KR20210131045A - Pine wood nematode secretory antigen PWN-SA571 specific antibodies and uses thereof - Google Patents

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to PWN-SA571, a pinewood nematode secreted protein, a composition for diagnosing the pinewood nematode (PWN) infection comprising the same, a kit for diagnosing the PWN infection comprising the composition, and a method for providing the information necessary for diagnosing the PWN using the composition or the kit. According to the present invention, the antibody or antigen-binding fragment thereof that specifically binds to PWN-SA571, a pinewood nematode secreted protein, can effectively detect a protein or antigen derived from the PWN, so that it can efficiently determine whether a pine tree is infected with the PWN.

Description

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 the pine wood nematode secretion protein PWN-SA571, a composition for diagnosing pine wilt nematode infection comprising the same, a kit for diagnosing pine wilt nematode infection comprising the composition, and pine 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. It is known that most trees of the genus Pine except for trifolium, which are native to North America, are infected with PWN. It is known that transmission of pathogenic PWN is made by long-horned beetles belonging to Genus Monochamus. More than 163 species of blue oxen belonging to the genus Apigenus are potential vectors of PWN that have a worldwide distribution. Therefore, there is a very high possibility that potential vectors of PWN exist even in the Old Continent, where PWN has not invaded so far. In fact, in the case of continental Europe where PWN is currently spreading, various baleen oxen exist, and these are spreading the spread of PWN.

Trees belonging to the genus of pines susceptible to PWN include Pinus densiflora, Pinus korainensis, Pinus parviflora, Pinus pinaster, Pinus radiate, Pinus thunbergii, and Guju. Pine (Pinus Sylvestris) and the like are known. Among the pines distributed in Europe, in the case of P. pinea , which is mainly distributed along the coast of Italy, it is known that PWN does not proliferate as much as other susceptible pines. And, CJFL in PWN-sensitive pine trees with abamectin is known to prevent PWN infection, so for the protection of important pine trees with historical or social significance, canopy injection using abamectin or its derivatives is important for PWN infection It is used as a preventive measure.

However, since it is currently impossible to perform canopy injection on all pine trees in the PWN-infected area, the best solution is to suppress the spread of PWN. It is known that PWN is transmitted to other pine trees by transferring an infected tree to a non-infected area by humans rather than by naturally being transmitted by alfalfa. Therefore, it is essential to develop a method to check 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 region, microsatellite, or general genes have been reported. There is a difficult side. In addition, a diagnostic method using loop-mediated isothermal amplification and recombinant polymerase amplification, which can amplify DNA at isothermal conditions, has been developed. These methods have the advantage of being able to quickly and quickly diagnose than the existing PCR-based diagnostic methods. Another diagnostic method is a method of diagnosing using an antigen/antibody reaction. However, until now, an antigen/antibody-based technology that can diagnose Pine wilt nematode-infected trees has not been developed.

Accordingly, the present inventors tried to develop a method for diagnosing PWN infection using an antigen/antibody reaction. As a result, they found PWN-SA571, one of the PWN-secreted antigens present in the PWN-infected tree, and produced a monoclonal antibody against it. The present invention was completed by developing a method for determining whether PWN infection is present.

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 secreted protein PWN-SA571.

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

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

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

This will be described in detail as follows. On the other hand, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description 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 secreted protein PWN-SA571.

As used throughout this specification, the term "Pine wood nematode (PWN; scientific name: Bursaphelenchus xylophilus )" is a thread-like nematode with a size of about 1 mm and inhabits in the body of vector insect vectors ( Monochamus alternatus, borealis, etc.) When gnawing at young shoots, they invade trees through wounds.The invading nematodes multiply rapidly and block the passage of moisture and nutrients to cause pine nematode disease, which kills the tree. The pine wilt nematode infects pine trees such as pine, sea pine, pine, and pine, and causes disease.

As used throughout this specification, the term "Pine wood nematode secretory antigen 571 (Pine wood nematode secretory antigen 571)" is one of the proteins derived from the pine wilt nematode, and the protein is a protein secreted by the pine nematode invading into 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 specifically, may include 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, and specifically, SEQ ID NO: 2 or sequence which is a part of the amino acid sequence of SEQ ID NO: 1 It may be one that specifically binds to a peptide comprising the amino acid sequence of number 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, and specifically, the amino acid sequence of SEQ ID NO: 1 It may be one using 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 consists of two each of a light chain and a heavy chain, and each chain consists of a variable domain having a variable amino acid sequence and a constant domain having a constant sequence. . In the antibody, an antigen-binding site is located at the end of the three-dimensional structure of the variable region, and this region is formed by a collection of three complementarity determining regions present in each of the light and heavy chains. The complementarity determining region is a region with particularly high amino acid sequence variability among the variable regions, and specific antibodies to various antigens can be found by such high variability. The scope of the present application includes not only complete antibody forms, but also antigen-binding fragments of such 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 However, it is not limited thereto.

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

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

In one embodiment of the present application, the amino acid sequence of SEQ ID NO: 4 to SEQ ID NO: 8 may be encoded by the nucleotide sequence of SEQ ID NO: 10 to 14, respectively.

A second aspect of the present application provides a composition for diagnosing Pine wilt nematode infection, comprising the anti-PWN-SA571 antibody or antigen-binding fragment thereof. The content overlapping with the first aspect is equally applied to the composition for diagnosis of pine wilt nematode infection of the second aspect.

In one embodiment of the present application, the diagnosis of the pine wilt nematode infection is to determine whether the pine wilt nematode is infected in the individual, and a disease caused by the pine wilt nematode infection, specifically, the pine wilt nematode disease includes confirming whether or not may be doing

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 be one that specifically binds to a peptide comprising the 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 the amino acid sequence of any one of SEQ ID NOs: 4 to 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 wilt nematode infection, comprising the composition for diagnosing pine wilt nematode infection. The contents overlapping with the first aspect and the second aspect are equally applied to the pine wilt nematode infection diagnosis kit of the third aspect.

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

In one embodiment of the present application, the kit may include an agent capable of detecting an antigen-antibody complex, specifically, the anti-PWN-SA571 antibody or antigen-binding fragment thereof of the present application and PWN-SA571 protein or a protein thereof 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 and reagents commonly used in the art used for immunological analysis. Such tools/reagents may include, but are not limited to, suitable carriers, labels capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling material is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. 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, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles 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) kit, complement fixation assay kit, FACS kit, protein chip kit, immunofluorescence kit, chemiluminescent material binding assay kit, chromatography kit, magnetic activated cell sorting, MACS) kit, but is not limited thereto, as long as it can detect a protein or antigen derived from Pine wilt nematode or the antigen-antibody complex.

Specifically, labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive isotopes, and must be limited thereto. it's not going to be Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxy Dase, 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 materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrine, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Microparticles include, but are not limited to, nano gold, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, viologen, 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- , and the like, but are not limited thereto. Radioisotopes 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, ¹⁸⁶ Re, and the like.

A fourth aspect of the present application is a method comprising: (A) obtaining a sample from a subject; And (B) comprising the step of reacting the obtained sample with the anti-PWN-SA571 antibody or antigen-binding fragment thereof of any one of claims 1 to 4, providing an information providing method for diagnosing Pine wilt nematode infection do. The contents overlapping with the first to third aspects are equally applied to the information provision method for diagnosing tree wilt 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 the Pine wilt nematode infection.

As used throughout this specification, the term "individual" refers to any organism that is infected or likely to be infected with the pine wilt nematode, and specifically, pine (Pinus densiflora), pine (Pinus korainensis), cypress (Pinus parviflora), French coastal pine (Pinus pinaster), radiata pine (Pinus radiate), gom pine (Pinus thunbergii), and may include pine trees such as sylvestris (Pinus Sylvestris), but is not limited thereto. In addition, the subject may refer to any organism that is infected with pine wilt nematode and has the potential to develop or develop pine wilt disease.

As used throughout this specification, the term "sample" refers to 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, or peptide. , antigens, antigen fragments, nucleic acids, and the like, as long as the PWN-SA571-derived protein can be specifically detected from the Pine wilt nematode-derived protein, and the type thereof is not limited thereto.

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

In one embodiment of the present application, the step of obtaining the 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 (C) detects 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 to form the complex It may further include the step of diagnosing as being infected with pine wilt 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 normal control sample with the anti-PWN-SA571 antibody or antigen-binding fragment thereof may be doing

In one embodiment of the present application, the anti-PWN-SA571 antibody or antigen-binding fragment thereof may include the amino acid sequence of any one of SEQ ID NOs: 4 to 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.

The antibody or antigen-binding fragment thereof that specifically binds to the pine wilt nematode secreted protein PWN-SA571 of the present application can effectively detect a protein or antigen derived from pine wilt nematodes, so that it can efficiently diagnose whether pine wilt nematodes are infected. can

1 is a diagram showing the similarity of PWN-SA571, which is a pine wilt nematode secreted protein, and proteins of similar nematodes.
2 is a diagram showing the results of signal peptide prediction analysis of PWN-SA571 protein.
3 is a result 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 nematodes [PWN ( Bursaphelenchus) xylophilus ), Bursaphelenchus mucronatus, Bursaphelenchus doui, Bursaphelenchus thailande ) extracts were analyzed for immunoreactivity by ELISA.
5 is a result of analyzing the immunoreactivity of the anti-PWN-SA571 monoclonal antibody of the present application by a nano-gold dot blot assay.
6 is a 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 application will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: Discovery of PWN-SA571, a protein derived from pine wilt nematodes

In order to develop a composition and method for diagnosing pine wilt nematode infection, the following experiments were performed to discover novel proteins derived from pine wilt nematodes and to prepare antigens for them.

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

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

PWN-SA571 was identified as a protein secreted when the pine wilt nematode decomposes pine trees, and a signal peptide may exist at the N-terminus. The location and the like were analyzed. As a result, amino acid sequences 1 to 17 of PWN-SA571 were predicted to be signal peptides ( FIG. 2 ), and it was confirmed that the molecular weight of PWN-SA571 excluding the signal peptide was 12.25 kDa and pI was 6.94.

Example 2: PWN-SA571 antigen peptide construction

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

Specifically, using various web-based antigenicity prediction tools, Antigenicity analysis was performed for PWN-SA571. 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 antigenicity and water solubility of the peptides, respectively (peptide 1, SEQ ID NO: 2). ) and 18 (peptide 2, SEQ ID NO: 3) amino acids were prepared as an antigenic peptide.

Antigenicity prediction
program
SVMTriP Immunomedicine Group EMBOSS explorer antigenic Peptide antigen 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 EDGTVNAGVN 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} GIVADT ¹⁰⁰ 7 ¹⁰¹ VDAGNHTFHG ^{110 118} FVNEVGDAVK ^{127 119} VNEVGDAVKS ¹²⁸ 8 ¹¹⁷ QFVNEVGDAV ¹²⁶

Example 3: Preparation of monoclonal antibody against PWN-SA571

The following experiment was performed to prepare a monoclonal antibody against PWN-SA571 using the two antigenic peptides prepared in Example 2 above.

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 an antigen for mouse immunization and ELISA (Enzyme linked) were conjugated. immunosorbent assay) antigens for screening were prepared, respectively.

Next, a mouse immunization method and ELISA were performed to prepare a monoclonal antibody. The experiment was performed under 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, two antigens each bound with KLH and two antigen peptides were mixed with Freund's complete or incomplete adjuvant (Merck KGaA, Darmstadt, Germany) and injected into four Balb/C mice to induce immune action in mice. did.

Next, in order to select antibody-secreting cells that recognize the two types of antigenic peptides, blood is collected from the mice receiving the antigen injection and the reactivity is confirmed using the BSA-conjugated antigen peptide, A fusion cell line library was prepared by fusion of splenocytes and the 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, 16 hybridoma cell clones showing high reactivity to peptide 1 were obtained, and then, 8 sub-clones showing high reactivity were selected by performing limiting dilution to establish monoclonal antibodies. In addition, after securing 10 hybridoma cell clones showing high reactivity to peptide 2, limiting dilution was performed to select 5 sub-clones showing high reactivity to establish 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 monoclonal antibodies against PWN-SA571 peptide 1 were IgG2b and the rest were all IgM (Table 2). 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 wilt 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 kinds of nematodes [PWN ( Bursaphelenchus xylophilus ), Bursaphelenchus mucronatus, Bursaphelenchus doui, Bursaphelenchus thailandae ) The reactivity to the extract was confirmed by ELISA.

Specifically, each antigen was diluted to 0.2 μg/ml using a coating solution (0.4M carbonate buffer, pH9.6), aliquoted into a 96-well plate, and reacted at 4°C overnight, 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-prepared cell culture medium was added, reacted at room temperature for 1 hour, and washed 3 times with PBS (PBST) containing 0.25% Tween-20. Next, 50 μl of the secondary antibody was added, and the reaction proceeded with stirring at room temperature for 1 hour, followed by washing 3 times with PBST. Next, the TMB solution for ELISA (ThermoFisher Scii.) was put and reacted at room temperature for 30 minutes, 2M sulfuric acid was added to stop the reaction, and the 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. The first group is monoclonal antibodies showing high reactivity to P. nematode-infected trees, including 7E4-E9, 4A4-G10, and 7E3-D7. The antibodies were not as high in sensitivity to pine wilt nematodes compared to the other three similar nematodes. The second group is monoclonal antibodies showing high reactivity to the pine wilt nematode, and includes 2E6-C11, 2C11-A6, and 9G6-E7. The antibodies showed a lower reactivity to pine wilt nematode-infected trees compared to the first group. The third group includes antibodies 8C6-D7 and 6E2-D7, which are not high but have selective reactivity to PWN and high reactivity to BSA-conjugated peptide 1 ( FIG. 4A ).

In addition, the monoclonal antibodies against PWN-SA571 peptide 2 showed high reactivity to the BSA-conjugated peptide compared to the free peptide in most cases, but there was a difference in reactivity to PWN or PWN infected trees. Specifically, among monoclonal antibodies, 4F12-H7-A3 and 5D4-H4-D4 showed selective reactivity against PWN and PWN-infected trees. 2H7-H8-B8, which showed the highest reactivity to BSA-conjugated peptide 2, had no selective reactivity against 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 pseudonematode, but showed more than twice the reactivity compared to the whole tree ( FIG. 4B ).

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

A method for producing high titer monoclonal antibodies is to prepare ascite produced by injecting hybridoma cells into the abdominal cavity of a mouse. Therefore, ascites of 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 ago, and the abdomen of the mouse swelled. waited until After carefully collecting ascite using a syringe from the inflated abdomen, it was immediately aliquoted and stored at -80°C. In addition, after removing impurities from the ascite with a 0.2 μm size scanning filter, ^{IgM or IgG present in high concentration was separated using Poros TM} CaptureSelect ^TM IgM Affinity Matrix (ThermoFisher Sci) or protein G-agarose packed in an FPLC column. Thus, the antibody was purified on ascite.

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

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

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

Example 6: Anti-PWN-SA571 Antigen Reactivity Confirmation of Anti-PWN-SA571 Monoclonal Antibody Using Nano Gold-Dot Blot Assay

In order to confirm the reactivity to the Pine wilt nematode antigen for the monoclonal antibodies prepared in Example 3, the following experiment was performed.

Specifically, in order to perform a dot blot assay using nano-gold (Nano-gold), 0.02 ~ 1.0 μg of BSA-conjugated PWN-SA571 peptide 1 or 2 was added to the lateral diffusion strip. strip) (Prime4dia, Anyang, Korea), ^{dried at 45 o} C for 30 minutes and fixed. 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), 20 μl of monoclonal antibody supernatant, and 20 μl of monoclonal antibody supernatant in a 96-well plate. After mixing in the wells, the side diffusion strips were dipped with the filter paper side up. When the monoclonal antibody and anti-mouse IgG nanogold complex move along the side diffusion strip and meet the antigen of peptide 1 or 2, it precipitates and develops 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, the 2C11-A6, 7E3-D7, 7F4-E9, 8C6-D7, 4A4-G10 monoclonal antibody showed high reactivity ( FIG. 5A ), and also, the antibody Among the 7E3-D7, 7F4-F9, and 8C6-D7 monoclonal antibodies, as a result of confirming the reactivity to the antigen, it was confirmed that all showed reactivity to the antigen 0.1 mg (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 ( Figure 5D).

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

In order to confirm the reactivity to the pine wilt nematode antigen for the monoclonal antibodies prepared in Example 3, a TMB-dot blot assay was performed as follows.

First, since the sensitivity can be improved by labeling the purified monoclonal antibody with biotin, the seven purified monoclonal antibodies against PWN-SA571 peptide 1 were labeled with biotin by the following method. Specifically, 6.7 μl of 20.0 mM N-Hydroxysulfosuccinimide (NHS)-polyethylene glycol 4 (PEG4)-Biotin Dimethylformamide (DMF) per 1 mg of purified antibody immunoglobulin was added, followed by stirring 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 order to select the optimal solution for extracting antigens from the pine wilt nematode in performing the pine wilt infection diagnosis method, PWN was extracted using the following five buffers.

1. RIPA Buffer: 50mM 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, mix with SDS Sample buffer (SDS-SB) in a ratio of 4:1 and heat 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, a TMB-dot blot assay was performed using the biotin-labeling 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 five solutions was mixed with a nitrocellulose membrane (0.45 μm, Merck Millipore, Burlington, MA, USA; 0.2 μm, Pall, Part Washington, NY, USA) and then ^{dried at 45 o} C for 30 minutes and fixed. The fixed NC membrane was blocked with TBST containing 5% non-fat dried skim milk or 1% BSA for 5 minutes, washed 3 times with TBST, and then a culture solution of monoclonal antibody-secreting cells diluted 1:2 (9G6-E6) or 1:200 diluted biotin-labeling PWN-SA571 peptide 1 antibody (the remaining 7) and reacted at room temperature for 20-30 minutes, and washed 3 times with TBST. The NC membrane reacted with the antibody was again blocked in the same manner as above, and then 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 3 times, it 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 above seven biotin-labeling antibodies recognized the BSA-conjugated-peptide 1 antigen well. In addition, in the case of 2C11-A6 and 4A4-G10 antibodies, PWN antigens extracted with various solutions were recognized, and among them, they showed high reactivity to antigens extracted with RIPA and Tris-Cyto (FIG. 6).

From the above description, those skilled in the art to which the present invention pertains will 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 restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pine wood nematode secretory antigen PWN-SA571 specific antibodies and uses them <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 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 tcccagatcc actgcctgtg aagcgatcag ggaccccaga ttccctagtg 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 acagatttg 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, characterized in that it specifically binds to PWN-SA571 secreted protein PWN-SA571.
According to claim 1,
The antibody or antigen-binding fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 4 to 8, anti-PWN-SA571 antibody or antigen-binding fragment thereof.
According to claim 1,
The antibody or antigen-binding fragment thereof is an anti-PWN-SA571 antibody or antigen-binding fragment thereof, characterized in that the antigen is a Pine wilt nematode secreted protein consisting of the amino acid sequence of SEQ ID NO: 1.
According to claim 1,
The antibody or antigen-binding fragment thereof is an anti-PWN-SA571 antibody or antigen-binding fragment thereof, characterized in that the antigen is a Pine wilt nematode secreted protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
A composition for diagnosis of Pine wilt nematode infection, comprising the antibody of any one of claims 1 to 4 or an antigen-binding fragment thereof.
A kit for diagnosing pine wilt nematode infection, comprising the composition of claim 5.
7. The method of claim 6,
The kit is a kit for diagnosing Pine wilt nematode infection, which includes an agent capable of detecting the antigen-antibody complex.
(A) obtaining a sample from the subject;
(B) reacting the obtained sample with the anti-PWN-SA571 antibody or antigen-binding fragment thereof of any one of claims 1 to 4
Including, information providing method for diagnosing pine wilt nematode infection.
9. The method of claim 8,
The step (B) is the step of forming an antigen-antibody complex by reacting the antigen-derived pine wilt nematode contained in the sample with the antibody or antigen-binding fragment thereof, an information providing method for diagnosing pine wilt nematode infection.
9. The method of claim 8,
The step of obtaining the sample of step (A) is to use a solution that does not contain Triton X-100, an information providing method for diagnosing pine wilt nematode infection.
9. The method of claim 8,
The method comprises the steps of (C) detecting the 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 Pine wilt nematode.
The method of providing information for diagnosing pine wilt nematode infection, further comprising a.

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