with horseradish peroxydase was reacted, and unreacted peroxidase-IgG was removed by washing. TMB (tetramethyl benzidine), which is a substrate of peroxidase, was added and developed, and the absorbance (A ₄₅₀ ) was measured according to the degree of reaction. The results were shown in FIG. Among the antibodies against hepatitis F antigen, Cs1, which has good reactivity, was used to produce the raw material for kit preparation of the present invention.

In addition, Klotz plot was performed through indirect competitive ELISA (Indirect competitive ELISA) to measure the dissociation constant for the antibody to be used in the production of the kit used in the present invention, and as a result, the monoclonal antibody of the present invention Dissociation constants were the same as in FIG. 3B.

6) Purification of Hepatitis B Specific Antigen Using Immunochromatography

Monoclonal antibody Cs1 was covalently bound to CNBr-activated Sepharose CL-6B (GE Healthcare, USA), respectively. Thereafter, they were packed into columns and used for antigen preparation. Hepatobacterium from Chosun University College of Medicine and Hanyang University College of Medicine was pulverized for 5 minutes using tissue grinder (PolyTron, Kinematica AG, Switzerland) with 5 ml of physiological saline per 10 mg of carcass, and 20 minutes at 10,000 rpm. The supernatant was taken by centrifugation. This supernatant was subjected to immunoaffinity chromatography using a column prepared in advance. The protein was eluted with 100 mM glycine (glycine, pH 2.8), and immediately eluted with 1M Tris (pH 8.0) and dialyzed with physiological saline.

The purification process using the immunoaffinity chromatography of the hepatobacilli specific antigen according to the present invention is shown in FIG. 4a, and the results of the separation and purification of the hepatobacilli specific antigen are shown in FIG. 4b.

Example  3: Hepatitis  Rapid with antigen Immunochromatography  Strip Shape Diagnosis Of kit  Produce

1) Immobilization of Antibodies

As shown in FIG. 5A, a specific position of the nitrocellulose membrane attached to the plastic card, that is, the protein A (test line) T, and the control line (C) position streptoavidin Each was dispensed and dried in a 30 ° C. incubator for 2 days to allow the monoclonal antibody to completely passivate onto the nitrocellulose membrane.

2) Preparation of Gold Condensate and Preparation of Gold Condensation Pads

Hepatitis antigens purified by immuno-affinity chromatography using monoclonal antibody Cs1 were mixed with about 40 nm colloidal gold particles and reacted for 1 hour in a 37 ℃ thermostatic water bath. And colloidal gold particles were bonded to each other. After 1 hour, bovine serum albumin (BSA) and sucrose were added at 3% and 1%, respectively, and then reacted for 1 hour to react with the sites of colloidal gold particles that the antibody did not bind. It was forever. Thereafter, centrifugation was performed at 10,000 rpm for 20 minutes to recover the condensed hepatitis antigen-conjugated gold condensate. The absorbance was measured at 540 nm and then refrigerated. The prepared gold condensate was injected into a microwell plate made of plastic, and then dried to prepare a gold conjugate well.

3) Assembly of strip kit

As illustrated in FIG. 5A, a strip-shaped kit is completed by attaching an absorbent pad to absorb a sample and a buffer to an upper end of a plastic card to which a protein A (Protein A) -bound nitrocellulose membrane is attached. It was.

Example  4 : Hepatitis  Rapid with antigen Immunochromatography device  Form diagnosis Of kit  Produce

1) Immobilization of Antibodies

As shown in FIG. 5A, a specific position of the nitrocellulose membrane attached to the plastic card, that is, the protein A (test line) T, and the control line (C) position streptoavidin Each was dispensed and dried in a 30 ° C. incubator for 2 days to allow the monoclonal antibody to completely passivate onto the nitrocellulose membrane.

2) Preparation of Gold Condensate and Preparation of Gold Condensation Pads

Hepatitis B antigen and biotin-bovine serum albumin, which were purified by immuno-affinity chromatography using monoclonal antibody Cs1, were mixed with colloidal gold particles of about 40nm in size, and then The reaction was carried out for 1 hour to allow the hepatotoxic antigen and the colloidal gold particles to bind to each other. After 1 hour, bovine serum albumin (BSA) and sucrose were added at 3% and 1%, respectively, and then reacted for 1 hour to react with the sites of colloidal gold particles that the antibody did not bind. It was forever. Thereafter, centrifugation was performed at 10,000 rpm for 20 minutes to recover the condensed hepatitis antigen-conjugated gold condensate. The absorbance was measured at 540 nm and then refrigerated. The prepared gold condensate was absorbed into a pad made of glass fiber, and then dried to prepare a gold condensation pad.

3) Assembly of Device Type Kit

As illustrated in FIG. 6A, an absorbent pad capable of absorbing a sample and a buffer is attached to an upper end of a plastic card to which a protein A (Protein A) -bound nitrocellulose membrane is attached. A dropping sample pad and a condensation pad were attached. In the case of the device form, the assembled semifinished product was placed in a plastic housing as indicated in FIG. 6A to complete the kit.

Example  5: strip form Of kit  method of inspection

One drop of wash solution was added to the gold condensation well provided with the kit. Thereafter, 3 microliters of serum specimens were added using a pipette and mixed. After that, the strip of the kit was plugged in so that the sample liquid was absorbed and left for 10 minutes. 3 drops of the washing solution was put in the washing well, and then the strip was inserted into the kit in which the sample solution was absorbed and left for 15 minutes to perform the washing reaction. After completion of the reaction, the strips were read for Gandistoma antibody according to the position of the red band on the test line as indicated in FIG. 5B.

Example  6: device  shape Of kit  method of inspection

The device kit contained in the aluminum pouch was taken out and placed on a flat floor. Three microliters of serum samples were taken and placed in a sample window as indicated in FIG. 6A to allow the sample to be absorbed. Thereafter, three drops of the assay buffer were dropped and left for 15 minutes. After completion of the reaction, the kit was read for the presence of Gandistoma antibody according to the position of the red band on the test line as indicated in FIG. 6B.

Example  7: Hepatitis  Rapid for Antibody Detection Immunochromatography  Strip form and device  shape Of kit  Efficacy test

1) Positive Sensitivity Efficacy Test

Using the diagnostic kit prepared in Examples 3 to 4, the sensitivity performance of the kit was tested for Gandistoma positive 100 samples. Clinical trials to determine the sensitivity of this diagnostic kit were conducted in the Department of Parasitology, Wonkwang University School of Medicine (Iksan, Korea). In addition, for the control test, the hepatic pneumonia-positive specimens were subjected to a confirmatory test using the Gendia hepatopulmonary dystomosis antibody ELISA (ELISA, Green Cross, Korea) kit, and then compared with the diagnostic kit achieved in the present invention. The results were as shown in Table 1.

Test results using positive 100 samples for sensitivity test Total Results (Total 100 Samples) ELISA results positivity voice Nanosine Gandistoma Antibodies positivity 93 0 voice 7 0 system 100 0 Sensitivity ¹ 93.0% NOTE ¹ Sensitivity = [(Number of Positive Reaction Kits) / (Total Positive Samples)] × 100 (%), (93/100) × 100 = 93.0%

2) negative specificity efficacy test

Specificity performance of the kit for the Gandistoma negative 100 specimens was tested using the diagnostic kits prepared in Examples 3-4 above. Clinical trials to confirm the specificity of this diagnostic kit were performed in the Department of Parasitology, Wonkwang University School of Medicine (Iksan, Korea). In addition, for the control test, the hepatic pneumonia-positive specimens were subjected to a confirmatory test using the Gendia hepatopulmonary dystomosis antibody ELISA (ELISA, Green Cross, Korea) kit, and then compared with the diagnostic kit achieved in the present invention. The results were as shown in Table 2.

Test results using negative 100 specimens for specificity testing Total Results (Total 100 Samples) ELISA results positivity voice Nanosine Gandistoma Antibodies positivity 0 8 voice 0 92 system 0 100 Specificity ¹ 92.0% NOTE ¹ Specificity = [(Number of Negative Reaction Kits) / (Total Negative Samples)] × 100 (%), (92/100) × 100 = 92.0% in the above case when measuring negative samples.

3) Conclusion through sensitivity and specificity test results

As described above, the diagnostic kit of the present invention was very easy to use compared to the existing diagnostic kit, despite the rapid examination without the help of expensive equipment, it was confirmed that the sensitivity and specificity is high and clinically effective.

The present invention purifies the hepatitis specific antigen from hepatitis and clones a fusion cell line obtained by fusion of splenocytes and myeloma cells of immunized mice using the same to produce monoclonal antibodies and to purify them to select highly reactive monoclonal antibodies. By using the immunochromatography method can provide a quick diagnosis kit of hepatitis, which is designed to include hepatitis specific antigen obtained by purifying the hepatitis, in particular, the antigen, using the hepatitis diagnosis kit is more specificity and sensitivity than the existing diagnostic kit, It is a very useful invention for the medical diagnosis industry because it is very easy to use in one step and can provide a diagnostic method that can be quickly point-of-care testing (POCT) without the help of expensive equipment. .

FIG. 1A is a diagram showing immunoblot results showing the reactivity of antigens of hepatitis isolated from hepatitis with hepatosipatitis antibodies in serum of patients (10).

Figure 1b is a diagram showing the results of electrophoresis of hepatitis antigens isolated from hepatitis.

Figure 2 is a flow chart illustrating a method for producing a monoclonal antibody for the isolation and purification of hepatomegaly antigens that specifically react to the hepatomegaly antibodies according to the present invention.

Figure 3a is a graph showing the results of measuring the reactivity of the monoclonal antibody Cs1 for hepatitis B antigen separation and purification according to the present invention.

3B is a graph of dissociation constant (K _d ) of monoclonal antibody Cs1 according to the present invention.

Figure 4a is a flow chart illustrating a process for separating and purifying hepatomegaly antibody specific antigen from hepatitis using a monoclonal antibody according to the present invention.

Figure 4b is a graph showing the results of the separation and purification of hepatitis B specific antigen according to the present invention.

Figure 5a is a diagram showing the configuration of a rapid immunochromatography strip form for the diagnosis of hepatotoxicosis antibody according to the present invention, Figure 5b is a test result of the form of a rapid immunochromatography strip diagnostic hepatitis patient according to the present invention It is a figure which shows a photograph.

Figure 6a is a diagram showing the configuration of the form of hepatotoxicity antibody diagnostic rapid immunochromatography device according to the present invention, Figure 6b is a photograph showing the test results of the form of hepatotoxicity antibody diagnostic rapid immunochromatography device according to the present invention Drawing.

Claims (15)

A hepatomegaly diagnosis kit comprising a hepatomegaly specific antigen isolated and purified from a monoclonal antibody specific for an antigen of a hepatomegaly. The diagnostic kit of claim 1, wherein the monoclonal antibody specific for the antigen of the hepatitis is a monoclonal antibody Cs1. The diagnostic kit according to claim 1, wherein the diagnostic kit is in strip form using immunochromatography. The diagnostic kit of claim 1, wherein the antigen-antibody complex is detected by chromosomal particle binding. The diagnostic kit according to claim 4, wherein an antigen specific for a hepatomegaly antibody is used as an antigen binding to the chromosomal particle. The diagnostic kit according to claim 5, wherein the antigen specific for the hepatomegaly antibody is an antigen purified through immunoaffinity chromatography using monoclonal antibody Cs1. The diagnostic kit according to claim 2 or 6, wherein the monoclonal antibody Cs1 is produced from a fusion cell line of Accession No. KCTC 11182BP. (a) purifying the antigen in the hepatic decay; (b) preparing a fusion cell producing a monoclonal antibody against the antigen of step (a) and producing a monoclonal antibody therefrom; (c) selecting monoclonal antibodies highly reactive to hepatotoxic antigens in the monoclonal antibody of step (b); (d) separating and purifying the specific antigen for the monoclonal antibody selected in step (c); And (e) comprising the step of preparing a diagnostic kit using the specific antigen of step (d), a method for producing a hepatosiosis diagnosis kit. The method of claim 8, wherein the fusion cell of step (b) is characterized in that the fusion cell of Accession No. KCTC 11182BP. The method of claim 8, wherein the monoclonal antibody selected in step (c) is monoclonal antibody Cs1. A composition for diagnosing hepatomegaly, comprising a hepatobacterium specific antigen isolated and purified from a monoclonal antibody specific for an antigen of a hepatomegaly. 12. The method of claim 11, wherein the monoclonal antibody specific for the antigen of the hepatic insect repellent is characterized in that the monoclonal antibody Cs1 diagnostic composition. The method of claim 12, wherein the monoclonal antibody Cs1 is a composition for diagnosing hepatomegaly, characterized in that produced from the fusion cell line of Accession No. KCTC 11182BP. Antibody Cs1 produced from a fusion cell line of Accession No. KCTC 11182BP. Fusion cell line of Accession No. KCTC 11182BP.

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