Diagnostic kit for skin comprising anti-staphylococcus aureus protein a antibodies

Abstract

This invention relates to an antibody specific for Staphylococcus aureus protein A, and a composition or kit for Staphylococcus aureus detection or skin condition diagnosis containing this antibody. The monoclonal antibody of this invention exhibits very high reactivity to the antigen. In particular, by combining two antibodies with different complementary determinant regions (CDRs) as a capture antibody and a detection antibody, the antigen can be detected rapidly and accurately even at low concentrations, thus enabling applications in rapid self-diagnostic kits, etc. Using this monoclonal antibody, the proliferation status of Staphylococcus aureus on the skin surface can be visually confirmed, thereby diagnosing abnormal skin conditions such as skin inflammation and allergic immune responses, predicting the incidence and persistence of related diseases, and ultimately determining subsequent treatment plans.

Description

Technical Field

[0001] This invention relates to an antibody specific to Staphylococcus aureus protein A, and a composition or kit for Staphylococcus aureus detection or skin condition diagnosis containing the antibody. Background Technology

[0002] In recent years, with the active development of microbiome research, the fields of gut microbiome and skin microbiome have become the focus of attention. In particular, regarding the skin microbiome, very detailed results and data can be obtained using next-generation sequencing (NGS) and gene testing, but there is still a lack of rapid diagnostic methods that can easily examine skin conditions.

[0003] On the other hand, recent studies have shown that Staphylococcal protein A (SPA) from Staphylococcus aureus induces epithelial cells in skin tissue to produce interleukin-18 (IL-18) cytokines, which in turn activate CD4+ T cells, causing them to secrete interferon-γ (IFN-γ), interleukin-3 (IL-3), interleukin-13 (IL-13), etc., thereby triggering histamine and mast cell infiltration, leading to inflammatory skin diseases such as atopic dermatitis.

[0004] Therefore, the inventors conducted research aimed at developing a kit for detecting Staphylococcus aureus, which is highly associated with symptoms of atopic dermatitis caused by skin inflammation and allergic immune responses. In particular, given the significant increase in consumer acceptance of rapid diagnostic kits due to COVID-19, this invention aims to develop a rapid self-diagnostic kit for measuring the skin surface concentration of protein A, a major protein of Staphylococcus aureus. This invention was accomplished by developing novel monoclonal antibodies against protein A and screening for the optimal combination, demonstrating the ability to rapidly and accurately detect Staphylococcus aureus. Summary of the Invention

[0005] The technical problem to be solved The present invention aims to provide an antibody specific to Staphylococcus aureus protein A and a composition or kit for detecting Staphylococcus aureus containing the antibody.

[0006] The technical problems of this invention are not limited to those mentioned above. Other technical problems not mentioned can be clearly understood by those skilled in the art based on the following description.

[0007] Technical solutions for solving the problem According to embodiments of the present invention, an antibody specific to Staphylococcus aureus protein A is provided, the antibody comprising: a heavy chain variable region comprising heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6.

[0008] Additionally, an antibody specific to Staphylococcus aureus protein A is provided, the antibody comprising: a heavy chain variable region comprising heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 14.

[0009] Invention Effects The monoclonal antibody of this invention is a novel antibody specific to Staphylococcus aureus protein A, exhibiting very high reactivity to the antigen. In particular, the monoclonal antibody according to this invention can rapidly and accurately detect the antigen even at low concentrations by combining two antibodies with different CDRs as a capture antibody and a detection antibody, thus enabling applications in rapid self-diagnostic kits, etc. Furthermore, this monoclonal antibody allows for direct visualization of the proliferation status of Staphylococcus aureus on the skin surface, thereby diagnosing skin abnormalities such as inflammation and hypersensitivity reactions, predicting the incidence and persistence of related diseases, and ultimately determining subsequent treatment plans. Attached Figure Description

[0010] To provide a fuller understanding of the accompanying drawings referenced in the detailed description of this invention, a brief description of each drawing is provided.

[0011] Figure 1 This is the result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirming the purified monoclonal antibody.

[0012] Figure 2The results were obtained by comparing the affinity of the screened monoclonal antibodies with commercially available antibodies for the protein A antigen using Western blotting.

[0013] Figure 3 The results were obtained by observing the reactivity of the monoclonal antibody combination of the present invention to the protein A antigen on a rapid test strip.

[0014] Figure 4 The results were observed on a rapid detection kit prepared using the monoclonal antibodies 7G1 and 6B5 of the present invention to observe the reactivity to protein A antigen.

[0015] Figure 5 The purpose is to confirm whether Staphylococcus aureus can be detected when actual skin samples are applied to a rapid test kit prepared using the monoclonal antibodies 7G1 and 6B5, which are the present invention. Detailed Implementation

[0016] According to embodiments of the present invention, an antibody specific to Staphylococcus aureus protein A is provided, the antibody comprising: a heavy chain variable region comprising heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6.

[0017] Additionally, an antibody specific to Staphylococcus aureus protein A is provided, the antibody comprising: a heavy chain variable region comprising heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 14.

[0018] According to another embodiment of the invention, a polynucleotide encoding the antibody is provided.

[0019] According to another embodiment of the present invention, an expression vector comprising the polynucleotide is provided.

[0020] According to another embodiment of the present invention, a host cell comprising the expression vector is provided.

[0021] According to another embodiment of the present invention, a composition for detecting Staphylococcus aureus containing the antibody is provided.

[0022] According to another embodiment of the present invention, a kit for detecting Staphylococcus aureus containing the antibody is provided. Specific Implementation Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used in this specification is well-known and commonly used in the art. Furthermore, in describing embodiments of the invention, detailed descriptions of well-known structures or functions are omitted when it is determined that such descriptions would hinder understanding of the embodiments. Furthermore, embodiments of the invention are described below, but the technical concept of the invention is not limited thereto or restricted thereto, and can be implemented in various ways by modifications by one of ordinary skill in the art.

[0024] In this specification, when a part contains a structural element, unless otherwise stated to the contrary, it means that other structural elements may be included, rather than excluded. In this specification, the term "and / or" includes a combination of multiple related items, or any one of multiple related items.

[0025] According to embodiments of the present invention, a novel antibody specific to Staphylococcus aureus protein A is provided.

[0026] In this invention, "antibody" refers to a protein molecule that acts as a receptor for specifically recognizing antigens and is contained in immunoglobulin molecules that are immunologically reactive to specific antigens, including polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments.

[0027] In this invention, "monoclonal antibody" refers to an antibody molecule composed of a single molecule obtained from substantially the same antibody population, and such monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope.

[0028] For the purposes of this invention, the antibody may be a monoclonal antibody that specifically binds to Staphylococcus aureus protein A.

[0029] In this invention, immunoglobulins have heavy and light chains, each of which contains constant and variable regions (referred to as domains). The variable regions of the heavy and light chains include three hypervariable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions. The primary function of the CDRs is to bind epitopes of antigens. The CDRs of each chain are sequentially designated CDR1, CDR2, and CDR3, starting from the N-terminus, and are also identified by the chain in which the specific CDR is located.

[0030] According to one embodiment of the present invention, an antibody specific to Staphylococcus aureus protein A is provided, the antibody comprising: a heavy chain variable region comprising heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6, the antibody being named 7G1.

[0031] The antibody may include a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7, and may include a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8.

[0032] According to another embodiment of the present invention, an antibody specific to Staphylococcus aureus protein A is provided, the antibody comprising: a heavy chain variable region comprising heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 14, the antibody being named 6B5.

[0033] The antibody may include a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.

[0034] The antibodies of the present invention may comprise variants of the amino acid sequences listed in the sequence listing, provided they fall within the range capable of specifically recognizing Staphylococcus aureus protein A. For example, the amino acid sequence of the antibody may be altered to improve the antibody's binding affinity and / or other biological properties. Such modifications include, for example, the deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid mutations are based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. By analyzing the size, shape, and type of amino acid side chain substituents, it can be seen that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on this, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically equivalent.

[0035] When introducing variations, the hydrophobic index of amino acids can be considered. Each amino acid is assigned a hydrophobic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0036] The hydrophobicity index of amino acids is crucial for conferring the interactive biological function of proteins. It is well known that similar biological activity can only be preserved by replacing amino acids with those having a similar hydrophobicity index. When introducing variations based on the hydrophobicity index, substitutions are preferably made between amino acids with a difference in hydrophobicity index within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0037] Furthermore, it is well known that substitutions between amino acids with similar hydrophilicity values ​​produce proteins with equivalent biological activity, assigning the following hydrophilicity values ​​to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0038] When introducing variations based on hydrophilicity values, substitutions are preferably performed between amino acids with hydrophilicity value differences within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0039] Furthermore, amino acid substitutions in proteins that do not completely alter molecular activity are well-known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions are between the following amino acid residues: Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0040] Therefore, the antibody according to the present invention can have 80% to 99% homology, 90% to 99% homology and 95% to 99% homology with the above-mentioned amino acid sequence.

[0041] According to another embodiment of the invention, a polynucleotide encoding the antibody is provided. The polynucleotide includes not only natural nucleotides but also analogues of modified sugar or base segments. The polynucleotide may be modified, and the modifications include the addition, deletion, or non-conservative or conserved substitution of nucleotides.

[0042] According to another embodiment of the present invention, an expression vector comprising the polynucleotide and a host cell comprising the expression vector are provided.

[0043] In this invention, a "vector" is used as a means of expressing a target gene in a host cell, including plasmid vectors; granular vectors; and viral vectors such as phage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. The polynucleotide encoding the antibody can be operatively linked to a promoter. "Operationally linked" refers to a functional link between a nucleic acid expression control sequence (e.g., an array of promoter or transcription factor binding sites) and another nucleic acid sequence, wherein the control sequence regulates the transcription and / or translation of the other nucleic acid sequence. The vector system of this invention can be constructed using various methods known in the art, and generally, vectors for cloning or for expression can be constructed. Additionally, the vectors of this invention can be constructed using prokaryotic or eukaryotic cells as host cells.

[0044] On the other hand, the expression vector of the present invention may contain an antibiotic resistance gene, which is commonly used in the art, as a selection marker gene, wherein the antibiotic resistance gene may be one or more selected from ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, genimycin, neomycin and tetracycline.

[0045] Furthermore, in this invention, the host cell can be a bacterium or an animal cell. The cell transformed by the vector is a host cell capable of stably and continuously cloning and expressing the vector of this invention, and any host cell known in the art can be used. For example, suitable eukaryotic host cells for the vector may be monkey kidney cells (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138 cells, baby hamster kidney (BHK) cells, MDCK cells, myeloma cell lines, HuT78 cells, and HEK-293 cells, but are not limited thereto.

[0046] According to another embodiment of the present invention, a composition or kit for detecting Staphylococcus aureus comprising the antibody is provided. The composition or kit can be used to diagnose skin conditions.

[0047] The compositions or kits according to the present invention can detect Staphylococcus aureus based on the detection of antigen-antibody complexes, wherein the antigen may contain Staphylococcus aureus protein A or the strain itself. Depending on the detection method, the antibodies may be one or more; for example, when there are two antibodies, one may be a capture antibody and the other a detection antibody. In one embodiment of the present invention, a rapid antigen diagnostic kit is manufactured using a 7G1 antibody as the capture antibody and a 6B5 antibody as the detection antibody.

[0048] In this invention, the "antigen-antibody complex" refers to a conjugate of a corresponding protein antigen in a sample and an antibody that recognizes it. The antigen-antibody complex can be detected using methods known in the art, such as optical methods, photochemical methods, biochemical methods, immunochemical methods, electrochemical methods, absorbance methods, chemical methods, and others. Specifically, it can be detected using any method selected from the group consisting of colorimetric methods, electrochemical methods, fluorometric methods, luminometry, particle counting methods, visual assessment methods, and scintillation counting methods. In addition, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, flow cytometry (FACS), protein microarrays, and other methods can be used, but are not limited to these. In this invention, various markers can be used to detect antigen-antibody complexes. Specific examples include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioisotopes, including colloidal gold particles or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, but are not limited to these.

[0049] In this invention, the kit may be an enzyme-linked immunosorbent assay (ELISA) kit, a sandwich ELISA kit, a protein chip kit, or a rapid kit. Specifically, it may be a sandwich ELISA kit or a rapid detection kit using two antibodies, but it is not limited to these and may be applicable to all kits using more than one antibody.

[0050] In this invention, "enzyme-linked immunosorbent assay (ELISA)" is also called an enzyme immunoassay method, and is a method for quantifying antigen-antibody complexes by binding an enzyme to an antibody, using absorbance based on the reaction of the enzyme with the substrate. The ELISA includes, for example, a direct ELISA, which utilizes a labeled antibody that recognizes an antigen attached to a solid support; an indirect ELISA, which utilizes a labeled secondary antibody that recognizes a capture antibody in an antibody complex, the capture antibody recognizing the antigen attached to the solid support; a direct sandwich ELISA, which utilizes another labeled antibody that recognizes an antigen in an antigen-antibody complex attached to a solid support; and an indirect sandwich ELISA, which utilizes a labeled secondary antibody that, after reacting with another antibody that recognizes an antigen in an antigen-antibody complex attached to a solid support, recognizes that other antibody.

[0051] In this invention, "rapid kit" is also referred to as rapid diagnostic test (RDT), rapid antigen detection, or immunochromatographic kit analysis. Rapid kit analysis is a method of analysis using an immunochromatographic test strip comprising a first sample pad, a membrane, and an absorbent pad. Users can easily detect analytes in biological or chemical samples in a short time without special skills or equipment. Compared to commercially available antibodies, the antibodies according to this invention exhibit superior reactivity with antigens, thus providing superior sensitivity and specificity. It is characterized by its applicability to rapid kits for detecting the presence of antigens in a short time. For example, the rapid kit can complete the detection within 60 minutes, specifically within 30 minutes, more specifically within 15 minutes, but is not limited thereto.

[0052] The specimens (samples) used in the kit may include tissue, whole blood, urine, saliva, etc., but specifically, according to the purposes of the invention, they may be skin tissue. For example, the specimen can be prepared by scraping the surface of the skin tissue with a scraper and then dissolving it in a buffer solution. Specifically, the kit according to the invention, by directly using the skin surface, i.e., epidermal tissue, can be used for rapid and convenient self-diagnosis of the presence and concentration of Staphylococcus aureus on the skin. More specifically, by measuring the concentration of Staphylococcus aureus on the skin surface, skin conditions can be diagnosed, where the bacterium is a pathogenic factor for inflammatory skin diseases such as atopic dermatitis or hypersensitivity immune response diseases, predicting the incidence and persistence of related diseases, and thus serving as a rapid self-diagnostic kit for determining subsequent treatment methods.

[0053] The present invention will now be described in more detail through embodiments, but the present invention is not limited thereto.

[0054] Example 1. Preparation of monoclonal antibody against Staphylococcus aureus protein A (Protein A) 1-1. Immunization of mice A mixture containing 150 μg of protein A in PBS was prepared with an equal volume of Freund's incomplete adjuvant (Sigma) to a total volume of 600 μl. This mixture was then injected into 6-week-old female BALB / c mice for primary immunization, with each mouse receiving 200 μl. Two weeks later, 150 μg of protein A was dissolved in PBS to a total volume of 600 μl, and this mixture was then injected into the same mice for secondary immunization, with each mouse receiving 200 μl.

[0055] 1-2. Cell fusion Four days after the second immunization, lymph nodes were aseptically harvested from mice and washed twice with Duchenne Modified Eagle Medium (DMEM). The washed lymph nodes were then prepared into single cells using a cell strainer (Falcon), washed again with DMEM, and resuspended in DMEM. Lymph node cells were mixed with SP2 / O cells at a 5:1 ratio, and 1 ml of PEG1500 (Polyethylene Glycol, Sigma) was slowly added over 2 minutes to induce cell confluence. DMEM was added, and the mixture was incubated at 37°C for 15 minutes, followed by centrifugation at 1200 rpm, discarding the supernatant. Cells were then divided into 1x102 cells. 6Cells were suspended at a concentration of 1 cell / ml in DMEM containing HAT (0.1 mM hypoxanthine, 0.4 M aminopterin, 16 μM thymidine, Sigma) and 20% FBS. The suspension was then added to 96-well plates at 100 μl and cultured in a cell culture incubator for 2 weeks to screen for fusion cells.

[0056] 1-3. Confirmation of antibody production in fusion cells The presence of antibodies in fused cells was observed using an indirect ELISA method with immunoantigen and fusion cell culture medium. Immunoantigen was diluted to a concentration of 0.5 μg / ml, and 50 μl was added to each well. The wells were then incubated overnight at refrigeration to coat the antigen, followed by washing three times with 0.05% phosphate-buffered saline (PBST) containing Tween 20. 180 μl of 1% bovine serum albumin / phosphate-buffered saline (BSA / PBS) was added to each well, and the mixture was incubated at room temperature for 1 hour for blocking. 50 μl of fusion cell culture medium was added to each well, and the mixture was incubated at room temperature for 1 hour, followed by washing three times with 0.05% PBST. 50 μl of HRP-conjugated anti-mouse IgG antibody (Sigma) was aliquoted into each well at a ratio of 1:10000, and the mixture was incubated at room temperature for 30 minutes, followed by washing three times with 0.05% PBST. Add 50 μl of TMB substrate (Surmodics) to each well and react at room temperature for 15 minutes in the dark. Stop the reaction by adding 50 μl of 1N sulfuric acid to each well and measure the OD (optical density) value using an ELISA reader.

[0057] 1-4. Antibody purification Antibody-producing fusion cells were transferred to 25T culture flasks for mass culture. The antibodies were then purified from the obtained cell supernatant using a Protein G affinity chromatography column. After isolating a total of 11 antibodies, they were loaded onto a gel for SDS-PAGE. Results are as follows: Figure 1 As shown.

[0058] like Figure 1 As shown, the expression of 11 monoclonal antibodies (1F2, 4C1, 5B7, 5G2, 6D7, 2E3, 2G2, 5D8, 6C12, 6B5, 7G1) was confirmed, thus confirming that good purification was achieved.

[0059] 1-5. Screening of candidate groups for monoclonal antibodies Antibodies were screened by detecting the reactivity of antibodies with antigens using a sandwich ELISA method. The sandwich ELISA method was performed as follows: First, a total of 11 monoclonal antibodies purified in Examples 1-4 were diluted to 1 μg / ml with carbonate buffer, and 100 μl was added to each well. The wells were then incubated overnight at refrigeration to coat the antibodies. After washing each well three times with 0.05% PBST, 300 μl of 1% BSA / PBS was added to each well, and the mixture was incubated at room temperature for 1 hour for blocking. Immunogens were diluted to concentrations of 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml, and 100 μl of each was added. After incubating at room temperature for 1 hour, the mixture was washed three times with 0.05% PBST. Each biotinylated antibody was diluted to 1 μg / ml, and 100 μl was added to each well. The mixture was incubated at room temperature for 1 hour. After washing three times with 0.05% PBST, horseradish peroxidase-labeled streptavidin (SA-HRP, Sigma) was diluted 1:8000 and 100 μl was added to each well. The reaction was carried out at room temperature for 30 minutes. After washing as described above, 100 μl of TMB (Surmodics) was added to each well. The reaction was carried out in the dark at room temperature for 15 minutes. Finally, 50 μl of 1N sulfuric acid was added to each well to terminate the reaction, and the OD (optical density) value was measured using a microplate reader. Monoclonal antibody candidate groups were screened using the antigen-antibody concentration-dependent reaction described above. The results are shown in Table 1.

[0060] Table 1

[0061] As shown in Table 1, two of the 11 monoclonal antibodies were selected and paired together for sandwich ELISA. The results confirmed that they were highly reactive to the antigen.

[0062] Example 2. Comparison of the affinity of the screened monoclonal antibodies and commercially available antibodies for protein A antigen. 2-1. Comparison using the Indirect ELISA method Protein A antigen was serially diluted with carbonate buffer to concentrations of 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, 0.625 ng / ml, 0.313 ng / ml, and 0.156 ng / ml. 100 μl of each solution was added to each well, and the wells were incubated overnight at refrigeration to coat the antigen. After washing all wells three times with 0.05% PBST, 300 μl of 1% BSA / PBS was added to each well, and the mixture was incubated at room temperature for 1 hour for blocking. After washing all wells three times with 0.05% PBST using the same method described above, the monoclonal antibodies screened in Examples 1-5, as well as the purchased antibodies from EastCoast Bio (J144) and Merck (SAB4200745), were diluted to a concentration of 1 μg / ml with 1% BSA / PBS. 100 μl of each well was added, and the mixture was incubated at room temperature for 1 hour, followed by three washes with 0.05% PBST. 100 μl of HRP-conjugated anti-mouse IgG antibody (Sigma) was added to each well at a ratio of 1:10000, and the mixture was incubated at room temperature for 30 minutes, followed by three washes with 0.05% PBST. 50 μl of TMB substrate (Surmodics) was added to each well, and the mixture was incubated at room temperature in the dark for 15 minutes. 50 μl of 1N sulfuric acid was added to each well to terminate the reaction, and the OD (optical density) value was measured using a microplate reader. The results are shown in Table 2.

[0063] Table 2

[0064] As shown in Table 2, the monoclonal antibodies screened by this invention exhibit superior reactivity compared to commercially available antibodies.

[0065] 2-2. Comparison using Western blotting Add 2 μg of protein A antigen to SDS sample buffer, heat at 95–100 °C for 10 minutes, then load 20 μl of sample and marker into a 12% SDS-PAGE gel, and electrophores at 80–120 V for 2 hours. After completion, peel off the gel and rinse with running water. Stack the gel on a transfer pad in the following order: 3M paper-PVDF-gel-3M paper. Place the gel in the transfer chamber and transfer at 185 mA for approximately 2 hours. Subsequently, block with 5% skim milk, and react overnight under refrigeration with the monoclonal antibody screened in Example 1 and the purchased Merck antibody (SAB4200745) as primary antibodies at a ratio of 1:1000. The membrane was washed three times with 1×TBST for 5 minutes each time. Then, secondary antibody was added at a ratio of 1:4000 and reacted at room temperature for 1 hour. The membrane was then washed three more times with 1×TBST for 5 minutes each time. ECL buffer was then sprayed evenly onto the membrane before detection. Results are as follows: Figure 2 As shown.

[0066] like Figure 2 As shown, the results confirm that the monoclonal antibodies 6B5 and 7G1 screened by this invention exhibit superior reactivity (size: 42kDa) compared to commercially available antibodies.

[0067] Example 3. Preparation of a rapid diagnostic kit using novel monoclonal antibodies 3-1. Antibody screening using rapid test strips A rapid detection kit containing the monoclonal antibodies screened in Example 2 was prepared, and its detection capability for protein A was confirmed. More specifically, the monoclonal antibody candidates screened by the ELISA method were diluted to a concentration of 1 mg / ml, and 1 μl of each was spotted onto a nitrocellulose membrane. Furthermore, the degree of color change at the spotting site was observed after mixing the antibody-gold nanoparticle conjugate with protein A (1 mg / ml) or a Staphylococcus aureus strain. The results are as follows: Figure 3 As shown.

[0068] like Figure 3 As shown, the results of the pairwise reactions of the monoclonal antibodies according to the present invention confirmed that they did not bind to the blank control, but reacted strongly with protein A and Staphylococcus aureus strains, and confirmed that combination 4, 6B5 and 7G1, showed the best results.

[0069] 3-2. Confirm the detection capability of the final screened antibodies on the rapid diagnostic kit. The detection capabilities of the monoclonal antibodies 7G1 and 6B5, ultimately screened in section 3-1, for protein A antigen were confirmed using a rapid test kit. Specifically, the 7G1 antibody was used as the capture antibody, and the 6B5 antibody was used as the detection antibody. Test strips measuring 27 × 300 mm were prepared by spotting the 6B5 antibody (detection line) and anti-Nus A antibody (control line) onto a nitrocellulose membrane. Next, the 7G1 antibody-gold nanoparticle conjugate and Nus A protein-gold nanoparticles were dispensed onto the conjugate pad and dried. The conjugate pad and sample pad were adhered to the lower end of the nitrocellulose membrane, and the absorbent pad was adhered to the upper end to prepare the rapid test kit. Samples of protein A prepared at different concentrations were then placed into the sample injection section, and the degree of color change of the detection line was observed. The results are as follows: Figure 4 As shown.

[0070] like Figure 4 As shown, this demonstrates that the two monoclonal antibodies, 7G1 and 6B5, can be used in a rapid diagnostic kit for detecting Staphylococcus aureus on the skin, particularly for rapidly and accurately detecting levels as low as approximately 0.001 μg / ml. Next, using samples prepared from actual skin epidermis containing Staphylococcus aureus, an experiment was conducted to determine whether the developed rapid diagnostic kit could detect Staphylococcus aureus. The results are as follows... Figure 5 As shown.

[0071] like Figure 5 As shown, it is confirmed that the monoclonal antibodies 7G1 and 6B5 described in this invention can be used to achieve rapid diagnosis of Staphylococcus aureus in actual skin.

[0072] Example 4. Sequence analysis of novel monoclonal antibodies 7G1 and 6B5 Sequence analysis was performed on the monoclonal antibodies 7G1 and 6B5 that were verified to be effective in Example 3.

[0073] The results confirmed that monoclonal antibody 7G1 consists of a heavy chain comprising SEQ ID NO: 7 (CDR1 to CDR3) of SEQ ID NO: 1 to SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 8 (CDR1 to CDR3) of SEQ ID NO: 4 to SEQ ID NO: 6. It was also confirmed that monoclonal antibody 6B5 consists of a heavy chain comprising SEQ ID NO: 15 (CDR1 to CDR3) of SEQ ID NO: 9 to SEQ ID NO: 11, and a light chain comprising SEQ ID NO: 16 (CDR1 to CDR3) of SEQ ID NO: 12 to SEQ ID NO: 14.

[0074] The above experimental results confirm that, by using the novel monoclonal antibodies 7G1 and 6B5, which specifically bind to protein A of Staphylococcus aureus, a rapid diagnostic kit can be prepared to diagnose skin conditions by directly determining the presence of Staphylococcus aureus on the skin.

[0075] Table 3

[0076] The foregoing has described specific parts of the present invention in detail; however, those skilled in the art should understand that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the invention. Therefore, the essential scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An antibody specific to Staphylococcus aureus protein A, characterized in that, Include: The heavy chain variable region comprises heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and The light chain variable region includes light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO:

6.

2. The antibody according to claim 1, characterized in that, The antibody contains a heavy chain variable region represented by the amino acid sequence of SEQ ID NO:

7.

3. The antibody according to claim 1, characterized in that, The antibody contains a light chain variable region represented by the amino acid sequence of SEQ ID NO:

8.

4. An antibody specific to Staphylococcus aureus protein A, characterized in that, Include: The heavy chain variable region comprises heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; and The light chain variable region includes light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO:

14.

5. The antibody according to claim 4, characterized in that, The antibody contains a heavy chain variable region represented by the amino acid sequence of SEQ ID NO:

15.

6. The antibody according to claim 4, characterized in that, The antibody contains a light chain variable region represented by the amino acid sequence of SEQ ID NO:

16.

7. A polynucleotide, characterized in that, Its encoding is an antibody according to any one of claims 1-6.

8. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 7.

9. A host cell, characterized in that, It comprises the expression vector according to claim 8.

10. A composition for detecting Staphylococcus aureus, characterized in that, It contains the antibody according to any one of claims 1-6.

11. A reagent kit for detecting Staphylococcus aureus, characterized in that, It contains the antibody according to any one of claims 1-6.

12. The kit according to claim 11, characterized in that, The kit is an enzyme-linked immunosorbent assay (ELISA) kit, a sandwich ELISA kit, a protein chip kit, or a rapid kit.

13. The kit according to claim 11, characterized in that, The kit includes two antibodies: a capture antibody and a detection antibody.

14. The kit according to claim 11, characterized in that, The kit is used to diagnose skin conditions.

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