KR100957675B1 - The rapid diagnostic kits for the detection of antibodies against Paragonimus westermani and monoclonal antibodies for production of Paragonimus westermani-specific antigens - Google Patents

Abstract

The present invention relates to a diagnostic kit comprising a pneumococcal specific antigen isolated and purified from a pneumococcal specific monoclonal antibody, and a method for diagnosing pneumonia by using the same. The diagnostic kit of the present invention is a quick, easy diagnostic kit that provides higher specificity and sensitivity than conventionally developed diagnostic kits. The present invention also relates to a composition for diagnosing pneumoconiosis, a monoclonal antibody, and a fusion cell producing the same, comprising a pneumococcal specific antigen used in the kit.

Pulmonary Insufficiency, Monoclonal Antibody, Fusion Cell, Pulmonary Insufficiency Diagnosis Kit

Description

The rapid diagnostic kits for the detection of antibodies against Paragonimus westermani and monoclonal antibodies for production of Paragonimus westermani-specific antigens}

The present invention relates to a diagnostic kit comprising a pneumococcal specific antigen isolated and purified from a pneumococcal specific monoclonal antibody, and a method for diagnosing pneumonia by using the same. The present invention also relates to a composition for diagnosing pneumoconiosis, a monoclonal antibody, and a fusion cell producing the same, comprising a pneumococcal specific antigen used in the kit.

According to the data conducted by the Ministry of Health and Welfare and the Korea Health Care Management Association every five years, the retention rate of roundworm eggs was 54.9% in 1971, but decreased to 0.06% in 1997, and in Seoul Asan Hospital for five years from 1997 to 2000. Of the 250,000 tests conducted, only 30 (0.012%) of the roundworm eggs were prone, and recent infection of the rotifers has almost disappeared. In contrast, insects such as pararagonimus westermani and colonornochis sinensis, and insects such as Taenia solium and Spargnum were the main causes of parasite infection. Diagnosis and treatment are more important because these insects and insects cause clinically serious infections and side effects.

Pulmonary dysplasia, also known as lung dystomosis, is a disease caused by ingestion of freshwater lobsters containing parasitic insects called paragonimus westermanis, day or night. It is distributed in the back, but unlike the soil-borne parasites such as roundworm, duodenal insects, worms, etc., despite a lot of efforts is a dull parasitic insects.

Pneumococcal infestation occurs after 15-20 days of growth in an outdoor insect worm (miracidum) breaks off the eggshell and parasitizes in a mollusk such as the first host, Daschalgi, converts to cercaria. Invade crustaceans, such as crayfish, to be converted into metacercaria, whereby humans and animals eat these infected crustaceans, such as freshwater dogs and lobsters, by invading the lungs through the intestinal mucosa through the intestinal mucosa ( Human Parasitology, So Jin-Tak 著, 1987, Shinkwang Publishing Co.).

Pulmonary pneumoconiosis symptoms include fever, shortness of breath, and pain, such as general pneumonia and pleurisy, and sputum of the same color as fishy fishy stalk. When a pulmonary vermin is injured in a pulmonary vessel, blood is mixed with phlegm. On the other hand, when the lungs invade the brain, epileptic symptoms such as vision impairment or general paralysis appear.

Therefore, in order to treat the above-mentioned symptoms, Emethine hydrochloride (trade name; Prazacantel), which is also used as a medicine for hepatic insufficiency, is administered, but this is a treatment selected without confirming the cause of the disease. Risk of lung disease and the presence of treatment may occur. Therefore, it is necessary to first diagnose an infection caused by pulmonary pneumoniae prior to treatment.

As a test method that has been used conventionally, there is a method of finding characteristic eggs of pulmonary pneumoniae in sputum or stool of a patient. In addition, there is an intradermal reaction test that was developed in the 1950s and has been widely used to date. However, detecting eggs from sputum or feces was inconvenient to collect samples and test methods, and the recovery rate of eggs was only 10-30%. In addition, the intradermal test also showed positive response in the past infected persons not infected with pulmonary repellent (Chung et al., 1995; Sandun et al., 1959; Walton and Ju, 1959; Kim Dong-chan et al., 1969; Hunter (Hunter et al. (1958) reported that 44.7% of the fact that also appear to be positive for patients with hepatitis, this method also has a problem of poor sensitivity and specificity.

Therefore, studies have been attempted to increase the sensitivity and specificity of intradermal reactions using purified pneumococcal antigens (Dong-Ik Choi, 1959; Sawada et al, 1964; Ahn, et al., 1975). The whole crude carcass extract extracted is prepared as antigen, and is a serological method using these antigens (ie, enzyme immunoassay, ELISA). However, the insecticide antigen has a problem of causing a nonspecific reaction with other parasites or a nonspecific reaction, and enzyme immunoassay has a disadvantage of having to perform at least 3 hours using highly skilled techniques, personnel, and equipment.

 Therefore, the present inventors prepared two types of monoclonal antibodies specific for pulmonary pneumococcal, and combined the monoclonal antibody and the crude extract with immuno-affinity chromatography to isolate and purify the pneumococcal specific antigen. By making and using a pneumoconiosis diagnostic kit containing these isolated and purified antigens, it was possible to reduce non-specific reactions with other parasites and to efficiently detect pneumonia in the field in a short time without high manpower and expensive equipment. It was confirmed that the diagnosis can be completed the present invention.

One object of the present invention is to provide a pneumococcal diagnosis kit comprising a pneumococcal specific antigen isolated and purified from a pneumococcal specific monoclonal antibody.

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

It is another object of the present invention to provide a pneumococcal specific monoclonal antibody used in the production of a pneumococcal specific antigen used in the diagnostic kit and a fusion cell for producing the same.

In one embodiment, the present invention relates to a pneumococcal diagnostic kit comprising a pneumococcal specific antigen purified from a pneumococcal specific monoclonal antibody.

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 confirm the development of pulmonary pneumoconiosis.

As used herein, the term "pneumonia" is also called pulmonary dystomoma, and refers to a disease or condition caused by infection of parasitic insects of the paragonimus westermani.

As used herein, “lungworm specific antigen” refers to an antigen isolated and purified from a novel pulmonary insect specific monoclonal antibody developed by the present inventors. The pneumococcal specific monoclonal antibodies are preferably monoclonal antibodies Pw1 and Pw2 produced by fusion cells (hybridoma) of accession number KCTC 11180BP and fusion cells (hybridoma) of accession number KCTC 11181BP, respectively.

As a specific example, the pneumococcal specific antigen was prepared by the following method. Pneumococcal antigens were prepared from pneumococcal worms isolated from the host and reacted with the sera of a number of pneumococcal patients to find pneumococcal antigens that react strongly with pneumococcal specific antibodies (FIG. 1). The antigens were extracted and injected into mice using immunogens to produce fusion cells that secrete two types of monoclonal antibodies that specifically react with the pneumococcal antigen. Monoclonal antibodies produced in each of these two fusion cells were purified by affinity chromatography using protein G-Sepharose filler. Purified monoclonal antibodies were reacted with CNBr-activated Sepharose to prepare monoclonal antibody-Sepharose fillers. It was packed into a column and used to purify pneumococcal specific antigens.

In order to purify the pneumonia-specific antigen, the tissues were first pulverized by adding phosphate buffered saline (PBS), and then immunoaffinity chromatography was performed using a column filled with monoclonal antibody-Sepharose. Lungworm specific antigens were purified and used in the diagnostic kit of the present invention.

Accordingly, in one specific embodiment, the present invention provides a method for preparing a diagnostic kit using the pneumococcal specific antigen. Specifically, first, highly reactive pulmonary insecticides were selected from the sera of pulmonary pneumoconiosis patients through immunoblot, and immunized mice and monoclonal antibodies were prepared. In addition, fusion cell lines were made using splenocytes and myeloma cells of immunized mice containing the monoclonal antibody, and antibodies were produced therefrom. The antibodies thus produced were purified, and among them, those having high reactivity to the pneumococcal antigen were selected and used for the production of kits.

Assay systems for use in the diagnostic 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. do. Preferably, a diagnostic kit in the form of a strip or a device using immunochromatography may be used. Diagnosis using an immunochromatographic assay (ICA) is also called a rapid test because of its speed and simplicity, in which pneumococcal antibodies in the serum of a sample are applied to colloidal gold particles. It reacts with the bound tracer antigen and then binds to a capture that is fixed to the inner surface of the micropore while traveling through the micropore of the nitrocellulose membrane by capillary action. It is a method for visually discriminating positive and negative by forming a color band.

In the diagnostic kit of the present invention, the antigen-antibody complex is detected by color particle binding, and the color particles include, for example, colloidal gold particles or color glass or plastic (e.g., polystyrene, polypropylene, latex, etc.). bead). In the present invention, gold colloids are preferably used.

In addition, in the diagnostic kit of the present invention, the capture agent that binds to the antigen-antibody complex includes, in addition to protein A, protein G or anti-human immunoglobulin. . In a specific embodiment of the present invention, Protein A was used as the capture body.

As a specific embodiment, the diagnostic kit for lung pneumoconiosis using the immunochromatography method of the present invention is a glass fiber pad 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. Two different antibodies are immobilized on an invisible line on the nitrocellulose membrane. As shown in Figures 9 and 10, the lower test line (T) is fixed to Protein A (Protein A), the upper control line (Control line, C) is fixed to Streptoavidin (Streptoavidin). The glass fiber pad contains the antigen-gold condensate that conjugates pneumococcal-specific antigen and biotin-BSA to gold particles, and the biotin-bovine albumin-gold condensate is absorbed by the glass fiber 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 at the same time through the micropores in the nitrocellulose membrane by capillary action. Move. 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 condensates even without antibodies to pulmonary pneumoniae, so it must be present every test, which confirms whether the test was performed properly. Can be. That is, if there is a pneumococcal antibody, the test line and the control line of the kit appear at the same time, and if there is no pneumococcal antibody, the test line does not appear and only the control line appears.

Sensitivity and specificity of the diagnosis of pneumoconiosis using the diagnostic kit of the present invention is 90% or more as compared to the commercially available diagnostic kit (eg, Gendia hepatic lung disease antibody Eliza kit (manufactured by Green Cross, Korea), etc.) And specificity, and could be diagnosed quickly and simply as compared to the conventional diagnostic kit (see Example 7). Therefore, the diagnostic kit of the present invention will be readily available for diagnosis of pneumoconiosis.

In addition, the present invention is 1) to purify the constituents in the pulmonary insufficiency, 2) to produce a fusion cell to produce a monoclonal antibody to the constituents of the above, 3) the reactivity to the pulmonary worms in the monoclonal antibody produced in the fusion cells A method for producing a pneumococcal diagnostic kit comprising the step of selecting the high antibody and 4) separating and purifying the specific antigen for the antibody, thereby making a diagnosis specific to the pneumococcal antigen and capable of excluding a false positive response. Kits may be provided.

In another aspect, the present invention relates to a method for diagnosing pneumoconiosis using the above diagnostic kit.

Specifically, the biological kit is allowed to be absorbed by contacting the sample dropping unit of the diagnostic kit in the diagnostic kit of the present invention, and then, whether the test kit is positive or not according to the presence or absence of a test line of the diagnostic kit is determined. That's how.

As used herein, the term "biological sample" refers to serum, plasma, and the like of a mammal including a human whose suspicious or suspected pulmonary pneumoconiosis is to be examined, and may also be referred to as "sample" or "sample" in the present invention. The biological sample may be used with or without dilution before contacting the sample drop of the diagnostic kit of the present invention.

If there is a pneumococcal antibody in the biological sample, as the pneumococcal specific antigen in the diagnostic kit of the present invention binds to the antibody and passes through the test line, the complex in which the pneumococcal 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 pneumococcal antibody in the biological sample, it does not bind to the pneumococcal antigen of the present invention and thus no change occurs in the test line on the membrane.

As another aspect, the present invention relates to a composition for diagnosing pulmonary pneumoconiosis comprising a pneumococcal specific antigen isolated and purified from monoclonal antibodies specific for pneumococcal.

The pneumococcal specific antigens are antigens isolated and purified from monoclonal antibodies specific for pneumococcal, and the monoclonal antibodies are specifically monoclonal antibodies Pw1 and Pw2 produced by fusion cells of Accession No. KCTC 11180BP and Fusion Cells of Accession No. KCTC 11181BP, respectively. .

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

In another aspect, the present invention relates to novel monoclonal antibodies specific for pneumococcal worms used for the production of pneumococcal specific antigens of the above diagnostic kits and to fusion cells producing them.

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.).

As one example, the monoclonal antibody immunizes a mouse using a member of a pulmonary worm that reacts strongly with the antibody in the blood of a pulmonary worm, and then splenocytes thereof fused with myeloma. Cells (hybridoma) can be produced and fused cells that specifically bind to pneumococcal antigen can be selected and prepared therefrom. More specifically, as illustrated in FIG. 2, the proteins were isolated by electrophoresis and reacted with various pneumococcal positive blood to perform immunoblot to identify antigens highly reactive with pneumococcal antibodies. . These were extracted from gels and used as immunogens.

Using the prepared antigen as an immunogen, two kinds of fusion cells secreting anti-pulmonary insecticidal monoclonal antibodies that specifically reacted only with the pneumococcal antigen were selectively cloned. Specifically, in order to select fusion cells that secrete anti-lungworm specific monoclonal antibodies that specifically react to the pneumococcal antigen, an ELISA plate combined with the pneumococcal antigen extracted through electrophoresis was prepared to react the monoclonal antibodies. Were screened. In the plate bound to the pneumococcal antigen, fusion cells secreting the monoclonal antibody to respond were selected, and the monoclonal antibodies secreted here were named Pw1 and Pw2 and used for the production of pneumococcal antigen of the diagnostic kit of the present invention. The selected fusion cell lines were then injected into the mouse abdominal cavity, and after a certain period of time, ascites was collected and monoclonal antibodies were isolated therefrom.

Fusion cells producing the monoclonal antibodies Pw1 and Pw2, which selectively recognize the pneumococcal antigen of the present invention, were collected in KCTC (Korean Collection for Type Cultures), 52, Eeun-dong, Yuseong-gu, Daejeon, September 4, 2007 Deposited with Accession No. KCTC 11180BP and KCTC 11181BP, respectively.

The diagnostic kit comprising the pneumococcal specific monoclonal antibody of the present invention and the pneumococcal specific antigen purified through the same is higher in specificity and sensitivity than the conventional diagnostic methods such as the egg test and the intradermal reaction test, and is more one-step than the enzyme immunoassay. This diagnostic kit is very simple and can be quickly point-of-care testing (POCT) without the help of expensive equipment. It overcomes the low sensitivity and specificity that is a problem in the existing diagnostic kits and results from enzyme immunoassay. The high degree of concordance with and can be easily used for the diagnosis of pneumoconiosis, which is more effective in treatment.

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 Antigen Purification of Pulmonary Insufficiency Reactive with Pneumococcal Specific Antibody

1-1. Identification of pneumococcal antigens reactive with pneumococcal specific antibodies

This test was performed using pulmonary pneumoconiosis antibody positive sera provided in the Department of Parasitology, Wonkwang University School of Medicine (Iksan, Korea). The positive serum has been prepared for the last several years as serum extracted from blood identified as a pneumoconiosis patient through an egg test and an intradermal reaction test. These sera were used for immunoblot.

Lung respiratory worms were provided from patients or dogs from the Department of Parasitology, Chosun University, Gwangju, Korea, and the Department of Parasitology, Hanyang University, Seoul, Korea. The lungs were washed three times with physiological saline, and then pulverized for 5 minutes using a tissue grinder (tissue grinder; PolyTron, Kinematica AG, Switzerland) by adding 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 placed in 10% acrylamide gel and subjected to electrophoresis. Protein antigens in the electrophoretic gels were transferred to nitrocellulose membrane and reacted with the blood of pneumoconiosis patients at 37 ° C. for 1 hour. This was washed with physiological saline containing 0.1% Tween 20 and then reacted with an anti-human IgG-HRP condensate. Thereafter, color development was performed using a membrane for membrane (Membrane 1 component TMB, BioFX, USA) (FIG. 1).

1-2. Purification of Pneumococcal Specific Antigen

Purification of pulmonary respiratory tract antigen isolated by electrophoresis was performed using electroelution. After performing SDS-PAGE as in FIG. 1, antigens of 46, 30, and 23 kDa molecular weights, which are strongly reacted with pneumoconiosis antibodies, were identified. Subsequently, as shown in FIG. 2, a large amount of pneumococcal antigens were subjected to electrophoresis under the same conditions to cut and collect gels having molecular weights of 46, 30, and 23 kDa, and then electrolyzed (ElectroEluter Model 422; Bio-Rad). Co., USA) was used for electroelution. The extracted pneumonia antigen solution was purified by dialysis with physiological saline containing 0.1% SDS and 0.1% Tween 20.

Example 2 Preparation of Monoclonal Antibodies Against Pneumococcal Antigens

2-1. Immunization of Antigens

Six to eight-week-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 pneumococcal antigen. ) Intraperitoneally. 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 vein, serum was obtained, diluted in phosphate buffer (1/1000), and the titer of the antibody was determined by ELISA (enzyme linked immunosorbant assay). If the titer is low, immunize again after 2 weeks.

2-2. Fusion of Splenocytes and Myeloma Cells

In the same manner as in Example 2-1, after removing the spleen from the immunized mice, the spleen was crushed by a tissue grinder, 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 × 10 ⁷ myeloma cells and 1 × 10 ⁸ splenocytes were transferred to a 50 ml container, and the cells were recovered by adding an appropriate amount of RPMI1640 and centrifuging at 200 × 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. The media was removed after centrifugation at 200 x g for 5 minutes. 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. Cells were then cultured in 37 ° C., 5% CO ₂ incubator.

2-3. Antibody Production Using Cloning and Ascites of Fusion Cell Lines

After about 10 days, when it is confirmed that the fused cells grow and form colonies, the medium is taken from a 96-well cell culture container, and the wells which produce antibodies against the pneumoconiosis antigens 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.

Intraperitoneal injections of 6-8 week old mice (BALB / C) were injected with 0.5 ml of incomplete Freund's adjuvant. 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 into the abdominal cavity. After 1-2 weeks, ascites were properly filled, the asctic fluid was collected using a syringe, and then stored frozen.

2-4. Purification of Monoclonal Antibodies

Ammonium sulfate was added to the ascites collected in the same manner as in Example 2-3 at a concentration of 10% and then 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 after all injections, the non-adsorbed substances were removed using a 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.

2-5. Selection of Monoclonal Antibodies and Calculation of Dissociation Constants (K _d )

Purified monoclonal antibodies were measured using the Eliza method to select antibodies highly reactive with pneumococcal antigen. Pneumococcal antigen extracted from the gel was coated on a 96 well ELISA plate at a concentration of 2 μg / ml. Purified monoclonal antibodies were each diluted in steps to react with the pneumococcal-coated plate and the unbound antibody was removed by washing. Goat anti-mouse immunoglobulin G (IgG) combined with horseradish peroxydase is reacted and washed to remove unreacted peroxidase-IgG. TMB (tetramethyl benzidine) was used as a substrate of peroxidase and the absorbance (A ₄₅₀ ) was measured according to the degree of reaction. Was used (FIG. 4).

In addition, in order to measure the dissociation constants for the two antibodies to be used in the manufacture of the kit used in the present invention, Klotz plot was performed through an indirect competitive ELISA, and the dissociation constants were determined. The dissociation constants of the monoclonal antibodies were as in FIGS. 5 and 6.

2-6. Purification of Pneumococcal Specific Antigens Using Immunochromatography

Monoclonal antibodies Pw1 and Pw2 were covalently bound to CNBr-activated Sepharose CL-6B (GE Healthcare, USA), respectively. Thereafter, they were packed into columns and used for antigen preparation. As mentioned in Example 1, 5 ml of pulmonary respiratory worms provided by Chosun University College of Medicine and Hanyang University College of Medicine were added using tissue grinder (Tissue Grinder; PolyTron, Kinematica AG, Switzerland). The supernatant was triturated for 20 minutes and centrifuged at 10,000 rpm for 20 minutes. 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 and purified by buffering with 1M Tris (pH 8.0) and dialyzed with physiological saline.

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

3-1. Immobilization of Antibodies

As shown in FIG. 6, protein A (Protein A) is placed at a specific position of the nitrocellulose membrane attached to the plastic card, that is, a test line (T), and streptoavidin (Control C) at the control line (C) position. 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.

3-2. Preparation of Gold Condensate and Preparation of Gold Condensation Pads

Pneumonia antigens purified by immuno-affinity chromatography using monoclonal antibodies Pw1 and Pw2 were mixed with colloidal gold particles of about 40nm size and reacted for 1 hour in a 37 ℃ thermostatic water bath. The pneumococcal antigen and colloidal gold particles were allowed 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, centrifuged at 10,000 rpm for 20 minutes to recover the gold condensate conjugated with the pneumococcal antigen, 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.

3-3. Assembly of the strip kit

As illustrated in FIG. 6, a strip-type 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: Rapid using pneumococcal antigen Immunochromatography device  Form diagnosis Of kit  Produce

4-1. Immobilization of Antibodies

As shown in FIG. 6, protein A (Protein A) is placed at a specific position of the nitrocellulose membrane attached to the plastic card, that is, a test line (T), and streptoavidin (Control C) at the control line (C) position. 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.

4-2. Preparation of Gold Condensate and Preparation of Gold Condensation Pads

Pneumonia antigen and biotin-bovine serum albumin purified by immuno-affinity chromatography using monoclonal antibodies Pw1 and Pw2 were mixed with colloidal gold particles having a size of about 40 nm, respectively, and then incubated at 37 ° C. The reaction was carried out in a water bath for 1 hour to bind the pneumococcal antigen and colloidal gold particles 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 prevent colloidal gold particles that the antibody did not bind. It was allowed to react with the site. Thereafter, centrifuged at 10,000 rpm for 20 minutes to recover the gold condensate conjugated with the pneumococcal antigen, 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.

4-3. Assembly of Device Shape Kits

As illustrated in FIG. 11, 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, and a sample attached to a lower end thereof. 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 housing made of plastic as shown in FIG. 11 to complete the kit.

Example  5: strip form Of kit  method of inspection

Add 1 drop of wash solution to the gold condensation well supplied with the kit. Then, 3 μl of serum sample solution is added using a pipette and mixed. After that, the strip of the kit is plugged in so that the sample solution is absorbed and left for 10 minutes. Put 3 drops of the washing solution into the washing well, insert the strip-type kit absorbed with the sample solution, and leave it for 15 minutes to perform the washing reaction. After completion of the reaction, the strip is read for pneumococcal antibody depending on the position of the red band on the test line as indicated in FIG. 10.

Example  6: device  shape Of kit  method of inspection

Remove the device kit from the aluminum pouch and place it on a flat floor. 3 μl of serum sample is collected and placed in a sample window as indicated in FIG. 11 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 pneumococcal antibody according to the position of the red band on the test line as indicated in FIG. 12.

Example  7: Rapid Detection for Pneumococcal Antibodies Immunochromatography  Strip form and device  Efficacy Test of Morphology Kit

7-1. Positive sensitivity efficacy test

Using the diagnostic kit prepared in Examples 3 to 4, the sensitivity performance of the kit was tested for 223 lung dystomoma positive samples. 100 clinical trials were conducted to confirm the sensitivity of this diagnostic kit at Chosun University College of Medicine, Parasitology Laboratory (Gwangju, Korea), 73 at Hanyang University College of Medicine (Seoul, Korea), and Wonkwang University School of Medicine. Fifty cases were performed in the parasite school (Iksan, Korea). In addition, for the control test, the pneumonia pneumonia positive samples were tested using the Gendia hepatopulmonary dystomosis antibody ELISA (ELISA, Green Cross, Korea) kit, and then compared with the diagnostic kit of the present invention. The results are as shown in Tables 1-4.

7-2. Voice specificity efficacy test

Specificity performance of the kit against the lung dystomoma 278 specimen was tested using the diagnostic kit prepared in Examples 3-4. 50 clinical trials were conducted in the Department of Parasitology, Chosun University, Gwangju, Korea, and 128 in the Department of Parasitology, Hanyang University, Seoul, Korea. In each case, 100 cases were used in the parasite school (Iksan, Korea). In addition, for the control test, the pneumonia-positive specimens were subjected to a confirmatory test using a Genedia hepatopulmonary dystomosis antibody ELISA (ELISA, Green Cross, Korea) kit, and then compared with the diagnostic kit achieved in the present invention. The results are as shown in Tables 5-8.

7-3. 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 need for expensive equipment, it was confirmed that the sensitivity and specificity is high and clinically effective. .

1 and 2 is a view showing the immunoblot (immunoblot) showing the reactivity of the pneumococcal antigens and pneumococcal antibodies in the serum of the patient according to an embodiment of the present invention.

FIG. 3 is a flowchart illustrating a method for preparing monoclonal antibodies for the isolation and purification of antigens of pulmonary pneumoniae that specifically reacts against pneumococcal antibodies according to the present invention.

Figure 4 is a view showing a graph measuring the reactivity of monoclonal antibody for pulmonary repellent antigen separation and purification according to the present invention.

5 and 6 are graphs showing dissociation constants (K _d ) graphs of these monoclonal antibodies according to the present invention.

7 is a flowchart of isolating and purifying pneumococcal antibody specific antigen from pulmonary pneumoniae using monoclonal antibody according to the present invention.

FIG. 8 is a result diagram showing a graph for isolating and purifying a pneumococcal specific antigen according to the present invention. FIG.

Figure 9 is a diagram showing the configuration of the rapid immunochromatography strip form for the diagnosis of pneumoconiosis antibodies according to the present invention.

10 is a view showing a photograph of the test results in the form of pneumoconiosis antibody diagnostic rapid immunochromatography strip according to the present invention.

Fig. 11 shows the schematic diagram of the configuration of a pneumoconiosis antibody diagnostic rapid immunochromatography device in accordance with the present invention.

12 is a photograph showing a test result in the form of a pneumoconiosis antibody diagnostic rapid immunochromatography device according to the present invention.

Claims (16)

A pneumoconiosis diagnostic kit comprising a pneumococcal specific antigen isolated and purified from a pneumococcal specific monoclonal antibody produced by Accession No. KCTC 11180BP and Accession No. KCTC 11181BP fusion cells, respectively. delete delete The diagnostic kit of a strip type according to claim 1, using immunochromatography. The diagnostic kit of a device type according to claim 1, wherein the immunochromatography method is used. The diagnostic kit of claim 1, wherein the antigen-antibody complex is detected by color particle binding. (a) isolating and purifying antigens that specifically bind to pneumococcal antibody positive sera from pneumonia; (b) preparing a fusion cell of Accession No. KCTC 11180BP or Accession No. KCTC 11181BP to produce a monoclonal antibody against the antigen of step (a) and producing a monoclonal antibody therefrom; (c) selecting an antibody having high reactivity to the pneumococcal antigen in the monoclonal antibody of step (b); (d) separating and purifying the specific antigen for the antibody of step (c); And (e) a method of manufacturing a pneumoconiosis diagnostic kit of claim 1 comprising the step of preparing a diagnostic kit using the specific antigen of step (d). delete delete A composition for diagnosing pulmonary pneumoconiosis comprising pneumococcal specific antigens isolated and purified from pneumococcal specific monoclonal antibodies produced by fusion cells of Accession No. KCTC 11180BP and Accession No. KCTC 11181BP. delete delete Monoclonal antibody produced in fusion cell line of Accession No. KCTC 11180BP. Monoclonal antibody produced in fusion cell line of Accession No. KCTC 11181BP. Fusion cell of Accession No. KCTC 11180BP. Fusion cell of Accession No. KCTC 11181BP.

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