KR102476515B1 - Antibody for periodontal disease and use thereof - Google Patents

Abstract

The present invention provides a novel antibody capable of providing rapid diagnostic information on periodontal disease. The antibody according to the present invention has high affinity for Arg-gingipain B, Lys-gingipain, or both antigens, and exhibits excellent effects as an in vitro diagnostic kit for periodontal disease.

Description

Periodontal disease specific antibody and use thereof {ANTIBODY FOR PERIODONTAL DISEASE AND USE THEREOF}

The present invention relates to a periodontal disease-specific antibody, and more particularly, to an antibody specific to RgpB as an Arg-specific gingipain and Kgp as a Lys-specific gingipain, and a method for diagnosing periodontal disease using the same.

The incidence of periodontal disease increases with age. When periodontal disease is confined to the gums, it is called gingivitis, and when this inflammation spreads to the gums and around the gums, it is called periodontitis. As periodontitis progresses, it damages the periodontal ligament and even the alveolar bone. One example of alveolar bone damage is alveolar bone osteodystrophy, which further subdivides into alveolar bone osteoporosis, alveolar bone osteomalacia, and alveolar bone osteopenia. When the alveolar bone is damaged, the teeth are eventually lost.

Approximately 7-15% of adults suffer from periodontitis, which is one of the most common diseases. A multifactorial disease is when the presence of pathogenic bacteria in the pocket between the teeth and gums is an essential but not sufficient criterion. The host inflammatory and immune systems also play an important role in the pathogenesis of the disease. The progression of periodontitis is thought to be a chronic inflammatory response to bacteria that leads to destruction of the supporting tissue of the teeth. Its behavior is cyclical and may remain overlooked at an early stage.

The destruction process is thought to be the result of complex interactions between the host's defense system and specific bacterial species in the periodontal pocket. Pathogenic bacteria involved in the development and progression of periodontal disease include, but are not limited to, Porphyromonas gingivalis gingivalis ), Bacteroides forcitus ( Bacteroides forsythus ), Actinobacillus Actinomycetem comitans ( Actinobacillus actinomycetemcomitans ), Triponema denticola ( Triponema denticola ) and Prevotella intermedia ( Prevotella intermedia ).

More recently, the importance of bacterial virulence products (mainly toxins and enzymes) has been widely studied and thought to play a major role in pathogenesis. Bacteria are thought to go through different phases of growth and become more or less destructive in periodontal pockets during these phases of growth. Active bacteria produce virulence products to aid their survival and nutrition in periodontal pockets. During periods of elevated bacterial activity, these virulence products contribute to the destruction of the tooth's supporting tissue and to a decrease in the effectiveness of the host's defense system.

Dentists today check for periodontitis by measuring the probing depth of the periodontal pocket, examining X-ray images of tooth attachment to alveolar bone, and noting bleeding on probing. Risk factors include smoking habits, stress, and a family history of periodontitis. The method relies heavily on the personal expertise of the dentist. Probe depth is only a measure of histological attachment loss and thus serves little to inform the actual occurrence of periodontitis or its future progression.

Occasionally, a microbial sample is taken and sent to a laboratory for analysis by culture or DNA-technology (the checkerboard DNA-DNA hybridization technique developed by Sokranski). However, the results show only the presence of certain bacteria, which are only noticeable after about a week and do not necessarily indicate periodontitis. In addition, a vast amount of work has to be done to discover compounds, mainly enzymes or proteins such as cytokines, in liquids from the patient's oral cavity, such as gingival fissure fluid (GCF), which can diagnose or predict the progression of periodontitis. this exists

For the treatment of periodontal disease, it is important to promptly diagnose the onset of the disease at the beginning, predict its risk, and appropriately distribute it to treatment, monitoring, and patient management targets. Therefore, there is a need for an accurate diagnosis that can be treated appropriately and early.

Republic of Korea Patent Publication No. 10-2020-0066749

In order to overcome the above problems, the present inventors invented a novel antibody for diagnosing periodontal disease, and completed the present invention by preparing a rapid diagnosis kit using the antibody.

An object of the present invention is to provide an antibody or antigen-binding fragment thereof specific for Arg-specific gingipain (RgpB) and/or Lys-specific gingipain (Kgp).

The present invention also provides a composition for diagnosing periodontal disease comprising an Arg-specific gingipain and/or Lys-specific gingipain-specific antibody or antigen-binding fragment thereof.

The present invention also provides a kit for diagnosing periodontal disease comprising an Arg-specific gingipain and/or Lys-specific gingipain-specific antibody or antigen-binding fragment thereof.

The present invention also relates to detection or quantification of antigens of Arg-gingipain B, Lys-gingipain or both, including reacting an Arg-specific gingipain and/or Lys-specific gingipain-specific antibody or antigen-binding fragment thereof with a sample. provides a way

The present invention also provides a method for diagnosing periodontal disease, comprising reacting an Arg-specific gingipain and/or Lys-specific gingipain-specific antibody or antigen-binding fragment thereof with a sample.

Accordingly, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Arg-gingipain B, Lys-gingipain, or both.

Specifically, the antibody or antigen-binding fragment thereof may be any one or more of the following:

CDR1 region composed of the amino acid sequence of SEQ ID NO: 1, CDR2 region composed of the amino acid sequence of SEQ ID NO: 2, and CDR3 region composed of the amino acid sequence of SEQ ID NO: 3 and CDR1 region composed of the amino acid sequence of SEQ ID NO: 4 , CDR2 region consisting of the amino acid sequence of SEQ ID NO: 5, and light chain variable region comprising a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 6; Arg-ginjipine B, Lys-ginjipine or an antibody for both of them, or an antigen-binding fragment thereof; or

A CDR1 region composed of the amino acid sequence of SEQ ID NO: 7, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 8, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 9 and a CDR1 region composed of the amino acid sequence of SEQ ID NO: 10 , CDR2 region consisting of the amino acid sequence of SEQ ID NO: 11, and light chain variable region comprising a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 12; Arg-ginjipine B, Lys-ginjipine or an antibody for both of them, or an antigen-binding fragment thereof.

Parts other than the parts corresponding to SEQ ID NOs: 1 to 6 and/or SEQ ID NOs: 7 to 12 in the antibody may be corresponding parts (other than CDR parts) of any antibody.

An "antibody" of the present invention may be used as a complete form having two full-length light chains and two full-length heavy chains, as well as fragments of antibody molecules. The term includes chimeric antibodies, humanized antibodies, human antibodies and bivalent or bispecific molecules (eg bispecific antibodies), diabodies, triabodies and tetrabodies. The term further includes single-chain antibodies, scabs, derivatives of antibody constant regions and artificial antibodies based on protein scaffolds that have a binding function to FcRn. A full antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes. The antibody fragment refers to a fragment having an antigen-binding function, and includes Fd, Fab, Fab', F(ab')2, and Fv. The Fd refers to the heavy chain part included in the Fab fragment. 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 single 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, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab') 2 fragments can be obtained by digestion with pepsin), specifically It can be produced through genetic recombination technology.

"Monoclonal antibody" refers to an antibody molecule of single molecular composition obtained from a population of substantially identical antibodies, such monoclonal antibodies displaying a single binding specificity and affinity for a particular epitope.

Typically, an immunoglobulin has a heavy chain and a light chain, each comprising a constant region and a variable region (the regions are also known as domains). The variable regions of the light and heavy chains include three variable regions called complementarity-determining regions (hereinafter referred to as “CDRs”) and four framework regions. The CDR mainly serves to bind to an antigenic epitope. The CDRs of each chain are typically called CDR1, CDR2, CDR3 sequentially starting from the N-terminus, and are also identified by the chain in which a particular CDR is located.

The "antigen" of the present invention is a molecule containing a site that binds to an antibody, and the antigen molecule has a three-dimensional molecular structure that determines antigen specificity, reacts with the antibody corresponding thereto, and converts the structural site into an epitope or epitope. It is said.

The term "antigen-binding portion" of an antibody (or simply 'antibody portion') refers to one or more fragments of an antibody that have the ability to specifically bind to an antigen. The antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term 'antigen-binding portion' of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., Nature 341:544, 1989); and (vi) an isolated complementarity determining region (CDR).

The antibody or antigen-binding fragment thereof comprising SEQ ID NOs: 1 to 6 as the CDR region comprises a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13 and/or a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14. It may be an antibody or antigen-binding fragment thereof comprising a.

The antibody or antigen-binding fragment thereof comprising SEQ ID NOs: 7 to 12 as the CDR region comprises a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15 and/or a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16 It may be an antibody or antigen-binding fragment thereof comprising the region.

SEQ ID NO: 13 and SEQ ID NO: 14 in the antibody; And / or parts other than the parts corresponding to SEQ ID NO: 15 and SEQ ID NO: 16 may be the corresponding parts (parts other than the light chain variable region and the heavy chain variable region) of any antibody.

The antibody according to the present invention has binding ability to specifically bind to Arg-gingipain B, Lys-gingipain or both.

Several types of proteolytic enzymes with trypsin-like protease activity produced by P. gingipalis are known, and among them, Lys-gingipain (abbreviated as “KGP”) and Arg-gingipain (Arg-gingipain) gingipain, abbreviated as "Rgp-B") is a major proteolytic enzyme, and these enzymes are known to have high decomposition activity for high-molecular kininogen or fibrinogen, and are used to treat bacterial colonization and periodontal disease. It is thought to be involved in the expression of or destruction of periodontal tissue.

Accordingly, the novel antibody or antigen-binding fragment thereof according to the present invention is both Lys-gingipain (abbreviated as "KGP") and Arg-gingipain (abbreviated as "Rgp-B"). By specifically binding to the pathogenic bacteria involved in the development and progression of periodontal disease, such as Porphyromonas gingivalis gingivalis ), Bacteroides forsytus ( Bacteroides forsythus ), Actinobacillus Actinomycetem comitans ( Actinobacillus actinomycetemcomitans ), Triponema denticola ( Triponema denticola ), Prevotella intermedia ( Prevotella intermedia ), etc., can provide information on infection status. In particular, Porphyromonas gingivalis ( Porphyromonas gingivalis ) can provide information on infection. It can also be used to diagnose periodontal disease or to detect Arg-gingipain B, Lys-gingipain or both. In addition, it can be used for the purpose of preventing and treating periodontal disease by using the antibody or antigen-binding fragment thereof.

The present invention provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof against Arg-gingipain B, Lys-gingipain, or both. The nucleic acid molecule may be RNA or cDNA for the RNA. In one embodiment, the nucleic acid molecule may be a nucleotide sequence represented by SEQ ID NOs: 17 to 20 based on each of the above-mentioned antibodies or antigen-binding fragments thereof, but is not limited thereto. Specifically, the base sequence encoding the light chain variable region of the Fab type KGP-G5 antibody may be represented by SEQ ID NO: 17, and the base sequence encoding the heavy chain variable region may be represented by SEQ ID NO: 18, and the light chain of the Fab type RgpB-A7 antibody. The nucleotide sequence encoding the variable region may be represented by SEQ ID NO: 19, and the nucleotide sequence encoding the heavy chain variable region may be represented by SEQ ID NO: 20.

The present invention provides a recombinant vector containing a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to Arg-gingipain B, Lys-gingipain, or both. The recombinant vector includes, but is not limited to, expression vectors suitable for eukaryotic hosts, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. This is included. Expression vectors that can be used in bacterial hosts include Escherichia coli, pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives. coli ), plasmids having a broader host range such as RP4, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as gt10, gt11, and NM989, and M13 and filamentous single-stranded Other DNA phage such as DNA phage are included. Expression vectors useful in yeast cells are the 2 plasmid and its derivatives. A useful vector for insect cells is pVL941.

A "vector" of the present invention means a DNA preparation containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles or simply latent genomic inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself. As the plasmid is currently the most commonly used form of vector, 'plasmid' and 'vector' are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) an origin of replication to ensure efficient replication, including several hundred plasmid vectors per host cell, and (b) a host cell transformed with the plasmid vector to be selected for selection. and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and the foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or linker according to a conventional method.

In the present invention, the antibody or fragment thereof of the present invention can be mass-produced by transforming a recombinant vector into an appropriate host cell, eg, yeast cell, animal cell, etc., and then culturing the transformed host cell.

Suitable host cells of the vector are Escherichia coli, Bacillus subtilis (Bacillus subtilis), Genus Streptomyces (Streptomyces sp.), Pseudomonas genus (Pseudomonas sp.), Proteus mirabilis (Proteus mirabilis) or Staphylococcus genus (Staphylococcus sp.). In addition, the genus Aspergillus (Aspergillus sp.), Pichia pastoris (Pichia pastoris), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Shijosakaromas genus (Schizosaccharomyces sp.) and Neurospora crassa (Neurospora crassa), other lower eukaryotic cells, and higher eukaryotic cells, such as cells from insects. It can also be derived from plants and mammals. Preferably, monkey kidney cells 7 (COS7) cells, NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells, etc. can be used, but are not limited thereto.

As a method of introducing the recombinant vector of the present invention into host cells and transforming them, conventionally known genetic engineering methods may be used. For example, as physical methods, there are microinjection (injecting DNA directly into cells), liposome, directed DNA uptake, receptor-mediated DNA transfer, or DNA delivery method using Ca ⁺⁺ , and recently, virus Gene delivery methods are widely used. One example is a method using a retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, poxvirus vector or lentiviral vector. In particular, retroviruses have high gene transfer efficiency and are resistant to gross deletion or It can be used in a wide range of cells without binding to host DNA by rearrangement (changing host DNA that resembles its own DNA, resulting in changes in host DNA function).

A nucleic acid is 'operably linked' when it is placed into a functional relationship with another nucleic acid sequence. It can be genes and regulatory sequence(s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) are bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a pre-protein that participates in secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. Generally, 'operably linked' means that the linked DNA sequences are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be in contact. Linkage of these sequences is accomplished by ligation (linkage) at convenient restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.

Antibodies that specifically bind to Arg-ginjipine B, Lys-ginjipine or both of them of the present invention are preferably obtained by expression and purification by genetic recombination, specifically, the heavy chain variable region or the entire heavy chain of the antibody. The gene sequence encoding the region and the gene sequence encoding the light chain variable region or the entire light chain region may be separately expressed in one vector or two vectors, but is not limited thereto.

Specifically, by culturing the transformant in which the recombinant vector is expressed in a nutrient medium, it is possible to produce large quantities of antibodies that specifically bind to Arg-ginjipine B, Lys-ginjipine or both of them, and the medium and culture conditions are Those tolerant to the host cell can be appropriately selected and used. Conditions such as temperature, medium pH, and incubation time may be appropriately adjusted so as to be suitable for cell growth and mass production of proteins during culture.

Peptides or proteins produced recombinantly as described above can be recovered from culture media or cell lysates. If membrane bound, it can be released from the membrane using a suitable surfactant solution (eg, Triton-X 100) or by enzymatic cleavage.

Cells used for antibody expression can be disrupted by various physical or chemical means such as freeze-thaw purification, sonication, mechanical disruption, or cell disintegrants, and can be separated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A Laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989);Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)). Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoadsorption chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various variations and complex methods thereof are used. Possible but not limited to this.

The present invention is a composition for detecting or diagnosing Arg-gingipain B, Lys-gingipain, or both, comprising an antibody or an antigen-binding fragment thereof that specifically binds to Arg-gingipain B, Lys-gingipain, or both thereof. provides

The present invention also provides a composition for diagnosing periodontal disease comprising an antibody or antigen-binding fragment thereof that specifically binds to the Arg- gingipain B, Lys- gingipain, or both.

"Diagnosis" or "detection" in the present invention refers to any action that confirms the presence or characteristics of a pathological condition. For the purpose of the present invention, the diagnosis may be interpreted as all actions to determine whether periodontal disease is onset, Arg- gingipain B, Lys- gingipain, etc., but is not particularly limited thereto.

The "composition for detection or diagnosis" of the present invention refers to a main means used for diagnosing a disease of interest, and may include substances for diagnosing whether or not a periodontal disease has occurred according to the purpose of the present invention. Diagnostic methods may include contacting an antibody or antibody fragment with a sample. Such a sample may be, for example, tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine isolated from an individual, and preferably saliva. As used herein, the term "saliva" refers to a secretion fluid secreted into the mouth from the salivary glands.

Diagnostic methods may also include detection of antigen/antibody complexes.

In the present invention, periodontal disease is also referred to as periodontal disease, and refers to a disease in which bacteria infect the gap between the gingiva (gum) and the tooth to damage the periodontal ligament and adjacent tissues. At the onset of the periodontal disease, inflammation progresses and more tissues are damaged, forming a periodontal pocket. As periodontitis worsens, the depth of the periodontal pocket deepens. is known to cause bone loss. In addition, the periodontal disease is gingivitis, periodontal inflammation, gingival bleeding, gingival retraction, periodontal pocket, alveolar bone breakage or alveolar bone formation disorder. bone osteodystrophy), but is not limited thereto.

The composition according to the present invention may include the above antibody alone or both of the above antibodies.

For example, when both antibodies are included, each can be used as a capture antibody and a detection antibody. Specifically, the CDR1 region composed of the amino acid sequence of SEQ ID NO: 1, the CDR2 region composed of the amino acid sequence of SEQ ID NO: 2, and the heavy chain variable region including the CDR3 region composed of the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4 A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 5, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 6; Arg-gingipain B, Lys-gingipain, or both A labeling material may be conjugated to an antibody or an antigen-binding fragment thereof and used as a detection antibody that specifically binds to Arg-gingipain B, Lys-gingipain, or both antigens.

In addition, a CDR1 region composed of the amino acid sequence of SEQ ID NO: 7, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 8, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 A CDR1 region, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising a CDR3 region composed of the amino acid sequence of SEQ ID NO: 12; Antibodies or antigen-binding fragments thereof can be used as capture antibodies.

"Antigen-antibody complex" means a combination of a corresponding protein antigen in a sample and an antibody recognizing it. Detection of the antigen-antibody complex can be performed using methods known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, spectroscopic, chemical and other methods. by colormetric method, electrochemical method, fluorescence method, luminometry, particle counting method, visual assessment and scintillation counting method It can be detected by any method selected from the group consisting of western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, and Ouchterlony immunity. Methods such as diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, FACS, protein chip, etc. may be used, but are not limited thereto. .

In the present invention, various markers can be used to detect antigen-antibody complexes. Specific examples may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and radioactive isotopes, but are not necessarily limited thereto.

Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, β-latamase, etc., and fluorescent substances include fluorescein Phosphorus, Eu ³⁺ , Eu ³⁺ chelate or ^cryptate , etc. are included, ligands include biotin derivatives, etc., luminous ^substances include acridinium esters and isoluminol derivatives, etc., and microparticles include colloids. gold, colored latex, and the like, and radioactive isotopes include, but are not limited to, ⁵⁷ Co, ³ H, ¹²⁵ I, ¹²⁵ I-Bolton Hunter reagent, and the like.

In the composition according to the present invention, the antibody for labeling may be, for example, an antibody-metal nanoparticle complex in which an antibody molecule is bound to a metal nanoparticle. The metal nanoparticles are characterized in that they are selected from the group consisting of gold (Au), platinum (Pt), silver (Ag), iron (Fe), and combinations thereof. For example, the metal nanoparticles may be gold (Au) or silver (Ag). These nanoparticles are particles with a diameter of 1 to 100 nanometers and can be confirmed with the naked eye or absorption/fluorescence equipment.

The present invention also includes a kit for diagnosing periodontal disease comprising an antibody or an antigen-binding fragment thereof specific to Arg-specific gingipain and/or Lys-specific gingipain.

For example, a kit for diagnosing periodontal disease including each of the above antibodies or all of them may be provided.

In the case of including all of these, the kit can attach an antibody or an antigen-binding fragment thereof that can be used as a capture antibody to a substrate, and detection and diagnosis can be performed using the detectably labeled detection antibody.

That is, for example, the kit of the present invention may be in the form of an enzyme-linked immunosorbent assay (ELISA) kit, and specifically, the kit may include the above-described antibody as a capture antibody and a detection antibody, and the ELISA is (i ) a capture antibody comprising any one of the antibodies according to the present invention; (ii) The other one of the antibodies according to the present invention may be labeled with a detection label and used as the detection antibody, but the embodiment is not limited thereto.

The term "ELISA (Enzyme-linked immunosorbent assay)" of the present invention is also referred to as an enzyme immunoassay method, and is a method of quantifying using absorbance through the reaction between an enzyme and a substrate by forming an antigen-antibody complex by binding an enzyme to an antibody. to be. The ELISA includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of attached antibody and antigen, followed by reaction with another antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support and then recognizing this antibody indirect sandwich ELISA using labeled secondary antibodies; and the like.

In addition, the optimal amount of reagents to be used in a particular reaction of the kit can be easily determined by a person skilled in the art having learned the disclosure herein. Typically, the kits of the present invention are manufactured in separate packages or compartments containing the aforementioned ingredients.

The composition or kit for detection or diagnosis of the present invention can also provide rapid diagnosis information on periodontal disease by using an antibody that specifically binds to Arg-specific gingipain and/or Lys-specific gingipain. In addition, the kit may further include instructions for use and other tools or equipment necessary for detection.

According to one embodiment of the present invention, the kit may be a rapid kit.

In a specific embodiment, the kit according to the present invention specifically binds to the antigens of Arg-gingipain B, Lys-gingipain, or both, and is attached to a substrate, a CDR1 region composed of the amino acid sequence of SEQ ID NO: 7, CDR2 region composed of the amino acid sequence of SEQ ID NO: 8, heavy chain variable region including a CDR3 region composed of the amino acid sequence of SEQ ID NO: 9, CDR1 region composed of the amino acid sequence of SEQ ID NO: 10, and CDR2 region composed of the amino acid sequence of SEQ ID NO: 11 , And a light chain variable region comprising a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 12; Arg-jinjipine B, Lys-jinjipine or an antibody or antigen-binding fragment thereof for both of them; and

Arg-gingipain B, Lys-gingipain, or a CDR1 region composed of the amino acid sequence of SEQ ID NO: 1 to which a marker is conjugated and specifically binding to antigens of both of them, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 2, and SEQ ID NO: 3 A CDR1 region composed of the amino acid sequence of SEQ ID NO: 4, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 5, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 6. It may be a kit for diagnosing periodontal disease, including; an antibody or antigen-binding fragment thereof for Arg-jinjipine B, Lys-jinjipine or both thereof, including; light chain variable region.

The present invention provides a method for diagnosing periodontal disease comprising the step of reacting a sample with an antibody or an antigen-binding fragment thereof that specifically binds to the Arg- gingipain B, Lys- gingipain, or both.

Here, the diagnosis of periodontal disease can be performed by confirming the presence of Arg-gingipain B, Lys-gingipain, or both. Therefore, when Arg-gingipain B, Lys-gingipain, or both are detected, it can be confirmed as the onset of periodontitis.

The present invention relates to Arg-gingipain B, Lys-gingipain, or an antibody or antigen-binding fragment thereof that specifically binds to both of them; Arg-gingipain B, Lys-gingipain or these comprising the step of reacting with a sample. All antigen detection or quantification methods are provided.

The diagnostic, detection or quantification methods include, for example, ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunospecific assay), western blotting, immunoprecipitation, immunohistochemistry, and immunohistochemistry. It may be any one selected from the group consisting of fluorescence staining (immunofluorescence).

In the present invention, ELISA (enzyme-lined immunosorbent assay, enzyme-linked immunospecific assay) method was preferably used, and the antibodies against Arg-gingipain B, Lys-gingipain, or both of them were used for capture or detection. It can be used as an antibody.

The present invention provides a novel antibody capable of providing rapid diagnosis information on periodontal disease, and the antibody has high affinity for Arg-gingipain B, Lys-gingipain, or both antigens, and is used as an in vitro diagnostic kit for periodontal disease. shows excellent effect.

1 is a diagram showing ELISA results for KGP, RgpA and RgpB of multivalent and monoclonal antibodies selected as a result of panning.
Figure 2 is a diagram showing the light chain variable region and heavy chain variable region sequences of each antibody of the present invention.
Figure 3 is a diagram showing the results of electrophoresis performed with the purified antibody of the present invention.
Figure 4 is a diagram showing the results of confirming the binding ability of the antibody of the present invention to KGP, RgpB by ELISA.
5 is a diagram showing the results of confirming the detection ability for each of KGP, RgpA and RgpB on the rapid kit diagnostic strip.
6 is a diagram showing the result of confirming the detection ability for each of the saliva and KGP mixture samples, KGP, RgpA and RgpB on the rapid kit diagnostic strip.
7 is a diagram showing the result of confirming the detection ability for each of these in a sample mixed with saliva and KGP, RgpA and RgpB.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Arg - Gingipain B ( RgpB ), Lys - Gingipain ( KGP ) Antibody selection that binds specifically

1-1. Manufacture of phage-displayed human antibody library

The human antibody library possessed by this laboratory was amplified by the following method.

After culturing the E. coli cells containing the human antibody library to OD600 = 0.6-0.7, adding helper phage, standing for 30 minutes and shaking for 30 minutes, kanamycin ( kanamycin) was added and incubated overnight. Then, the amplified library phages were recovered from the culture medium.

1-2. biopanning

100 μg each of KGP or RgpB was coated in a tube (immunotube), and the library phage prepared in 1-1 was added to the tube, placed at 37° C. for 1 hour, and then washed 10 times with 0.5% PBST. After washing, the remaining phages were diluted with 0.1 M glycine-HCl (pH 2.5), and E. coli The TG1 mutant strain was infected and amplified. At this time, the panning method was the same, and the number of washing with 0.5% PBST was increased by 10 times each time the number of panning increased.

Input and output titers from the first panning to the fourth panning were specifically described in the Bio-panning table in FIG. 1 .

1-3. Arg - Gingipain B ( RgpB ), Lys - Gingipain ( KGP ) Specific antibody clone selection

The clones amplified in the 1st to 4th panning were randomly selected and the cells infected with the E. coli TG1 mutant were plated to form about 1000 single colonies, and each single colony was OD600 = After incubation until 0.6-0.7, the supernatant (sup) induced at 37 ° C with IPTG (isopropyl-1-thio-β-D-galactopyranoside) was placed in a 96-well plate coated with 100 ng/well of KGP or RgpB and 37 After 1 hour at °C, it was washed three times with 0.05% PBST.

ELISA was then performed. Specifically, the amplified clone was added to a 96-well plate coated with KGP or RgpB, and the primary antibody (Goat F(ab')2 Anti-Human IgG (Fab')2 (HRP), Purchased from abcam (cat.ab98535)) was diluted 1:3000 in 0.05% PBST, bound at 37°C for 1 hour, washed, reacted with TMB-conjugated secondary antibody, washed, and TMB substrate solution was added to detect clones binding to Arg -gingipain B ( RgpB ) and Lys -gingipain ( KGP ) . BSA was used as a negative control.

The results are shown in Figure 1.

As can be seen in Figure 1 , two clones ( KGP - G5 and KGP / RgpB-A7) was obtained.

These clones were sequenced and two antibody clones were selected among them. As a result of sequencing, the heavy chain variable region sequence of SEQ ID NO: 13 and the light chain variable region sequence of SEQ ID NO: 14 were obtained for KGP-G5, and the heavy chain variable region sequence of SEQ ID NO: 15 and light chain variable region sequence of SEQ ID NO: 16 were obtained for RgpB-A7. sequence was obtained.

In addition, for the KGP-G5, the CDR1 region of the heavy chain variable region was SEQ ID NO: 1, the CDR2 region was SEQ ID NO: 2, and the CDR3 region was SEQ ID NO: 3, the CDR1 region of the light chain variable region was SEQ ID NO: 4, and the CDR2 region was SEQ ID NO: 4. SEQ ID NO: 5, and the CDR3 region were SEQ ID NO: 6, the CDR1 region of the heavy chain variable region for RgpB-A7 was SEQ ID NO: 7, the CDR2 region was SEQ ID NO: 8, and the CDR3 region was SEQ ID NO: 9, and the CDR1 region of the light chain variable region was SEQ ID NO: 10, CDR2 region SEQ ID NO: 11, and CDR3 region SEQ ID NO: 12.

This is shown in Figure 2.

In FIG. 2, the portion marked with red letters corresponds to the CDR region.

As described above, CDR1 represented by SEQ ID NO: 1, CDR2 represented by SEQ ID NO: 2, CDR3 represented by SEQ ID NO: 3, and CDR1 represented by SEQ ID NO: 4, CDR2 represented by SEQ ID NO: 5 , and a light chain variable region comprising CDR3 represented by SEQ ID NO: 6, the antibody was named KGP-G5,

CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, CDR3 represented by SEQ ID NO: 9, CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11, and SEQ ID NO: The antibody containing the light chain variable region including the CDR3 described in No. 12 was named RgpB-A7 (KGP+RgpB A7).

Example 2. of selected antibody clones pre-antibodyization

In order to more precisely analyze the selected clones, they were converted to whole Ig forms according to the following method.

The base sequence encoding the light chain variable region of the Fab type KGP-G5 antibody is SEQ ID NO: 17, and the base sequence encoding the heavy chain variable region can be represented by SEQ ID NO: 18, and the light chain variable region of the Fab type RgpB-A7 antibody The nucleotide sequence encoding SEQ ID NO: 19 and the nucleotide sequence encoding the heavy chain variable region SEQ ID NO: 20 were amplified through a polymerase chain reaction. Sequences for amplification used the sequences in Table 1 below.

amplified sequence
(KGP-G5) primer light chain leader sequence forward (SEQ ID NO: 21)
5'- TGC AAA GCT TCG GCA CGA GCA -3' reverse (SEQ ID NO: 22)
5'-TCC TTC AAC ACC AGA CAA C -3' heavy chain leader sequence forward (SEQ ID NO: 23)
5'-GAC GAA TTC ACT CTA ACC ATG GAA -3' reverse (SEQ ID NO: 24)
5'- GGA GTG GAC ACC TGT AG -3' amplified sequence
(RgpB-A7) primer light chain variable region forward (SEQ ID NO: 25)
5'-TTG TCT GGT GTT GAA GGA GAC ATC CAG ATG ACC CAG -3' reverse (SEQ ID NO: 26)
5'- GCA GCC GCC GTA CGT TT GAT CTC CAC CTT GGT CCC TTG -3' heavy chain variable region forward (SEQ ID NO: 27)
5'- ACT ACA GGT GTC CAC TCC GAA GTG CAG CTG GTG GAG -3' reverse (SEQ ID NO: 28)
5'- CAC CGG TTC GGG GAA GT -3'

The amplified sequence was separated through a 1.5% agarose gel (Cambrex), purified, and confirmed by electrophoresis.

The results are shown in Figure 3.

In FIG. 3, it can be seen that bands of all antibodies and heavy and light chains of KGP-G5 (KGP) and RgpB-A7 (RgpB) appear, and no other bands appear. When the antibody was reduced, protein bands having molecular weights of 50 KDa and 25 KDa, which are known as the molecular weights of the heavy and light chains of the antibody, were observed. Therefore, it was found that total antibodyization was successfully performed.

Example 3. Arg-jinjipine B ( RgpB), Lys-jinjipine (KGP) K _D value (K _D value) and binding capacity measurement

KD values were measured by performing three repetitions of the experiment using ELISA with each of the pre-antibodies _. Absorbance was measured after ELISA, and the results are shown in FIG. 4 .

As can be seen in Figure 4, both KGP-G5 (G5) and RgpB-A7 (A7) antibodies showed excellent binding ability to Arg-jinjipain B (RgpB) and Lys-jinjipain (KGP), respectively.

Example 4. Confirmation of periodontal disease diagnostic ability using Rapid diagnostic kit

The detection ability for Arg-gingipain B (RgpB) and Lys-gingipain (KGP) was confirmed using the rapid kit.

Specifically, the RgpB-A7 (A7) antibody was used as a capture antibody, and the KGP-G5 (KGP) antibody was used as a detection antibody. A 4 mm X 50 mm strip was prepared in which the A7 antibody (test line) and the anti-human IgG antibody (control line) were applied on a nitrocellulose membrane. Then, the G5 antibody-gold nanoparticle condensate was dispensed on a conjugate pad and dried to prepare. A rapid kit was prepared by attaching a conjugate pad and a sample pad to the bottom of the nitroreulose membrane and an absorption pad to the top, then inserting the sample containing Kgp and RgpA into the sample inlet and measuring the degree of color change of the test line. Observed.

The detection ability was tested for Kgp, RgpA (Arg-jinjipine A), and RgpB using the prepared rapid kit. In the experiment of FIG. 5, direct detection of Kgp, RgpA (Arg-jinjipain A), and RgpB was confirmed, and in the experiment of FIG. 6, the case of mixing with saliva was compared, and in the case of FIG. The detection ability was confirmed using a mixture of Arg-jinjipine A) and RgpB.

As can be seen from the results of FIGS. 5 to 7, when a blank containing no protein was added as a sample to the rapid kit, a line appeared only in the control line (C) and in the test line (T). no line was visible. However, when the buffer containing Kgp and RgpB was added to the rapid kit, colored lines appeared in both the control line (C) and the test line (T). When color development lines appear at both the control line (C) and inspection line (T) positions of the inspection window, it can be determined that Kgp and RgpB are present in the sample. This was especially confirmed in samples contained in saliva, and it was verified from the above results that periodontal disease can be rapidly diagnosed using the rapid kit of the present invention.

From the above description, those skilled in the art to which the present invention belongs will be able to understand that the present invention can be implemented in other specific forms without changing its technical spirit or essential features. 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> Korea Research Institute of Bioscience and Biotechnology Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> ANTIBODY FOR PERIODONTAL DISEASE AND USE THEREOF <130> KRIBB164P <160> 28 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> artificial sequence <220> <223> KGP-G5_H_CDR1 <400> 1 Thr Asp Gly Tyr One <210> 2 <211> 9 <212> PRT <213> artificial sequence <220> <223> KGP-G5_H_CDR2 <400> 2 Tyr Gly Thr Asp Gly Ala Thr Asn Tyr 1 5 <210> 3 <211> 9 <212> PRT <213> artificial sequence <220> <223> KGP-G5_H_CDR3 <400> 3 Val Asn Glu Ile Pro Asp Val Tyr Val 1 5 <210> 4 <211> 5 <212> PRT <213> artificial sequence <220> <223> KGP-G5_L_CDR1 <400> 4 Asp Val Asn Ser Trp 1 5 <210> 5 <211> 5 <212> PRT <213> artificial sequence <220> <223> KGP-G5_L_CDR2 <400> 5 Trp Ala Ser Ser Arg 1 5 <210> 6 <211> 6 <212> PRT <213> artificial sequence <220> <223> KGP-G5_L_CDR3 <400> 6 Cys Gln Gln Gly Ser Leu 1 5 <210> 7 <211> 4 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_H_CDR1 <400> 7 Ser Asp Ser Trp One <210> 8 <211> 9 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_H_CDR2 <400> 8 Asp Gly Tyr Ser Gly Asp Thr Tyr Tyr 1 5 <210> 9 <211> 9 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_H_CDR3 <400> 9 Asp Pro Tyr Phe Val Asp Leu Tyr Ala 1 5 <210> 10 <211> 5 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_L_CDR1 <400> 10 Ile Ser Ser Ser Tyr 1 5 <210> 11 <211> 5 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_L_CDR2 <400> 11 Ala Ala Ser Ser Leu 1 5 <210> 12 <211> 6 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_L_CDR3 <400> 12 Arg Tyr Asn Leu Pro Arg 1 5 <210> 13 <211> 124 <212> PRT <213> artificial sequence <220> <223> KGP-G5_H <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Gly 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Tyr Gly Thr Asp Gly Ala Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Trp Val Asn Glu Ile Pro Asp Val Tyr Val Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 <210> 14 <211> 104 <212> PRT <213> artificial sequence <220> <223> KGP-G5_L <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Leu Thr Phe Gly 85 90 95 Gln Gly Thr Lys Val Glu Ile Lys 100 <210> 15 <211> 124 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_H <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Asp Gly Tyr Ser Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Trp Asp Pro Tyr Phe Val Asp Leu Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 <210> 16 <211> 107 <212> PRT <213> artificial sequence <220> <223> KGP+RgpB A7_L <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ile Ser Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Asn Leu Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 372 <212> DNA <213> artificial sequence <220> <223> KGP-G5_H <400> 17 gaagtgcagc tggtggagtc tgggggaggc ttggttcagc ctggagggtc cctgcgtctc 60 tcctgtgcag cctctggatt caccttcact gacggttata tgagctgggt tcgccaggct 120 ccagggaagg ggctggagtg ggtttctgat atttatggta ctgacggtgc tactaactac 180 gcagactctg tgaagggccg tttcaccatc tcccggggaca actctaagaa cactctgtat 240 ctgcaaatga acagcctgcg tgccgaggac acggctgtct attactgtgc gaaatgggtc 300 aacgagatcc ccgacgtcta tgttatggat tattggggcc aaggcaccct ggtcaccgtc 360 tcctcggcca gc 372 <210> 18 <211> 312 <212> DNA <213> artificial sequence <220> <223> KGP-G5_L <400> 18 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtcggaga ccgcgtcacc 60 atcacttgcc gggcaagtca ggatgttaac tcttggctgg cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctactgg gcttccagca gggaaagtgg ggtcccatca 180 cgcttcagtg gcagtggatc tgggacggat ttcactctca ccatcagcag tctacaacct 240 gaagattttg caacttacta ctgtcaacag ggttccctga cgttcggcca aggtaccaag 300 gtggagatca aa 312 <210> 19 <211> 372 <212> DNA <213> artificial sequence <220> <223> KGP+RgpB A7_H <400> 19 gaagtgcagc tggtggagtc tgggggaggc ttggttcagc ctggagggtc cctgcgtctc 60 tcctgtgcag cctctggatt caccttctct gactcttgga tgagctgggt tcgccaggct 120 ccagggaagg ggctggagtg ggtttctgat attgatggtt atagcggtga tacttactac 180 gcagactctg tgaagggccg tttcaccatc tcccggggaca actctaagaa cactctgtat 240 ctgcaaatga acagcctgcg tgccgaggac acggctgtct attactgtgc gaaatgggac 300 ccgtacttcg tcgacctcta tgctatggat tattggggcc aaggcaccct ggtcaccgtc 360 tcctcggcca gc 372 <210> 20 <211> 321 <212> DNA <213> artificial sequence <220> <223> KGP+RgpB A7_L <400> 20 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtcggaga ccgcgtcacc 60 atcacttgcc gggcaagtca gatttcttct tcttacctgg cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctacgcg gcttccagct tggaaagtgg ggtcccatca 180 cgcttcagtg gcagtggatc tgggacggat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag cgttacaacc ttcctcggac gttcggccaa 300 ggtaccaagg tggagatcaa a 321 <210> 21 <211> 21 <212> DNA <213> artificial sequence <220> <223> KGP-G5_L_Forward <400> 21 tgcaaagctt cggcacgagc a 21 <210> 22 <211> 19 <212> DNA <213> artificial sequence <220> <223> KGP-G5_L_Reverse <400> 22 tccttcaaca ccagacaac 19 <210> 23 <211> 24 <212> DNA <213> artificial sequence <220> <223> KGP-G5_H_Forward <400> 23 gacgaattca ctctaaccat ggaa 24 <210> 24 <211> 17 <212> DNA <213> artificial sequence <220> <223> KGP-G5_H_Reverse <400> 24 ggaggtggaca cctgtag 17 <210> 25 <211> 36 <212> DNA <213> artificial sequence <220> <223> RgpB-A7_L_Forward <400> 25 ttgtctggtg ttgaaggaga catccagatg acccag 36 <210> 26 <211> 38 <212> DNA <213> artificial sequence <220> <223> RgpB-A7_L_Reverse <400> 26 gcagccgccg tacgtttgat ctccaccttg gtcccttg 38 <210> 27 <211> 36 <212> DNA <213> artificial sequence <220> <223> RgpB-A7_H_Forward <400> 27 actacaggtg tccactccga agtgcagctg gtggag 36 <210> 28 <211> 17 <212> DNA <213> artificial sequence <220> <223> RgpB-A7_H_Reverse <400> 28 caccggttcg gggaagt 17

Claims (18)

It relates to an antibody or antigen-binding fragment thereof that specifically binds to Arg-gingipain B, Lys-gingipain, or both,
The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and sequence comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 1, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 2, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 3 A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 4, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 5, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 6; containing Arg-ginjipine B, Lys- An antibody or antigen-binding fragment thereof directed against gingipine or both, or
A CDR1 region composed of the amino acid sequence of SEQ ID NO: 7, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 8, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 9 and a CDR1 region composed of the amino acid sequence of SEQ ID NO: 10 , CDR2 region consisting of the amino acid sequence of SEQ ID NO: 11, and light chain variable region comprising a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 12; Arg-ginjipine B, Lys-ginjipine or an antibody for both of them, or an antigen-binding fragment thereof. delete The heavy chain variable region and SEQ ID NO: 4 according to claim 1, comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 1, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 2, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 3. A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 5, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 5, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 6; Or an antibody or antigen-binding fragment thereof for all of them, comprising the amino acid sequence of SEQ ID NO: 13 as a heavy chain variable region, antibody or antigen-binding fragment thereof. The heavy chain variable region and SEQ ID NO: 4 according to claim 1, comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 1, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 2, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 3. A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 5, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 5, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 6; Or an antibody or antigen-binding fragment thereof for all of them, comprising the amino acid sequence of SEQ ID NO: 14 as a light chain variable region, antibody or antigen-binding fragment thereof. The heavy chain variable region and SEQ ID NO: 4 according to claim 1, comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 1, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 2, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 3. A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 5, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 5, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 6; Or an antibody or antigen-binding fragment thereof for all of them, comprising the amino acid sequence of SEQ ID NO: 13 as a heavy chain variable region and the amino acid sequence of SEQ ID NO: 14 as a light chain variable region, antibody or antigen-binding fragment thereof. The heavy chain variable region and SEQ ID NO: 10 according to claim 1, comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 7, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 8, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 9. A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 11, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 11, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 12; Or an antibody or antigen-binding fragment thereof for all of them, comprising the amino acid sequence of SEQ ID NO: 15 as a heavy chain variable region, antibody or antigen-binding fragment thereof. The heavy chain variable region and SEQ ID NO: 10 according to claim 1, comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 7, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 8, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 9. A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 11, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 11, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 12; Or an antibody or antigen-binding fragment thereof for all of them, comprising the amino acid sequence of SEQ ID NO: 16 as a light chain variable region, antibody or antigen-binding fragment thereof. The heavy chain variable region and SEQ ID NO: 10 according to claim 1, comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 7, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 8, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 9. A light chain variable region comprising a CDR1 region composed of the amino acid sequence of SEQ ID NO: 11, a CDR2 region composed of the amino acid sequence of SEQ ID NO: 11, and a CDR3 region composed of the amino acid sequence of SEQ ID NO: 12; Or an antibody or antigen-binding fragment thereof for all of them, comprising the amino acid sequence of SEQ ID NO: 15 as a heavy chain variable region and the amino acid sequence of SEQ ID NO: 16 as a light chain variable region, antibody or antigen-binding fragment thereof. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 and 3 to 8. A recombinant vector containing the nucleic acid molecule of claim 9. The nucleic acid molecule of claim 10 or a recombinant vector containing the nucleic acid molecule is introduced into a host cell Arg-jinjipain B, Lys-jinjipine or an antibody against both of them, or a recombinant cell having the ability to produce antigen-binding fragments thereof. A composition for diagnosing periodontal disease comprising an antibody or an antigen-binding fragment thereof that specifically binds to any one of claims 1 and 3 to 8 of Arg- gingipain B, Lys- gingipain, or both. Arg-gingipain B comprising the antibody or antigen-binding fragment thereof specifically binding to Arg-gingipain B, Lys-gingipain or both of claims 1 and 3 to 8, A composition for detecting or diagnosing Lys-gingipain or both. A kit for diagnosing periodontal disease comprising an antibody or antigen-binding fragment thereof that specifically binds to any one of claims 1 and 3 to 8 of Arg-gingipain B, Lys-gingipain, or both. The method of claim 14, wherein the capture antibody or antigen-binding fragment thereof specifically binds to Arg-gingipain B, Lys-gingipain or both of them attached to the substrate, and detectably labeled Arg-gingipain B, Lys - A kit for diagnosing periodontal disease, comprising a detection antibody or an antigen-binding fragment thereof that specifically binds to gingipain or both. The kit for diagnosing periodontal disease according to claim 14, wherein the kit is in the form of an enzyme-linked immunosorbent assay (ELISA). A step of reacting an antibody or an antigen-binding fragment thereof specifically binding to Arg-gingipain B, Lys-gingipain or both of claims 1 and 3 to 8 with a sample, A method for detecting or quantifying an antigen of Arg-gingipain B, Lys-gingipain, or both. (a) any one of claims 1 and 3 to 8, Arg-jinjipine B, Lys-jinjipine, or an antibody or antigen-binding fragment thereof that specifically binds to both of them is isolated from a subject, tissue, Reacting with any one sample selected from cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine:
(b) confirming whether the antibody or antigen-binding fragment thereof binds to Arg-gingipain B or Lys-gingipain; and
(c) when the antigen-antibody binding between the antibody or antigen-binding fragment thereof and Arg-jinjipain B or Lys-jinjipain is confirmed, determining that periodontal disease has occurred; diagnosing periodontal disease, including How to provide information for.

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