KR102542593B1 - Pine wood nematode secretory antigen PWN-SA571 specific antibodies and uses thereof - Google Patents

Abstract

The present application provides an antibody or antigen-binding fragment thereof that specifically binds to PWN-SA571, a pine wood nematode secretory protein, a composition for diagnosing pine wood nematode infection comprising the same, a kit for diagnosing pine wood nematode infection comprising the composition, and a pine wood nematode using the composition or kit. It relates to a method of providing information for diagnosing infection.
Antibodies or antigen-binding fragments thereof that specifically bind to PWN-SA571, a protein secreted by pine wood nematodes, of the present application can effectively detect proteins or antigens derived from pine wood nematodes, and thus can efficiently diagnose whether pines are infected with pine wood nematodes. can

Description

Pine wood nematode secretory antigen PWN-SA571 specific antibodies and uses thereof {Pine wood nematode secretory antigen PWN-SA571 specific antibodies and uses thereof}

The present application provides an antibody or antigen-binding fragment thereof that specifically binds to PWN-SA571, a pine wood nematode secretory protein, a composition for diagnosing pine wood nematode infection comprising the same, a kit for diagnosing pine wood nematode infection comprising the composition, and a pine wood nematode using the composition or kit. It relates to a method of providing information for diagnosing infection.

Pine wood nematode (PWN) is a pine parasitic nematode native to the Americas. Most Pine trees are known to be infected with PWN, except trifolium, which is native to North America. It is known that transmission of pathogenic PWN is carried out by long-horned beetles belonging to the Genus Monochamus . More than 163 species of longhorn beetles are potential vectors capable of disseminating PWN worldwide. Therefore, it is very likely that potential vectors of PWN exist even in areas of the Old Continent where PWN has not invaded so far. In fact, in the case of continental Europe, where PWN is currently spreading, various bearded alternatus exist, and they are spreading the spread of PWN.

Trees belonging to the pine genus susceptible to PWN include pine ( Pinus densiflora), pine nut (Pinus korainensis), island pine (Pinus parviflora), French maritime pine (Pinus pinaster), radiata pine (Pinus radiate), black pine (Pinus thunbergii), Kuju Pine (Pinus sylvestris) and the like are known. Among pine trees distributed in Europe, it is known that P. pinea, which is mainly distributed in coastal areas of Italy, does not grow as well as other susceptible pines. In addition, it is known that CJFL infection of PWN can be prevented by applying abamectin to PWN-susceptible pine trees, so canopy injection using abamectin or its derivatives is important to protect important pine trees having historical or social significance from PWN infection. It is used as a prophylaxis.

However, since it is currently impossible to perform canopy injections on all pine trees in PWN-infected areas, the best strategy is to suppress the spread of PWN. It is known that human-infected trees are moved to non-infected areas and PWN is transmitted to other pines rather than naturally by bearded alternatus. Therefore, it is essential to develop a method that can confirm whether pine trees are infected with PWN. To achieve this goal, various diagnostic methods have been researched and developed.

First of all, various PCR-based diagnostic methods using PWN genes such as ITS regions, microsatellites, or general genes have been reported, but these PCR-based methods require precise PCR machines, so application in forest fields is not possible. There is a difficult side. In addition, diagnostic methods using loop-mediated isothermal amplification and recombinant polymerase amplification, which can amplify DNA at isotherm, were developed. These methods have the advantage of being able to diagnose faster and faster than conventional PCR-based diagnostic methods. Another diagnostic method is a method of diagnosis using an antigen/antibody reaction. However, no antigen/antibody-based technology has been developed to diagnose Pine Wilt Nematode-infected trees.

As a result of efforts to develop a method for diagnosing PWN infection using an antigen/antibody reaction, the present inventors discovered PWN-SA571, one of the PWN-secreted antigens present in PWN-infected trees, and prepared monoclonal antibodies against it. The present invention was completed by developing a method for determining PWN infection.

Republic of Korea Patent Publication No. 10-2019-0113337

A first aspect of the present application is to provide an anti-PWN-SA571 antibody or antigen-binding fragment thereof, characterized in that it specifically binds to PWN-SA571, a protein secreted by Pine nematode.

A second aspect of the present application is to provide a composition for diagnosing pine wood nematode infection, comprising an anti-PWN-SA571 antibody or an antigen-binding fragment thereof.

A third aspect of the present application is to provide a kit for diagnosing pine wood nematode infection, including a composition for diagnosing delayed ischemia, including the composition for diagnosing pine wood nematode infection.

A fourth aspect of the present application includes: (A) obtaining a sample from an individual; and (B) reacting the obtained sample with the composition for diagnosing pine wood nematode infection of claim 5, to provide an information providing method for diagnosing pine wood nematode infection.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

A first aspect of the present application provides an anti-PWN-SA571 antibody or antigen-binding fragment thereof, characterized in that it specifically binds to PWN-SA571, a protein secreted by Pine nematode.

The term "Pine wood nematode (PWN; scientific name: Bursaphelenchus xylophilus )" used throughout the specification of this application is a thread-like nematode of about 1 mm in size, living in the body of an insect vector (Monochamus alternatus, Northern mustache alternatus, etc.) When gnawing new shoots, they infiltrate trees through wounds.The invading wilt nematodes multiply rapidly and block the passage of water and nutrients, causing pine wilt nematode disease that kills trees. The pine wood nematode infects pine trees such as pine, pine, pine, pine, and pine and causes disease.

The term used throughout the present specification, "Pine wood nematode secretory antigen 571 (PWN-SA571)" is one of the proteins derived from the pine wood nematode, and the protein is a protein secreted by the pine wood nematode invading a pine tree.

In one embodiment of the present application, the PWN-SA571 protein may include the amino acid sequence of SEQ ID NO: 1, and may specifically consist of the amino acid sequence of SEQ ID NO: 1. In addition, the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 9.

In one embodiment of the present application, the anti-PWN-SA571 antibody or antigen-binding fragment thereof may specifically bind to the PWN-SA571 protein, specifically SEQ ID NO: 2 or sequence that is part of the amino acid sequence of SEQ ID NO: 1 It may specifically bind to a peptide containing the amino acid sequence of No. 3.

In one embodiment of the present application, the anti-PWN-SA571 antibody or antigen-binding fragment thereof may have the PWN-SA571 protein consisting of the amino acid sequence of SEQ ID NO: 1 as an antigen, specifically, the amino acid sequence of SEQ ID NO: 1 It may be a peptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 as an antigen.

As used throughout this specification, the term “antibody” refers to a polypeptide that specifically binds to and recognizes an antigen or protein. The antibody is an immunoglobulin selected from the group consisting of IgA, IgE, IgM, IgD, IgY, IgNAR, heavy chain antibody and IgG, and can specifically bind to a target antigen. The antibody is composed of two light chains and two heavy chains each, and each chain consists of a variable domain with a variable amino acid sequence and a constant domain with a constant sequence. . In the antibody, an antigen binding site is located at the end of the variable region in the three-dimensional structure, and this site is formed by gathering three complementarity determining regions present in each of the light chain and the heavy chain. The complementarity-determining region is a region with particularly high amino acid sequence variability among variable regions, and specific antibodies against various antigens can be found by such high variability. The scope of the present application includes complete antibody forms as well as antigen-binding fragments of the antibody molecules.

In one embodiment of the present application, the anti-PWN-SA571 antibody or antigen-binding fragment thereof may be a monoclonal antibody, a polyclonal antibody, or a multispecific antibody (eg, a bispecific antibody), specifically a monoclonal antibody. It may be, but is not limited thereto.

As used throughout the present specification, the term "antigen-binding fragment" refers to an antibody fragment that retains an antigen-binding function, and includes scFv, dsFv, Fab, Fab', F(ab')2, sdAb and nanobodies. (nanobody), etc., and combinations thereof may correspond to antigen-binding fragments, and the antigen-binding fragments may include antigen recognition sites. The Fab has a structure having light and heavy chain variable regions, a light chain constant region, and a first constant region (CH1 domain) of a heavy chain, and has one antigen binding site. Fab' is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable region and light chain variable region are linked by a disulfide bond, and in short chain Fv (scFv), the heavy chain variable region and light chain variable region are generally covalently linked through a peptide linker. . Such antibody fragments can be obtained using proteolytic enzymes (for example, Fabs can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), preferably Preferably, it can be produced through genetic recombination technology. In addition, the sdAbs and Nanobodies are single variable domain antibody fragments, for example, those of heavy chain antibodies comprising a naturally occurring single variable domain (VH) and two constant domains (CH2 and CH3). It may include, but is not limited to, antibody fragments prepared by protein hydrolysis or genetic recombination technology and single domain antibody fragments prepared by artificially modifying antibody light or heavy chain variable domains.

In one embodiment of the present application, the antibody or antigen-binding fragment thereof may include an amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 8, and specifically, the antibody or antigen-binding fragment thereof is SEQ ID NO: 4 to SEQ ID NO: 8 It may consist of any one amino acid sequence of number 8.

In one embodiment of the present application, the amino acid sequences of SEQ ID NOs: 4 to 8 may be encoded by the nucleotide sequences of SEQ ID NOs: 10 to 14, respectively.

A second aspect of the present application provides a composition for diagnosing pinewood nematode infection, comprising the anti-PWN-SA571 antibody or antigen-binding fragment thereof. The contents overlapping with the first aspect are also applied to the composition for diagnosing pine wood nematode infection of the second aspect.

In one embodiment of the present application, the diagnosis of the pinewood nematode infection is to determine whether the individual is infected with the pinewood nematode, which includes confirming whether a disease caused by the pinewood nematode infection, specifically, whether the pinewood nematode disease has occurred. it may be

In one embodiment of the present application, the anti-PWN-SA571 antibody or antigen-binding fragment thereof may specifically bind to the PWN-SA571 protein consisting of the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 It may specifically bind to a peptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

In one embodiment of the present application, the anti-PWN-SA571 antibody or antigen-binding fragment thereof may include an amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 8, and specifically, the antibody or antigen-binding fragment thereof It may consist of any one amino acid sequence of SEQ ID NO: 4 to SEQ ID NO: 8.

A third aspect of the present application provides a kit for diagnosing pine wood nematode infection, including the composition for diagnosing pine wood nematode infection. The contents overlapping with the first and second aspects are also applied to the kit for diagnosing pine wood nematode infection of the third aspect.

In one embodiment of the present application, the kit may include the anti-PWN-SA571 antibody or an antigen-binding fragment thereof.

In one embodiment of the present application, the kit may include an agent capable of detecting an antigen-antibody complex, and specifically, the anti-PWN-SA571 antibody or antigen-binding fragment thereof and the PWN-SA571 protein or its It may include an agent capable of detecting a complex of fragments.

In one embodiment of the present application, the kit includes not only the anti-PWN-SA571 antibody or antigen-binding fragment thereof of the present application, but also tools, reagents, etc. commonly used in the art for immunological analysis. Such tools/reagents may include, but are not limited to, suitable carriers, labels capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminating agent. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, Polyacrylonitrile, fluorine resin, cross-linked dextran, polysaccharide, polymers such as magnetic microparticles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

In one embodiment of the present application, the kit is a radioimmunoassay (RIA) kit, an enzyme immunoassay (ELISA) kit, a Western blotting kit, an immunoprecipitation assay kit, an immunodiffusion assay (Immunodiffusion assay) assay kit, complement fixation assay kit, FACS kit, protein chip kit, immunofluorescence kit, chemiluminescent material binding method kit, chromatography kit, magnetic activated cell sorting, MACS) kit, etc., but is not limited thereto as long as it can detect a protein or antigen derived from Pine wilt nematode or detect the antigen-antibody complex.

Specifically, labels capable of qualitatively or quantitatively measuring the formation of an antigen-antibody complex include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, radioisotopes, etc., and are necessarily limited thereto. it is not going to be Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholine esterase, glucose oxy Multiple drugs, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphophenolpyruvate decarboxylase, β- Latamase and the like, but are not limited thereto. Fluorescent substances include fluorescein, isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanine, o-phthalaldehyde, fluorescamine, and the like, but are not limited thereto. Ligands include biotin derivatives and the like, but are not limited thereto. Luminescent substances include acridinium ester, luciferin, luciferase, and the like, but are not limited thereto. Microparticles include nano gold, colloidal gold, colored latex, and the like, but are not limited thereto. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W(CN)8, [Os(bpy) ₃ ] ²⁺ , [RU(bpy) ₃ ] ²⁺ , [MO(CN) ₈ ] ^{4- ,} etc., but are not limited thereto. Radioactive isotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ^{51 Cr, 57 Co} , ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, and ¹⁸⁶ Re.

A fourth aspect of the present disclosure includes (A) obtaining a sample from an individual; and (B) reacting the obtained sample with the anti-PWN-SA571 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, providing an information providing method for diagnosing infection with pine wood nematode. do. The contents overlapping with the first to third aspects are also applied to the information provision method for diagnosing wood nematode infection of the fourth aspect.

In one embodiment of the present application, the method may be to use the composition or kit for diagnosing pine wood nematode infection.

As used throughout the present specification, the term "subject" refers to all organisms that are infected or likely to be infected with the pine wood nematode, and specific examples include pine trees ( Pinus densiflora), pine nuts (Pinus korainensis), pine pine trees (Pinus parviflora), Pine trees such as French pine pine (Pinus pinaster), radiata pine (Pinus radiate), black pine (Pinus thunbergii), and pinus sylvestris may be included, but are not limited thereto. In addition, the object may mean all organisms that are infected with pine wood nematodes and are likely to develop or develop pine wood nematode disease.

As used throughout the present specification, the term "sample" means a material derived from an individual infected with or likely to be infected with the pine wilt nematode, and a genetic sample can be obtained from these samples, and the genetic sample is a protein, protein fragment, peptide , antigens, antigen fragments, nucleic acids, etc., and the type thereof is not limited thereto, as long as it is possible to detect the pine wood nematode-derived protein, specifically PWN-SA571 secretion antigen.

In one embodiment of the present application, step (B) may include forming an antigen-antibody complex by reacting the antibody or antigen-binding fragment thereof with the antigen derived from Pine wilt nematode contained in the sample.

In one embodiment of the present application, the step of obtaining a sample of step (A) may be to use a solution that does not contain Triton X-100.

In one embodiment of the present application, the method comprises (C) detecting the antigen-antibody complex of the antibody or antigen-binding fragment thereof and the antigen in the sample to measure the level of PWN-SA571 protein, and a sample forming the complex It may be to further include the step of diagnosing as being infected with the pine wood nematode.

In one embodiment of the present application, the step (C) further comprises measuring the level of PWN-SA571 protein by comparing the result of reacting the sample of a normal control with the anti-PWN-SA571 antibody or antigen-binding fragment thereof. it may be

In one embodiment of the present application, the anti-PWN-SA571 antibody or antigen-binding fragment thereof may include an amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 8, and specifically, the antibody or antigen-binding fragment thereof It may consist of any one amino acid sequence of SEQ ID NO: 4 to SEQ ID NO: 8.

Antibodies or antigen-binding fragments thereof that specifically bind to PWN-SA571, a protein secreted by pine wood nematodes, of the present application can effectively detect proteins or antigens derived from pine wood nematodes, and thus can efficiently diagnose whether pines are infected with pine wood nematodes. can

1 is a diagram showing the similarity between PWN-SA571, a protein secreted by pine wood nematodes, and proteins of similar nematodes.
Figure 2 is a diagram showing the signal peptide prediction analysis results of the PWN-SA571 protein.
Figure 3 shows the results of analyzing the immunoreactivity of the anti-PWN-SA571 monoclonal antibody prepared using ascite by ELISA.
4 is a BSA-conjugated peptide of the anti-PWN-SA571 monoclonal antibody of the present application, a healthy pine tree extract, a PWN-infected pine tree extract, and four species of nematodes [PWN ( Bursaphelenchus xylophilus ), Bursaphelenchus mucronatus, Bursaphelenchus doui, Bursaphelenchus thailandae ) immunoreactivity to the extracts was analyzed by ELISA.
Figure 5 is the result of analyzing the immunoreactivity of the anti-PWN-SA571 monoclonal antibody of the present application by nano-gold dot blot assay.
Figure 6 is the result of analyzing the immunoreactivity according to the extraction solution of the anti-PWN-SA571 monoclonal antibody of the present application by TMB dot blot assay.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: Discovery of protein PWN-SA571 derived from pine wood nematode

In order to develop a composition and method capable of diagnosing pine wood nematode infection, the following experiments were performed to discover new proteins derived from pine wood nematodes and prepare antigens therefor.

Specifically, in order to discover proteins derived from pine wilt nematodes that are likely to be secreted by pine wilt nematodes invading pine trees, antigens were extracted with PBS from pine wilt nematode-infected trees, and then proteomic analysis of the extracted antigens was performed using the pine wilt nematode proteomic database. , the BUX_s s00579.571 protein was found. Next, based on the results of the NCBI Blast search ( https://blast.ncbi.nlm.nih.gov/ Blast.cgi ), a functional study of the discovered protein was performed, and the protein was identified as PWN secretory antigen 571 (PWN secretory antigen 571). antigen 571, PWN-SA571) (SEQ ID NO: 1)

As a result of performing NCBI Blast using the amino acid sequence of PWN-SA571, it was confirmed that a homologue having 29-34% homology and 48-57% similarity existed in various nematodes (Fig. 1). ). However, nothing is currently known about the function of PWN-SA571 and its homologues.

Since PWN-SA571 was identified as a protein secreted when the pine wilt nematode decomposes pine trees, a signal peptide may exist at the N-terminus, so a signal peptide was detected using a web-based signal peptide prediction tool (SignalP-5.0 Server). The position of was analyzed. As a result, the amino acid sequence of positions 1 to 17 of PWN-SA571 was predicted as a signal peptide (FIG. 2), and it was confirmed that the molecular weight of PWN-SA571 excluding the signal peptide was 12.25 kDa and the pI was 6.94.

Example 2: Construction of PWN-SA571 antigen peptide

In order to prepare a PWN-SA571 antigen peptide for preparing a monoclonal antibody against PWN-SA571, the following experiment was performed.

Specifically, antigenicity analysis for PWN-SA571 was performed using various web-based antigenicity prediction tools. As a result of the analysis, two peptides containing amino acids showing high antigenicity were designed (Table 1), and 16 amino acids were added to the peptides designed to improve the antigenicity and water solubility of the peptides, respectively (peptide 1, SEQ ID NO: 2 ) and an antigenic peptide consisting of 18 amino acids (peptide 2, SEQ ID NO: 3) was prepared.

Antigenic Prediction
program
SVMTrip Immunomedicine Group EMBOSS explorer antigenic Peptide antigens used in this example No. One ⁵ VFLFAVVGVAITYA ^{14 4} KVFLFAVVGVAITYAFKIPLPFGQ ^{27 4} KVFLFAVVGVAITYAFKIPLPFGQI ²⁸ - 2 ¹⁶ TYAFKIPLPF ^{25 28} INVDKE EDGTVNAGVN ⁴³
(SEQ ID NO: 2) 3 ³⁴ EDGTTVNAGVN ⁴³ - - 4 ⁵⁵ SGLEVTSKNG ^{64 64} GSLALKPQAAI ^{74 65} SLALKPQAAIL ^{75 51} YGGS SGLEVTSKNG SLAL ⁶⁸
(SEQ ID NO: 3) 5 ⁷² AAILANNTAY ⁸¹ - -

- 6 ⁸⁹ VDKKEGIVAD ^{98 93} EGIVADT ^{99 94} GIVADTD ¹⁰⁰ 7 ¹⁰¹ VDAGNHTFHG ^{110 118} FVNEVGDAVK ^{127 119} VNEVGDAVKS ¹²⁸ 8 ¹¹⁷ QFVNEVGDAV ¹²⁶

Example 3: Construction of monoclonal antibodies against PWN-SA571

In order to prepare a monoclonal antibody against PWN-SA571 using the two antigenic peptides prepared in Example 2, the following experiments were performed.

First, KLH (keyhole limpet hemocyanin) and BSA (Bovine serum albumin) were conjugated to each of the two antigenic peptides (peptides 1 and 2) prepared in Example 2, and mouse immunization antigen and ELISA (Enzyme linked immunosorbent assay) Each antigen for screening was prepared.

Next, a mouse immunization method and ELISA were performed to prepare monoclonal antibodies. The experiment was performed according to the permission and guidance of the Animal Ethics Committee of Hallym University, and the experiment was performed according to the previously reported monoclonal antibody production method. Specifically, 4 Balb/C mice were injected with 2 antigens conjugated with KLH and 2 antigenic peptides, respectively, mixed with Freund's complete or incomplete adjuvant (Merck KGaA, Darmstadt, Germany) to induce immunity in mice. did

Next, in order to select antibody-secreting cells recognizing the two antigenic peptides, blood was collected from mice that had received antigen injection, and reactivity was confirmed using BSA-conjugated antigenic peptides. A fusion cell line library was constructed by fusing splenocytes with mouse myeloma cell line SP2/0. Next, ELISA was performed using the free peptide and the BSA-conjugated antigen peptide as antigens. As a result, after securing 16 hybridoma cell clones showing high reactivity to peptide 1, 8 sub-clones showing high reactivity were selected by limiting dilution and established as monoclonal antibodies. In addition, after securing 10 hybridoma cell clones showing high reactivity to peptide 2, performing limiting dilution, 5 sub-clones showing high reactivity were selected and established as monoclonal antibodies.

Next, in order to utilize the monoclonal antibody more efficiently, the isotype of the monoclonal antibody of the 13 subclones was confirmed. Specifically, the isotypes of immunoglobulins secreted by the 13 established hybridoma cell lines were analyzed using a rapid ELISA mouse mAb isotype kit (ThermoFisher Sci.). As a result, two of the monoclonal antibodies against PWN-SA571 peptide 1 were identified as IgG2b and the rest were all IgM (Table 2), and the monoclonal antibodies against PWN-SA571 peptide 2 were IgG1 except for one IgG2b. confirmed (Table 3).

Mab PWN-SA571 peptide 1 antibody 7F4-E9 9G6-E7 2E6-C11 7E3-D7 8C6-D7 4A4-G10 2C11-A6 6E2-D7 isotype IgM IgM IgM IgM IgG2b IgM IgM IgG2b

Mab PWN-SA571 peptide 2 antibody 2H7-H8-B8 4F12-H7-A3 1A4-B11-A2 4B9-F9-G11 5D4-H4-D4 isotype IgG1 IgG1 IgG2b IgG1 IgG1

Example 4: Confirmation of antigen reactivity of anti-PWN-SA571 monoclonal antibody

In order to confirm the reactivity of the monoclonal antibodies prepared in Example 3 to the pine wood nematode antigen, the following experiment was performed.

First, in order to confirm the antigen reactivity of the monoclonal antibody secreting cell line (subclone) selected in Example 3, free peptide, BSA-conjugated peptide, healthy pine tree extract, pine wood nematode infected tree (PWN- infected pine tree) extract, and four types of nematode [PWN ( Bursaphelenchus xylophilus ), Bursaphelenchus mucronatus, Bursaphelenchus doui, Bursaphelenchus thailandae ) extracts were confirmed by ELISA.

Specifically, each antigen was diluted to 0.2 μg/ml using a coating solution (0.4M carbonate buffer, pH9.6), dispensed into a 96-well plate, reacted overnight at 4° C., and StabilCoat® ( Surmodics IVD, Inc. Eden Prairie, MN, USA) was added and reacted at room temperature for 2 hours. Next, after removing the solution as much as possible, 50 to 100 μl of monoclonal antibody production cell culture medium was added, reacted at room temperature for 1 hour, and washed three times with PBS (PBST) containing 0.25% Tween-20. Next, after adding 50 μl of the secondary antibody, the reaction was carried out while stirring at room temperature for 1 hour, and then washed three times with PBST. Next, TMB solution for ELISA (ThermoFisher Scii.) was added and reacted at room temperature for 30 minutes, then 2M sulfuric acid was added to stop the reaction, and absorbance was measured at 450 nm using an Epoch spectrophotometer. , antigen reactivity was confirmed.

As a result, monoclonal antibodies against PWN-SA571 peptide 1 were largely divided into three groups. Group 1 includes 7E4-E9, 4A4-G10, and 7E3-D7, which are monoclonal antibodies showing high reactivity against pine wilt nematode-infected trees. The antibodies were not as high in sensitivity to the pine wood nematode as compared to the other three pseudonematodes. Group 2 includes 2E6-C11, 2C11-A6, and 9G6-E7 as monoclonal antibodies showing high reactivity against pine wood nematode. The antibodies showed lower reactivity to the pine wood nematode infected tree than the first group. The third group includes 8C6-D7 and 6E2-D7 as antibodies having selective reactivity to PWN and high reactivity to BSA-conjugated peptide 1, although not high (FIG. 4A).

In addition, in most cases, monoclonal antibodies against PWN-SA571 peptide 2 showed higher reactivity to BSA-conjugated peptides than free peptides, but there was a difference in reactivity to PWN or PWN infected trees. Specifically, among the monoclonal antibodies, 4F12-H7-A3 and 5D4-H4-D4 showed selective reactivity to PWN and PWN-infected trees. 2H7-H8-B8, which showed the highest reactivity to BSA-conjugated peptide 2, had no selective reactivity to PWN and PWN-infected trees. In the case of 1A4-B11-A2 and 4B9-F9-G11, the reactivity to PWN was not high compared to that of the nematode, but it showed more than twice the reactivity compared to the healthy tree (Fig. 4B).

Example 5: Construction of anti-PWN-SA571 monoclonal antibody using ascite and confirmation of antigen reactivity of the antibody

A method for producing a high-titer monoclonal antibody is to prepare ascite produced by injecting hybridoma cells into the peritoneal cavity of a mouse. Therefore, ascites of the 13 monoclonal antibodies identified in Example 3 were prepared by the following method. Specifically, a total of 13 monoclonal antibody-secreting hybridoma cells (1 x 10 ⁷ ) established in Example 3 were injected into the abdominal cavity of a mouse injected with pristane 10 days earlier, and the mouse's abdomen expanded. waited until After carefully collecting ascite from the distended abdomen using a syringe, it was immediately aliquoted and stored at -80°C. In addition, after removing impurities from the ascite with a 0.2 μm size scan filter, IgM or IgG present in high concentration is separated using Poros ^TM CaptureSelect ^TM IgM Affinity Matrix (ThermoFisher Sci) or protein G-agarose packed in an FPLC column Then, the antibody was purified from ascite.

Next, the antigen reactivity of a total of 13 ascites obtained above was confirmed. Specifically, ELISA was performed after diluting ascite to 1:1,000, 1:10,000, and 1:100,000 using PBS to confirm the reactivity to the free peptide and the BSA-conjugated peptide 1 or 2.

As a result, among the monoclonal antibodies against PWN-SA571 peptide 1, 6E2-D7 and 8C6-D7 showed high reactivity against the two peptide antigens even when diluted up to 1:100,000. In contrast, monoclonal antibodies against PWN-SA571 peptide 2 showed high reactivity at a 1:1,000 dilution (FIG. 3A).

In addition, looking at the reactivity of the 12 diluted ascites to PWN-infected pine tree (PWNIPT) extract and healthy pine tree (HT), all showed high reactivity at 1:1,000 dilution. , most of the antibodies showed low or almost no reactivity to healthy trees compared to PWN-infected trees (Fig. 3B). These results suggest that the use of two types of monoclonal antibodies against PWN-SA571 can be used to detect PWN-infected pine trees by ELISA method.

Example 6: Confirmation of antigen reactivity of anti-PWN-SA571 monoclonal antibody using nano gold-dot blot assay

In order to confirm the reactivity of the monoclonal antibodies prepared in Example 3 to the pine wood nematode antigen, the following experiment was performed.

Specifically, in order to perform a dot blot assay using nano-gold, 0.02 to 1.0 μg of BSA-conjugated PWN-SA571 peptide 1 or 2 was added to a lateral diffusion strip. strip) (Prime4dia, Anyang, Korea), and dried at 45 ^° C for 30 minutes to fix. For the lateral diffusion assay, 2.0 μl goat anti-mouse IgG-nanogold 40 nm (Prime4dia) was mixed with 20 μl of lateral diffusion buffer (Primer4dia) and 20 μl of the monoclonal antibody supernatant in a 96-well plate. After mixing in the well, the side spread strip was immersed filter paper side up. When the monoclonal antibody and the anti-mouse IgG nanogold complex migrated along the side diffusion strip and met the antigen of peptide 1 or 2, they precipitated and developed color. Based on this, the antigen reactivity of the monoclonal antibodies was confirmed.

As a result of confirming the titer of the monoclonal antibody against PWN-SA571 peptide 1, 2C11-A6, 7E3-D7, 7F4-E9, 8C6-D7, and 4A4-G10 monoclonal antibodies showed high reactivity (FIG. 5A). In addition, the antibody As a result of confirming the reactivity to the antigen of the 7E3-D7, 7F4-F9, and 8C6-D7 monoclonal antibodies, it was confirmed that all showed reactivity to the 0.1 mg antigen (FIG. 5B).

As a result of confirming the titer of the monoclonal antibody against PWN-SA571 peptide 2, 2H7-H8-B8, 1A4-B11-A2 and 4F12-H7-A3 monoclonal antibodies were recognized up to 0.2 mg (FIG. 5C). In addition, as a result of confirming the reactivity to 0.1 ~ 0.02 mg using three highly reactive monoclonal antibodies, 1A4-B11-A2 monoclonal antibody showed the highest reactivity, and 4F12-H7-A3 showed the lowest reactivity ( Fig. 5D).

Example 7 Confirmation of antigen reactivity of anti-PWN-SA571 monoclonal antibody using TMB-dot blot assay

In order to confirm the reactivity of the monoclonal antibodies prepared in Example 3 to the pine wood nematode antigen, TMB-dot blot assay was performed in the following manner.

First, since labeling purified monoclonal antibodies with biotin can improve sensitivity, seven purified monoclonal antibodies against PWN-SA571 peptide 1 were labeled with biotin in the following manner. Specifically, after adding 6.7 μl of 20.0 mM N-Hydroxysulfosuccinimide (NHS)-polyethylene glycol 4 (PEG4)-Biotin Dimethylformamide (DMF) per 1 mg of purified antibody immunoglobulin, the mixture was stirred at room temperature for 2 hours. In order to remove NHS-PEG4-Biotin that did not react with immunoglobulin, the reaction solution was subjected to dialysis overnight at 4°C using PBS. The immunoglobulin concentration of the dialyzed solution was measured by BCA.

Next, in the method for diagnosing pine wood nematode infection, in order to select an optimal solution for extracting antigens from pine wood nematodes, PWN was extracted using the following 5 buffers.

1. RIPA buffer: 50 mM Tris pH 8.0, 0.1% Nonidet P(NP)-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.15M NaCl

2. RIPA-SDS-SB: After extraction using RIPA buffer, mixing with SDS Sample buffer (SDS-SB) at a ratio of 4:1, heating for 10 minutes

3. Tris-Cytoskeleton Buffer (Tris-Cyto): 10mM Tris, pH 7.4, 0.1M NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 015 glycerol, 0.5% sodium deoxycholate

4. Tris-Triton X-100 buffer (Tris-T-100): 10 mM Tris-HCl pH 8.0, 1% SDS, 1% Triton X-100, 1% Tween 20, 1% NP-40

5. PBS-Triton X-100 (PBS-T-100): 10 mM PBS pH 7.0, 1% SDS, 1% Triton X-100, 1% Tween 20, 1% NP-40

Next, TMB-dot blot assay was performed using the biotin-labeled antibody prepared above and proteins extracted using various solutions. Specifically, 0.3 μl of PWN antigen (control, 0.5 μg/μl BSA-conjugated peptide 1 antigen) extracted with the above 5 solutions was transferred to a nitrocellulose membrane (0.45 μm, Merck Millipore, Burlington, MA, USA; 0.2 μm, Pall, Part Washington, NY, USA), and then dried at 45 ^° C for 30 minutes to fix. The fixed NC membrane was blocked with 5% non-fat dried skim milk or TBST containing 1% BSA for 5 minutes, washed three times with TBST, and then diluted 1:2 in monoclonal antibody-secreting cell culture medium (9G6-E6) or 1:200 diluted biotin-labeled PWN-SA571 peptide 1 antibody (remaining 7) at room temperature for 20 to 30 minutes, and washed three times with TBST. The NC membrane reacted with the antibody was blocked again in the same manner as above, and HPR-labeled goat anti-mouse IgA, IgG, IgM or Streptavidin was diluted 1:3,000 and 1:5,000 for 15 minutes. reacted After the reaction, after washing three times, the antibody was reacted with TMB solution for dot blot (ThermoFisher Sci.), and the reactivity of the antibody to the antigen was confirmed by color development.

As a result, all of the 7 biotin-labeled antibodies well recognized the BSA-conjugated-peptide 1 antigen. In addition, the 2C11-A6 and 4A4-G10 antibodies recognized PWN antigens extracted with various solutions, and showed high reactivity to antigens extracted with RIPA and Tris-Cyto among them (FIG. 6).

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pine wood nematode secretory antigen PWN-SA571 specific antibodies and uses its <130> 1 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 136 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 <400> 1 Met Tyr Ser Lys Val Phe Leu Phe Ala Val Val Gly Val Ala Ile Thr 1 5 10 15 Tyr Ala Phe Lys Ile Pro Leu Pro Phe Gly Gln Ile Asn Val Asp Lys 20 25 30 Glu Glu Asp Gly Thr Val Asn Ala Gly Val Asn Ser Asn Ile Asn Ile 35 40 45 Leu Gly Tyr Gly Gly Ser Ser Gly Leu Glu Val Thr Ser Lys Asn Gly 50 55 60 Ser Leu Ala Leu Lys Pro Gln Ala Ala Ile Leu Ala Asn Asn Thr Ala 65 70 75 80 Tyr Gly Ala Asn Ser Thr Tyr Ser Val Asp Lys Lys Glu Gly Ile Val 85 90 95 Ala Asp Thr Asp Val Asp Ala Gly Asn His Thr Phe His Gly Gly Val 100 105 110 Gly Lys Glu Ser Gln Phe Val Asn Glu Val Gly Asp Ala Val Lys Ser 115 120 125 Lys Lys Asn Lys Arg Gly Arg Ala 130 135 <210> 2 <211> 16 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 PEPTIDE 1 <400> 2 Ile Asn Val Asp Lys Glu Glu Asp Gly Thr Val Asn Ala Gly Val Asn 1 5 10 15 <210> 3 <211> 18 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 PEPTIDE 2 <400> 3 Tyr Gly Gly Ser Ser Gly Leu Glu Val Thr Ser Lys Asn Gly Ser Leu 1 5 10 15 Ala Leu <210> 4 <211> 206 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 Mab 6E2-D7 <400> 4 Ser Ser Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys 1 5 10 15 Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala 20 25 30 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp 35 40 45 Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 50 55 60 Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Met Lys Ala Glu Asp 65 70 75 80 Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Tyr Tyr Pro Phe Thr Phe 85 90 95 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr 100 105 110 Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala 115 120 125 Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val 130 135 140 Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser 145 150 155 160 Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr 165 170 175 Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys 180 185 190 Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 <210> 5 <211> 199 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 Mab 7E3-D7 <400> 5 Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Leu Thr Cys Ser 1 5 10 15 Ala Ser Ser Ser Ile Gly Tyr Met His Trp Asn Arg Gln Lys Pro Gly 20 25 30 Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly 35 40 45 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 50 55 60 Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His 65 70 75 80 Gln Arg Ser Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 85 90 95 Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser 100 105 110 Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn 115 120 125 Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser 130 135 140 Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys 145 150 155 160 Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu 165 170 175 Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser 180 185 190 Thr Ser Pro Ile Val Lys Ser 195 <210> 6 <211> 130 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 Mab 8C6-D7 <400> 6 Met Leu Lys Thr Trp Gln Phe Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr 1 5 10 15 Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp 20 25 30 Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr 35 40 45 Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys 50 55 60 Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly 65 70 75 80 Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser 85 90 95 Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn 100 105 110 Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val 115 120 125 Lys Ser 130 <210> 7 <211> 200 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 Mab 2H7-H8-B8 <400> 7 Ala Ser Leu Ser Val Ala Thr Gly Glu Lys Val Thr Ile Arg Cys Ile 1 5 10 15 Thr Ser Thr Asp Ile Asp Asp Lys Met Asn Trp Tyr Gln Gln Lys Pro 20 25 30 Gly Glu Pro Pro Lys Leu Leu Ile Ser Glu Gly Ser Thr Leu Arg Pro 35 40 45 Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Val 50 55 60 Phe Thr Ile Glu Asn Thr Leu Ser Glu Asp Val Ala Asp Tyr Tyr Cys 65 70 75 80 Leu Gln Ser Asp Asn Leu Pro Leu Thr Phe Gly Thr Gly Thr Lys Leu 85 90 95 Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 100 105 110 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 115 120 125 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 130 135 140 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 145 150 155 160 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 165 170 175 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 180 185 190 Ser Thr Ser Pro Ile Val Lys Ser 195 200 <210> 8 <211> 200 <212> PRT <213> artificial sequence <220> <223> PWN-SA571 1A4-B11-A2 <400> 8 Ala Ser Leu Ser Val Ala Thr Gly Glu Lys Val Thr Ile Arg Cys Ile 1 5 10 15 Thr Ser Thr Asp Ile Asp Asp Lys Met Asn Trp Tyr Gln Gln Lys Pro 20 25 30 Gly Glu Pro Pro Lys Leu Leu Ile Ser Glu Gly Ser Thr Leu Arg Pro 35 40 45 Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Val 50 55 60 Phe Thr Ile Glu Asp Thr Leu Ser Glu Asp Val Ala Asp Tyr Tyr Cys 65 70 75 80 Leu Gln Ser Asp Asn Leu Pro Leu Thr Phe Gly Thr Gly Thr Lys Leu 85 90 95 Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 100 105 110 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 115 120 125 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 130 135 140 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 145 150 155 160 Arg Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 165 170 175 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 180 185 190 Ser Thr Ser Pro Ile Val Lys Ser 195 200 <210> 9 <211> 411 <212> DNA <213> artificial sequence <220> <223> PWN-SA571 <400> 9 atgtactcca aggtgttcct ctttgccgtt gtcggcgttg caatcacata tgcattcaaa 60 attccccttc catttggcca gataaacgtc gacaaagaag aagacgggac ggtgaatgct 120 ggtgtcaact cgaatataaa tattcttgga tacggtggaa gttccggtct tgaagtgact 180 tcgaagaatg gcagcttggc tttaaaaccc caagcagcaa tcctcgctaa caacaccgcc 240 tatggagcca attcaactta ctctgtggac aagaaagagg gaattgtggc tgacactgat 300 gtagatgctg gaaatcacac cttccatggt ggagttggaa aagaatctca attcgtcaac 360 gaagtgggtg acgccgtcaa gtccaagaag aataagagag gtcgcgcgta a 411 <210> 10 <211> 618 <212> DNA <213> artificial sequence <220> <223> PWN-SA571 Mab 6E2-D7 <400> 10 tcctccctag ctgtgtcagt tggagagaag gttactatga gctgcaagtc cagtcagagc 60 cttttatata gtagcaatca aaagaactac ttggcctggt accagcagaa accagggcag 120 tctcctaaac tgctgattta ctgggcatcc actagggaat ctggggtccc tgatcgcttc 180 acaggcagtg gatctgggac agatttcact ctcaccatca acagtatgaa ggctgaagac 240 ctggcagttt attactgtca gcaatattat tactatccat tcacgttcgg ctcggggaca 300 aagttggaaa taaaacgggc tgatgctgca ccaactgtat ccatcttccc accatccagt 360 gagcagttaa catctggagg tgcctcagtc gtgtgcttct tgaacaactt ctaccccaaa 420 gacatcaatg tcaagtggaa gattgatggc agtgaacgac aaaatggcgt cctgaacagt 480 tggactgatc aggacagcaa agacagcacc tacagcatga gcagcaccct cacgttgacc 540 aaggacgagt atgaacgaca taacagctat acctgtgagg ccactcacaa gacatcaact 600 tcacccattg tcaagagc 618 <210> 11 <211> 597 <212> DNA <213> artificial sequence <220> <223> PWN-SA571 Mab 7E3-D7 <400> 11 gcaatcatgt ctgcatctcc aggggagaag gtcaccctga cctgcagtgc cagctcaagt 60 ataggttaca tgcactggaa ccgtcagaag ccaggcacct cccccaaaag atggatttat 120 gacacatcca aactggcttc tggagtccct gctcgcttca gtggcagtgg gtctgggacc 180 tcttattctc tcacaatcag cagcatggag gctgaagatg ctgccactta ttactgccat 240 cagcggagta gttacccatg gacgttcggt ggaggcacca agctggaaat caaacgggct 300 gatgctgcac caactgtatc catcttccca ccatccagtg agcagttaac atctggaggt 360 gcctcagtcg tgtgcttctt gaacaacttc taccccaaag acatcaatgt caagtggaag 420 attgatggca gtgaacgaca aaatggcgtc ctgaacagtt ggactgatca ggacagcaaa 480 gacagcacct acagcatgag cagcaccctc acgttgacca aggacgagta tgaacgacat 540 aacagctata cctgtgaggc cactcacaag acatcaactt cacccattgt caagagc 597 <210> 12 <211> 619 <212> DNA <213> artificial sequence <220> <223> PWN-SA571 Mab 8C6-D7 <400> 12 gctcttgaca atgggtgaag ttgatgtctt gtgagtggcc tcacaggtat agctgttatg 60 tcgttcatac tcgtccttgg tcaacgtgag ggtgctgctc atgctgtagg tgctgtcttt 120 gctgtcctga tcagtccaac tgttcaggac gccattttgt cgttcactgc catcaatctt 180 ccacttgaca ttgatgtctt tggggtagaa gttgttcaag aagcacacga ctgaggcacc 240 tccagatgtt aactgctcac tggatggtgg gaagatggat acagttggtg cagcatcagc 300 ccgttttatt tccagcttgg tcccccctcc gaacgtgtac ggatagctat aatattgctg 360 acagtaataa aactgccagg tcttcagcct tcacactgct gatggtgaga gtgaaatctg 420 tccccagatcc actgcctgtg aagcgatcag ggaccccaga ttcccctagtg gatgcccagt 480 aaatcagcag tttaggagac tgccctggtt tctgctggta ccaggccaag tagttctttt 540 gattgctact atataaaagg ctctgactgg acttgcagct catagtaacc ttctctccaa 600 ctgacacagc tagggagga 619 <210> 13 <211> 600 <212> DNA <213> artificial sequence <220> <223> PWN-SA571 Mab 2H7-H8-B8 <400> 13 gcatccctgt ccgtggctac aggagaaaaa gtcactatca gatgcataac cagcactgat 60 attgatgata aaatgaactg gtaccagcag aagccagggg aacctcctaa gctccttatt 120 tcagaaggca gtactcttcg tcctggagtc ccatcccgat tctccagcag tggctctggc 180 acagattttg tttttacaat tgagaacacg ctctcagaag atgttgcaga ttactactgt 240 ttgcaaagtg ataacttgcc tctcacgttc ggtactggga ccaagctgga gctgaaacgg 300 gctgatgctg caccaactgt atccatcttc ccaccatcca gtgagcagtt aacatctgga 360 ggtgcctcag tcgtgtgctt cttgaacaac ttctacccca aagacatcaa tgtcaagtgg 420 aagattgatg gcagtgaacg acaaaatggc gtcctgaaca gttggactga tcaggacagc 480 aaagacagca cctacagcat gagcagcacc ctcacgttga ccaaggacga gtatgaacga 540 cataacagct atacctgtga ggccactcac aagacatcaa cttcacccat tgtcaagagc 600 600 <210> 14 <211> 600 <212> DNA <213> artificial sequence <220> <223> PWN-SA571 1A4-B11-A2 <400> 14 gctcttgaca atgggtgaag ttgatgtctt gtgagtggcc tcacaggtat agctgttatg 60 tcgttcatac tcgtccttgg tcaacgtgag ggtgctgctc atgctgtagg tgctgtctct 120 gctgtcctga tcagtccaac tgttcaggac gccattttgt cgttcactgc catcaatctt 180 ccacttgaca ttgatgtctt tggggtagaa gttgttcaag aagcacacga ctgaggcacc 240 tccagatgtt aactgctcac tggatggtgg gaagatggat acagttggtg cagcatcagc 300 ccgtttcagc tccagcttgg tcccagtacc gaacgtgaga ggcaagttat cactttgcaa 360 acagtagtaa tctgcaacat cttctgagag cgtgtcctca attgtaaaaa caaaatctgt 420 gccagagcca ctgctggaga atcgggatgg gactccagga cgaagagtac tgccttctga 480 aataaggagc ttaggaggtt cccctggctt ctgctggtac cagttcattt tatcatcaat 540 atcagtgctg gttatgcatc tgatagtgac tttttctcct gtagccacgg acagggatgc 600 600

Claims (11)

An anti-PWN-SA571 antibody or antigen-binding fragment thereof, which specifically binds to the PWN-SA571 fragment of the pine wood nematode secretory protein consisting of the amino acid sequence of SEQ ID NO: 2 or 3.
According to claim 1,
The PWN-SA571 is a protein secreted when the pine wood nematode decomposes pine trees, the anti-PWN-SA571 antibody or antigen-binding fragment thereof.
delete delete A composition for diagnosing pine wood nematode infection, comprising the antibody or antigen-binding fragment thereof of claim 1 or 2.
A kit for diagnosing pine wood nematode infection, comprising the composition of claim 5.
delete (A) obtaining a sample from the subject;
(B) comprising the step of reacting the obtained sample with the antibody or antigen-binding fragment thereof of claim 1 or 2, a method for diagnosing pine wood nematode infection.
According to claim 8,
The step (B) is a step of forming an antigen-antibody complex by reacting the antibody or antigen-binding fragment thereof with the pinewood nematode-derived antigen contained in the sample.
According to claim 8,
The step of obtaining a sample of step (A) is to use a solution that does not contain Triton X-100, a method for diagnosing pine wood nematode infection.
According to claim 8,
The method comprises the steps of (C) detecting an antigen-antibody complex of the antibody or antigen-binding fragment thereof and the antigen in the sample, and diagnosing the sample forming the complex as being infected with the pine wilt nematode.
A method for diagnosing pine wood nematode infection, which further comprises a.

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