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WO2009069942A2 - Trousses de diagnostic rapide pour la détection d'anticorps dirigés contre paragonimus westermani et anticorps monoclonaux destinés à la production d'antigènes spécifiques de paragonimus westermani - Google Patents

Trousses de diagnostic rapide pour la détection d'anticorps dirigés contre paragonimus westermani et anticorps monoclonaux destinés à la production d'antigènes spécifiques de paragonimus westermani Download PDF

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Publication number
WO2009069942A2
WO2009069942A2 PCT/KR2008/006979 KR2008006979W WO2009069942A2 WO 2009069942 A2 WO2009069942 A2 WO 2009069942A2 KR 2008006979 W KR2008006979 W KR 2008006979W WO 2009069942 A2 WO2009069942 A2 WO 2009069942A2
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WIPO (PCT)
Prior art keywords
paragonimus westermani
monoclonal antibodies
antigens
diagnostic kit
paragonimiasis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/KR2008/006979
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English (en)
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WO2009069942A3 (fr
Inventor
Hyun Park
Chom Kyu Chong
Ho-Joon Shin
Suk-Il Kim
Kwang Myun Cheong
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Industry Academic Cooperation Foundation of Wonkwang University
Hyundai Bioland Co Ltd
Original Assignee
Bioland Ltd
Industry Academic Cooperation Foundation of Wonkwang University
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Publication of WO2009069942A2 publication Critical patent/WO2009069942A2/fr
Publication of WO2009069942A3 publication Critical patent/WO2009069942A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to a diagnostic kit comprising
  • Paragonimus westermani-specific antigens that are separated/purified from Paragonimus westermani-specific monoclonal antibodies, and a method for diagnosing paragonimiasis using the same.
  • the present invention relates to a composition for diagnosing Paragonimiasis, comprising Paragonimus westermani-specific antigens used in the kit; monoclonal antibodies; and hybridomas producing the same.
  • Trematodes such as Paragonimus westermani and Clonorchis sinensis, or Cestodes such as Taenia solium and Spargnum are major causes of parasitic infections, and they are still serious concerns due to their relatively high prevalence and severe complications.
  • Paragonimiasis also known as pulmonary distomatosis, is a disease caused by ingestion of raw or incompletely cooked freshwater crab infected with the fluke, Paragonimus westermani, which is found in Asia, Africa, and South America as well as in Korea, and is not easily eradicated even though many efforts have been made, unlike soil-transmitted parasites such as Ascaris, Ancylostoma, and Trichuris.
  • Infection of Paragonimus westermani occurs as follows. Eggs hatch (incubate?) in vitro for 15-20 days and release miracidia, which swim and penetrate the intermediate host, a mollusc such as snails, to form cercariae. Cercariae then penetrate the next intermediate host, a Crustacea such as crawfish, to form metacercariae . Humans or animals ingest raw freshwater crab or crawfish infected with the metacercariae, which penetrate through the intestinal wall into the lungs, leading to infection of Paragonimus westermani (Human Parasitology, Chin-Thack Soh, Shinkwang Publishing Co. , 1987) .
  • Paragonimiasis is accompanied by general symptoms of pneumonia andpleurisy, including fever, dyspnea, and chest pain, and fishy-smelling, rust-coloredphlegm. Invasion of pulmonary blood vessels by Paragonimus westermani occasionally causes hemoptysis. Additionally, brain invasion of Paragonimus westermani causes visual disturbances or epilepsy-like symptoms such as general paresis.
  • Emetine hydrochloride product name: Praziquantel
  • Praziquantel which is generally used for the treatment of clonorchiasis
  • this treatment is only determined by the symptoms, not based on the causes of disease .
  • pulmonary symptoms due to other diseases there is a problem in that it is difficult to make therapeutic decisions. Therefore, it is required to make a diagnosis of Paragonimus westermani infection before giving treatment.
  • the intradermal test also has problems in terms of sensitivity and specificity, since persons exposed but not infected with Paragonimus westermani show a positive reaction to the intradermal test (Chung et al., 1995; Sandunet al., 1959; Walton and Chyu, 1959; Dong-Chan Kim et al., 1969; Ho-Yeon Jung et al., 1989), and patients infected with Clonorchis sinensis also show a positive reaction at a rate of 44.7% (Hunter et al., 1958) .
  • the present inventors have prepared two kinds of monoclonal antibodies specific to Paragonimus westermani, and separated/purified Paragonimus westermani-specific antigens by immuno-affinity chromatography using the monoclonal antibodies and crude worm extract.
  • the separated/purified antigens are used for the production of a diagnostic kit for Paragonimiasis. They found that nonspecific reaction with other parasites can be reduced and rapid and effective diagnosis of Paragonimiasis can be achieved on-site without requiring highly skilled technicians and expensive equipment, thereby completing the present invention.
  • the Paragonimus westermani-specificmonoclonal antibodies of the present invention and a diagnostic kit comprising Paragonimus westermani-specific antigens purified therefrom are of high specificity and sensitivity compared to the conventional diagnostic methods, stool examination and intradermal test, and can be more easily performed in a one-step manner than ELISA.
  • the diagnostic kit is suitable for rapid point-of-care testing (POCT) without the aid of expensive instruments, advantageous over the known diagnostic kit in terms of sensitivity and specificity, and provides results highly consistent with those of ELISA, thereby being used for effective diagnosis of Paragonimiasis prior to treatment.
  • POCT point-of-care testing
  • FIGs. 1 and 2 are the results of immunoblot assay, which show the reactivity of Paragonimus westermani antigens according to Examples of the present invention with Paragonimus westermani antibodies in the sera of patients;
  • FIG. 3 is a flow chart showing processes of preparing monoclonal antibodies for the separation/purification of Paragonimus westermani antigens being specific to Paragonimus westermani antibodies;
  • FIG. 4 is a graph showing the reactivity of monoclonal antibodies used for the separation/purification of Paragonimus westermani antigens according to the present invention
  • FIGs. 5 and 6 are graphs showing the dissociation constant
  • FIG. 7 is a flow chart showing the process of separating/purifying Paragonimus westermani antibody-specific antigens from Paragonimus westermani using the monoclonal antibodies according to the present invention
  • FIG. 8 is a graph showing the results of separation/purification of Paragonimus westermani-specific antigens according to the present invention
  • FIG. 9 is a schematic diagram showing the structure of a strip-type kit for diagnosing Paragonimus westermani antibody based on rapid immunochromatography
  • FIG. 10 shows diagnosis results from a strip-type kit for diagnosing Paragonimus westermani antibody based on rapid immunochromatography
  • FIG. 11 is a schematic diagram showing the structure of a device-type kit for diagnosing Paragonimus westermani antibody based on rapid immunochromatography
  • FIG. 12 shows diagnosis results from a device-type kit for diagnosing Paragonimus westermani antibody based on rapid immunochromatography.
  • the present invention relates to a diagnostic kit for paragonimiasis, comprising Paragonimus westermani-specific antigens that are separated/purified from Paragonimus westermani-specific monoclonal antibodies.
  • diagnosis refers to the detection of the presence or properties of pathological states. With respect to the objects of the present invention, the diagnosis is to determine the incidence of Paragonimiasis.
  • paragonimiasis also called pulmonary distomatosis, refers to a disease caused by infection of the lung fluke, Paragonimus westermani.
  • Paragonimus westermani-specific antigens refers to antigens that are separated/purified from the novel Paragonimus westermani-specific monoclonal antibodies developed by the present inventors.
  • the Paragonimus westermani-specific monoclonal antibodies are preferably monoclonal antibodies, PwI and Pw2 that are produced by hybridomas identified by accession numbers of KCTC 11180BP and KCTC 11181BP, respectively.
  • the Paragonimus westermani-specific antigens are prepared by the following method.
  • Paragonimus westermani antigens are prepared from the Paragonimus westermani worm isolated from a host, and reacted with sera from a large number of patients with Paragonimiasis to screen the Paragonimus westermani antigens that strongly react with Paragonimus westermani-specific antibodies (FIG. 1) .
  • the antigens are extracted, and mice are injected with the antigens as an immunogen, so as to prepare hybridomas producing two kinds of monoclonal antibodies which are specifically reacted with the Paragonimus westermani antigens.
  • Each monoclonal antibody produced by two kinds of the hybridomas is purified by affinity chromatography using a protein G-Sepharose filler.
  • the purified monoclonal antibodies are reacted with CNBr-activated Sepharose to prepare a monoclonal antibody-Sepharose filler.
  • Acolumn is packedwith themonoclonal antibody-Sepharose filler, andused for the purification of Paragonimus westermani-specific antigens .
  • Paragonimus westermani-specific antigens The purification of Paragonimus westermani-specific antigens is performed as follows. First, adult worms of
  • Paragonimus westermani were harvested from the dogs' lungs.
  • Phosphate buffered saline PBS
  • PBS Phosphate buffered saline
  • the present invention provides amethod for constructing a diagnostic kit using the Paragonimus westermani-specific antigens.
  • Paragonimus westermani crude antigens showing high reactivity are first selected from the sera of patients with Paragonimiasis by immunoblot assay, and mice are immunized with the selected antigens to prepare monoclonal antibodies.
  • the splenocytes and myeloma cells of immunized mice which contain the monoclonal antibodies are used to prepare hybridomas, and then antibodies are produced using the hybridomas , The produced antibodies are purified, and antibodies showing high reactivity against Paragonimus westermani antigens are selected, which are used for the production of raw material to be used in the kit.
  • Assay systems for use in the diagnostic kit of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips and radial partition immunoassay devices, and flow-through devices.
  • the diagnostic kit of the present invention is preferably of a strip type or a device type using immunochromatography .
  • Immunochromatographic assay (ICA) is also referred to as rapid test kit due to its rapidity and simplicity.
  • ICA Immunochromatographic assay
  • Paragonimus westermani antibodies in the serum of a biological sample react with tracer antigens on colloidal gold particles, and then migrate through micropores of a nitrocellulose membrane by capillary action. During migration, the antibodies bind to captures on the internal surface of micropores to form a color band, thereby determining with the naked eye whether the test result is positive or negative.
  • the antigen-antibody complex can be detected by a colored particle immunoassay, in which a colored particle may be a colloidal gold particle, a colored glass or a plastic (polystyrene, polypropylene, latex etc.) bead, more preferably a colloidal gold particle.
  • a colored particle may be a colloidal gold particle, a colored glass or a plastic (polystyrene, polypropylene, latex etc.) bead, more preferably a colloidal gold particle.
  • the capture binding with the antigen-antibody complex may include Protein G or anti-human IgG, in addition to Protein A.
  • Protein A was used as the capture.
  • the Paragonimiasis diagnostic kit using immunochromatography of the present invention there are two major constituents in the Paragonimiasis diagnostic kit using immunochromatography of the present invention.
  • One is the nitrocellulose membrane which has two invisible lines on the surface and the other is a glass fiber pad containing antigen-gold particle conjugates in a dry state.
  • Two kinds of antibodies are immobilized on the invisible lines of the nitrocellulose membrane.
  • Protein A is immobilized on the lower test line (position marked as T)
  • streptavidin is immobilized on the upper control line (position marked as C) .
  • the antigen-gold conjugate and Biotin-BSA (biotin-bovine serum albumin) -gold conjugate in which each of Paragonimus westermani-specific antigen and Biotin-BSA (biotin-bovine serum albumin) is conjugated with gold particles, are absorbed and dried on the glass fiber pad.
  • the antigen-gold conjugates on the glass fiber pad in a dry state are rehydrated and then bound to antibodies in the sample, after which the formed complexes migrate through microspores of the nitrocellulose membrane by capillary action.
  • Protein A immobilized on the invisible line is reacted with the antibodies linked to the antigen-gold conjugate, resulting in developing a visible red color due to the red color of antigen-gold conjugate .
  • the upper line develops a color because streptavidin immobilized on the upper line may react with the biotin-BSA-gold conjugates although no antibody against Paragonimus westermani is present, thus the upper line always develops a color in each run of the test and may serve as a control. That is, when antibodies against Paragonimus westermani exist in the sample, both the test line and the control line of the kit become visible but only the control line becomes visible, when antibodies against Paragonimus westermani are not present .
  • Diagnosis of Paragonimiasis using the diagnostic kit of the present invention was found to show 90% or more sensitivity and specificity, compared to a commercially available diagnostic kit (e.g., Genedia Clonorchiasis and Paragonimiasis Ab ELISA kit manufactured by Green Cross Inc., Korea, etc), and can be rapidly and readily performed, as compared to the known diagnostic kit (see Example 7) .
  • the diagnostic kit of the present invention can be easily used for the diagnosis of Paragonimiasis .
  • the present invention provides a method for constructing the diagnostic kit for paragonimiasis, comprising the steps of 1) purifying crude antigens from Paragonimus westermani, 2) preparing hybridomas that produce monoclonal antibodies against the crude antigens, 3) selecting antibodies, which show high reactivity with the Paragonimus westermani antigens, from the monoclonal antibodies produced by the hybridoma, and 4 ) separating/purifying antigens specific to the antibodies, thereby providing a diagnostic kit that is specific to Paragonimus westermani antigens and able to exclude false-positive reactions.
  • the present invention relates to a method for diagnosing Paragonimiasis using the diagnostic kit.
  • a biological sample is applied and absorbed onto the sample pad of the diagnostic kit of the present invention. After completing the reaction, diagnostic test results for
  • Paragonimiasis are interpreted based on the presence or absence of test line.
  • biological sample means a serum or blood plasma from mammals including human suspected of having
  • Paragonimiasis For the sake of convenience, this may be referred to as “specimen” or “sample” in the present invention.
  • the biological sample may be diluted or undiluted prior to being added dropwise to the sample pad of the diagnostic kit of the present invention.
  • Paragonimus westermani antibodies are present in the biological sample, while they bind with Paragonimus westermani-specific antigens in the diagnostic kit of the present invention, and migrate through the test line, the complex of Paragonimus westermani-specific antigens and antibodies in the biological sample binds with the capture of test line to make color change on the test line, which can be observed with the naked eye.
  • the Paragonimus westermani antigens of the present invention cannot bind with the antibodies not to make color change on the test line.
  • the present invention relates to a composition for diagnosing Paragonimiasis, comprising Paragonimus westermani-specific antigens that are separated/purified from Paragonimus westermani-specific monoclonal antibodies.
  • the Paragonimus westermani-specific antigens are antigens that are separated/purified from Paragonimus westermani-specific monoclonal antibodies, and the monoclonal antibodies are monoclonal antibodies, PwI and Pw2 that are produced by hybridomas, which are identified by accession numbers of KCTC 11180BP and KCTC 11181BP, respectively.
  • the Paragonimus westermani-specific antigens contained in the composition of the present invention are antigens that are separated/purified from the monoclonal antibodies, which are selected from hybridomas secreting anti Paragonimus westermani-specific monoclonal antibodies that specifically react with the Paragonimus westermani antigens prepared by the present inventor, and thus the diagnostic composition for Paragonimiasis comprising the same shows much higher sensitivity and specificity to Paragonimiasis, compared to other known compositions (see Example 7) .
  • the present invention relates to novel Paragonimus westermani-specific monoclonal antibodies that are used for the production of Paragonimus westermani-specific antigens of the diagnostic kit and hybridomas producing the same.
  • the term "monoclonal antibody” means a highly specific antibody which is directed against a single antigenic site, as known in the art. Typically, unlike a polyclonal antibody including different antibodies directed against different epitopes, a monoclonal antibody is directed against a single epitope on an antigen.
  • the monoclonal antibody has an advantage that selectivity and specificity are improved in diagnosis and analysis using the antigen-antibody binding.
  • the monoclonal antibody has another advantage that it is produced by a fusion method to avoid contamination with other immunoglobulins .
  • the monoclonal antibody can be produced by a hybridoma method well known in the art (Kohler and Mislstein (1976) European Journal of Immunology 6:511-519) or phage antibody library (Clackson et al . , Nature, 352:624-628, 1991, etc. ) .
  • the monoclonal antibodies can be prepared as follows: mice are immunized with the crude antigens of Paragonimus westermani that strongly react with antibodies in the blood of patients with Paragonimiasis, and then their splenocytes are fused with the myeloma cells to produce hybridomas. Thereafter, hybridomas which specifically bind with Paragonimus westermani antigens are selected, and the monoclonal antibodies are prepared therefrom. More specifically, as shown in FIG.2, Paragonimus westermani antigens are subjected to electrophoresis to separate proteins, and the proteins are reacted with various Paragonimiasis-positive bloods, followed by immunoblot assay .
  • antigens which show high reactivity with Paragonimiasis antibodies are examined, and they are eluted from the gel to be used as immunogens .
  • the prepared antigens are injected to mice as immunogens, so as to selectively clone two kinds of hybridomas, which secrete anti Paragonimus westermani monoclonal antibodies that specifically react with Paragonimus westermani antigens.
  • the reactivity of the monoclonal antibodies are screened using an ELISA plate that binds with Paragonimus westermani antigens extracted by electrophoresis.
  • Hybridomas secreting monoclonal antibodies that show reactivity in the antigen-binding ELISA plate are selected, and monoclonal antibodies secreted therefrom are designated as PwI and Pw2, which are used for the production of Paragonimus westermani antigens of the diagnostic kit of the present invention.
  • the selected hybridomas are intraperitoneally injected into mice. After a predetermined period of time, ascites fluid is taken from the mice, and monoclonal antibodies are separated therefrom.
  • the hybridomas producing monoclonal antibodies, PwI and Pw2 that specifically recognize the Paragonimus westermani antigens of the present invention are deposited in KCTC (Korean Collection for Type Cultures, #52, Oun-dong, Yusong-ku, Taejon) with Accession Nos. KCTC 11180BP and KCTC 11181BP on Sep. 4, 2007, respectively.
  • KCTC Korean Collection for Type Cultures, #52, Oun-dong, Yusong-ku, Taejon
  • Example 1 Purification of Paragonimus westermani antigens being reactive with Paragonimus westermani-specific antibody
  • the present experiment was performed using Paragonimiasis-positive sera, provided by Department of Parasitology, Wonkwang University School of Medicine (Iksan, Korea) .
  • the positive sera which were prepared from the bloods of patients diagnosed with Paragonimiasis by stool examination and intradermal test, had been in use for several years.
  • the sera were used in immunoblot assay.
  • Paragonimus westermani taken from patients or dogs with Paragonimiasis was provided by Department of Parasitology, Chosun University School of Medicine (Gwangju, Korea) and Department of Parasitology, Hanyang University School of Medicine (Seoul, Korea) .
  • Paragonimus westermani was washed with a saline solution three times, and then 5 ml of saline solution per 10 mg of worm were added, and the worms were homogenized using a tissue grinder (PolyTron, Kinematica AG, Swiss) for 5 min, followed by centrifugation at 10,000 rpm for 20 min. The obtained supernatant was loaded in 10% acrylamide gel, followed by electrophoresis.
  • the protein antigens in the electrophoresed gel were transferred to nitrocellulose membrane, and then reacted with the bloods of Paragonimiasis patients at 37 ° C for 1 hr.
  • the nitrocellulose membrane was washed with a saline solution containing 0.1% Tween 20, and then reacted with anti-human IgG-HRP conjugates. Subsequently, color development was performed using Membrane 1 component TMB (BioFX, USA) (FIG. 1) .
  • the Paragonimus westermani antigens separated by electrophoresis were purified by electro-elution. As shown in Fig. 1, SDS-PAGE was performed to confirm the antigens having a molecular weight of 46, 30, and 23 kDa, which strongly reacted with Paragonimiasis antibodies. As shown in FIG. 2, a large amount of Paragonimus westermani antigens were then subjected to electrophoresis under the same conditions. Regions of the gels corresponding to the molecular weight of 46, 30, and 23 kDa were cut out and electroeluted in an ElectroEluter Model 422 (Bio-Rad, USA) .
  • Example 2 Production of monoclonal antibodies against Paragonimus westermani antigens
  • Example 2-2 Fusion of splenocytes with myeloma cells
  • the spleen was excised from a mouse immunized in the same manner as in Example 2-1 and homogenized using a tissue grinder.
  • the cell suspensions were put into a container, and centrifuged to recover the cells.
  • Myeloma cells were recovered from cell culture flasks, and suspended in RPMI1640. Then, the number of cells was counted. The number of splenocytes was also counted. l?
  • an RPMI 1640 medium was slowly added in an amount of 1 ml over 30 sec, again in an amount of 3 ml over 30 sec, again in an amount of 17 ml over 1 min, and finally in an amount of
  • the cells were allowed to stand for 5 min. After centrifugation at 200 ⁇ g for 5 min, the medium was removed and the cells were carefully suspended in 50 ml of an RPMI1640 containing 1% HAT (hypoxathine-aminopterin-thymidine) and 100 ⁇ i of the cell suspension was layered on feeder cells in each well of 96-well plates before incubation at 37 ° C in a 5% CO2 incubator .
  • HAT hypoxathine-aminopterin-thymidine
  • 0.5 ml of an incomplete Freund' s adjuvant was injected into the peritoneal cavity of a mouse (BALB/C) 6 - 8 weeks old. 1 week after the injection, the hybridomas were suspended at a density ofl.5 x 10 ⁇ cellsin0.5mlofPBS, and this cell suspension was peritoneally injected into the mouse. After 1 - 2 weeks, the ascites fluid was sampled with a syringe and freeze-stored.
  • the suspension was dialyzed in 20 mM PBS for 18 hrs or longer and loaded on a protein G-coupled column equilibrated with 20 mM PBS (pH 7.0) , followed by washing with PBS to remove unattached materials.
  • the antibodies bound to the column were eluted with a 100 mM glycine solution (pH 2.8) .
  • 1 M Tris pH 9.0 was added at a 1/10 volume of the eluent so as to adjust the pH.
  • the eluted antibody solution was concentrated. After dialysis in 150 mM PBS, the antibody fraction was quantified for antibody activity using a Bradford assay, and frozen and stored until use. 2-5. Selection of monoclonal antibody and calculation of dissociation constant (K d )
  • the reactivity of the purified monoclonal antibodies was measured by ELISA.
  • the Paragonimus westermani antigens extracted from the gel were applied to 96-well ELISA plate at a concentration of 2 ⁇ g/ml.
  • Each of the purified monoclonal antibodies was serially diluted and reacted with the plate coated with the Paragonimus westermani antigens. Then, the plate was washed, so that unbound antibodies were removed. The plate was reacted with Horseradish peroxidase-conjugated goat anti-mouse IgG, and then washed to remove unreacted peroxidase-IgG.
  • TMB tetramethyl benzidine
  • a 450 absorbance
  • PwI and Pw2 antibodies that showed high reactivity with Paragonimus westermani antigens were used for the production of raw materials used in the kit of the present invention (FIG. 4) .
  • the dissociation constant of two kinds of the antibodies to be used for the construction of the kit was determined using a Klotz plot through indirect competitive ELISA.
  • the dissociation constant of the monoclonal antibodies of the present invention is shown in FIGs. 5 and ⁇ . 2-6.
  • Purification of Paragonimus westermani-specific antigens by immunochromatography Monoclonal antibodies, PwI and Pw2 were covalently linked to CNBr-activated Sepharose CL-6B (GE Healthcare, USA) , respectively. Then, each column was packed with the monoclonal antibodies, and used for the production of antigens.
  • Example 1 a saline solution was added to Paragonimus westermani, provided by Chosun University School of Medicine and Hanyang University School of Medicine, at a volume of 5 ml per 10 mg of worm, and the worms were homogenized using a tissue grinder (PolyTron, Kinematica AG, Swiss) for 5 min, followed by centrifugation at 10,000 rpm for 20 min. The supernatant was subjected to immuno-affinity chromatography using the prepared column. The proteins were eluted using 100 mM glycine
  • Example 3 Construction of strip-type diagnostic kit using rapid immunochromatography assay with Paragonimus westermani antigen
  • Protein Aand streptavidin were allocated at respective specific positions, that is, a test line (T) and control line (C) , on a nitrocellulose membrane layered on a plastic card, followed by incubation under dry conditions at 30 ° C for 2 days in an incubator to completely immobilize the monoclonal antibody onto the nitrocellulose membrane.
  • BSA bovine serum albumin
  • sucrose sucrose
  • a strip-type plastic card was overlaid with the protein A-immobilized nitrocellulose membrane for a half region thereof while an absorption pad capable of absorbing a specimen and a buffer was attached onto the other half region of the plastic card to prepare a strip-type kit.
  • Example 4 Construction of device-type diagnostic kitusing rapid immunochromatography assay with Paragonimus westermani antigen
  • Protein Aand streptavidin were allocated at respective specific positions, that is, a test line (T) and control line (C) , on a nitrocellulose membrane layered on a plastic card, followed by incubation under dry conditions at 30 ° C for 2 days in an incubator to completely immobilize the monoclonal antibody onto the nitrocellulose membrane.
  • Paragonimus westermani antigens which were purified by immuno-affinity chromatography using monoclonal antibodies PwI and Pw2, and biotin-BSA were incubated along with colloidal gold particles 40 nm in size in a 37 °C water bath for 1 hr, respectively, so as to associate them with each other. After 1 hr, BSA (bovine serum albumin) and sucrose were added at concentrations of 3 % and 1 %, respectively, followed by incubation for an additional
  • the protein A-immobilized nitrocellulose membrane was attached onto the plastic card while an absorption pad capable of absorbing a specimen and a buffer was attached onto the top portion of the plastic card, and a sample pad serving to load a sample and a conjugate pad were attached onto the bottom portion of the plastic card.
  • This strip construct was assembled with a plastic housing, as shown in FIG. 11, to prepare a diagnostic kit.
  • One drop of a washing buffer was placed in the gold conj ugate well provided along with the kit. This was mixed with 3 ⁇ i of a blood sample using a pipette. The absorption pad attached to the strip-type kit was put into the gold conjugate well for 10 min so as to absorb the sample solution sufficiently, and then into a washing well containing three drops of an assay buffer for 15 min so as to wash the membrane . The presence of Paragonimus westermani antibody couldbe read according to the red appearance of the test line, as illustrated in FIG. 10.
  • a device-type kit was unpacked from an aluminum pouch and placed on a flat surface. 3 [ ⁇ , of a blood sample was loaded on a sample window, as shown in FIG. 11, so that it was absorbed by the absorption pad. Thereafter, three drops of an assay buffer were loaded. 15 min later, the presence of Paragonimus westermani antibody could be read according to the red appearance of the test line, as illustrated in FIG. 12.
  • Example 7 Efficacy test of strip- and device-type diagnostic kits using rapid immunochromatography for detection of Paragonimus westermani antibody
  • Sensitivity Number of Positively reacted kits/Number of Tested Positive Samples x 100 (%)
  • the diagnostic kits of the present invention are more convenient for use and allow more rapid diagnosis without the aid of expensive instruments, compared to conventional diagnostic kits.
  • the diagnostic kits of the present invention are of high sensitivity and specificity, and thus show highly useful clinical applicability.
  • the Paragonimus westermani-specificmonoclonal antibodies of the present invention and a diagnostic kit comprising Paragonimus westermani-specific antigens purified therefrom are of high sensitivity and specificity compared to the conventional diagnostic methods, stool examination and intradermal test, and can be more easily performed in a one-step manner than ELISA.
  • the diagnostic kit is suitable for rapid point-of-care testing (POCT) without the aid of expensive instruments, advantageous over the known diagnostic kit in terms of sensitivity and specificity, and provides highly consistent results with those of ELISA, thereby being used for effective diagnosis of Paragonimiasis prior to treatment.
  • POCT point-of-care testing

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Abstract

L'invention se rapporte à une une trousse de diagnostic qui comprend des antigènes spécifiques de Paragonimus westermani séparés et purifiés à partir d'anticorps monoclonaux spécifiques de Paragonimus westermani, et à un procédé qui permet de diagnostiquer une paragonimiase à l'aide de la trousse. La trousse de diagnostic précitée permet d'établir un diagnostic commode et rapide d'une spécificité et d'une sensibilités supérieures à celles des trousses de diagnostic classiques. L'invention porte également sur une composition destinée au diagnostic d'une paragonimiase, ladite composition comprenant les antigènes spécifiques de Paragonimus westermani utilisés dans la trousse; sur des anticorps monoclonaux; et sur des hybridomes permettant de produire ces derniers.
PCT/KR2008/006979 2007-11-26 2008-11-26 Trousses de diagnostic rapide pour la détection d'anticorps dirigés contre paragonimus westermani et anticorps monoclonaux destinés à la production d'antigènes spécifiques de paragonimus westermani Ceased WO2009069942A2 (fr)

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KR10-2007-0121106 2007-11-26
KR1020070121106A KR100957675B1 (ko) 2007-11-26 2007-11-26 폐흡충 특이 항원의 생산을 위한 단클론 항체 및 폐흡충증신속 진단 키트

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WO2009069942A2 true WO2009069942A2 (fr) 2009-06-04
WO2009069942A3 WO2009069942A3 (fr) 2009-08-27

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Cited By (2)

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CN102279259A (zh) * 2010-06-10 2011-12-14 中国疾病预防控制中心寄生虫病预防控制所 一种检测并殖吸虫病的诊断试剂盒及其制备方法
CN107058512A (zh) * 2017-02-23 2017-08-18 浙江工商大学 用于小龙虾、溪蟹中肺吸虫rpa快速检测的引物

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CN111296338A (zh) * 2020-03-03 2020-06-19 昆明医科大学 一种实验室溪蟹感染并殖吸虫的方法

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Publication number Priority date Publication date Assignee Title
US5854051A (en) * 1997-09-15 1998-12-29 Heska Corporation Parasitic helminth asparaginase proteins, nucleic acid molecules, and uses thereof
KR100439731B1 (ko) * 2001-05-02 2004-07-12 신풍제약주식회사 간흡충, 폐흡충, 유구낭미충 및 고충 감염증의효소면역진단 키트
KR20040063281A (ko) * 2003-01-06 2004-07-14 (주) 디엠 폐흡충 유래의 재조합 시스테인 단백분해효소 및 이를이용한 폐흡충 진단용 키트
KR100982064B1 (ko) * 2006-03-28 2010-09-13 원광대학교산학협력단 말라리아 원충에 대한 단일클론 항체 및 이를 이용한말라리아의 진단방법

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102279259A (zh) * 2010-06-10 2011-12-14 中国疾病预防控制中心寄生虫病预防控制所 一种检测并殖吸虫病的诊断试剂盒及其制备方法
CN107058512A (zh) * 2017-02-23 2017-08-18 浙江工商大学 用于小龙虾、溪蟹中肺吸虫rpa快速检测的引物
CN107058512B (zh) * 2017-02-23 2023-04-18 浙江工商大学 用于小龙虾、溪蟹中肺吸虫rpa快速检测的引物

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KR100957675B1 (ko) 2010-05-12
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