Diagnostic kit for skin comprising antibody for lactobacillus plantarum LDH1

Abstract

The present invention relates to an antibody specific to Lactobacillus plantarum LDH1, and to a composition or kit comprising same for detecting Lactobacillus plantarum or diagnosing skin conditions. The monoclonal antibody according to the present invention is highly reactive to antigens, and, particularly, can detect antigens rapidly and accurately even at low concentrations due to two antibodies of different CDRs having been combined and used as capture and detection antibodies, thus allowing the monoclonal antibody to be used in rapid self-diagnostic kits and the like. In using the monoclonal antibody, the degree of proliferation of Lactobacillus plantarum on the skin surface and the like can be visually checked, abnormal states of the skin can be diagnosed, the incidence probability and persistence of related diseases can be predicted, and subsequent treatment methods can be determined.

Description

Skin diagnostic kit containing antibodies to Lactobacillus plantarum LDH1

[0001] The present invention relates to an antibody specific for LDH1 of Lactobacillus plantarum and a composition or kit for detecting Lactobacillus plantarum or diagnosing skin conditions comprising the same.

[0002] With the recent surge in research on the microbiome, the fields of the gut microbiome and skin microbiome are gaining attention. While NGS and genetic testing can provide highly detailed results and data on the skin microbiome, a rapid diagnostic method for easily assessing skin condition remains lacking.

[0003] Meanwhile, recent studies have shown that the diversity of the skin microbiome increases with age, while the abundance of Lactobacillus and Cutibacterium decreases, suggesting that the skin microbiome may be related to skin aging. Among these, lactic acid bacteria (LAB) are commonly known as probiotics, and related human studies are actively underway. Although the skin application of lactic acid bacteria is still in its infancy compared to oral intake, various beneficial effects on the skin are being discovered. In particular, Lactobacillus plantarum is known to alleviate symptoms of atopic dermatitis in adults due to its immunomodulatory effects, and there are also research results showing its effectiveness in psoriasis, acne, and seborrheic dermatitis.

[0004] Accordingly, the present inventors sought to develop a kit for detecting Lactobacillus plantarum, which can have a positive effect on skin health. In particular, given the greatly increased consumer accessibility to rapid diagnostic kits due to COVID-19, the inventors sought to develop a rapid self-diagnosis kit for measuring the skin surface concentration of LDH1, a major protein of Lactobacillus plantarum. They developed a novel monoclonal antibody against LDH1 and selected an optimal combination thereof to confirm that it could rapidly and accurately detect Lactobacillus plantarum, thereby completing the present invention.

[0005] The purpose of the present invention is to provide an antibody specific for the LDH1 protein of Lactobacillus plantarum and a composition or kit for detecting Lactobacillus plantarum comprising the same.

[0006] The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0007] According to an embodiment of the present invention, an antibody specific for the LDH1 protein of Lactobacillus plantarum is provided, comprising a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6.

[0008] Also provided is an antibody specific for the LDH1 protein of Lactobacillus plantarum, comprising a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 14.

[0009] The monoclonal antibody according to the present invention is a novel antibody specific for the LDH1 protein of Lactobacillus plantarum, and has a very high reactivity to the antigen. In particular, the monoclonal antibody according to the present invention can be applied to rapid self-diagnosis kits, etc., by combining two antibodies with different CDRs as a capture antibody and a detection antibody, enabling rapid and accurate detection even at low concentrations. In addition, by using this, the proliferation status of Lactobacillus plantarum on the skin surface can be intuitively confirmed, thereby diagnosing skin conditions, predicting the likelihood and persistence of related diseases, and subsequently determining treatment methods.

[0010] In order to more fully understand the drawings cited in the detailed description of the present invention, a brief description of each drawing is provided.

[0011] Figure 1 shows the SDS-PAGE results confirming the purified monoclonal antibody.

[0012] Figure 2 shows the results of comparing the affinity of selected monoclonal antibodies and commercialized antibodies for the LDH1 antigen through indirect ELISA (A: Biorbyt (orb847579)).

[0013] Figure 3 shows the results of comparing the affinity of selected monoclonal antibodies and commercialized antibodies for the LDH1 antigen using Western blot.

[0014] Figure 4 shows the results of observing the reactivity of a monoclonal antibody combination according to the present invention to the LDH1 antigen on a rapid kit strip.

[0015] Figure 5 shows the results of observing the reactivity to the LDH1 antigen on a rapid kit using 2H1 and 3D5, which are monoclonal antibodies according to the present invention.

[0016] Figure 6 shows the results confirming that detection of Lactobacillus plantarum is possible when an actual skin sample is applied to a rapid kit manufactured using monoclonal antibodies 2H1 and 3D5 according to the present invention.

[0017] According to an embodiment of the present invention, an antibody specific for the LDH1 protein of Lactobacillus plantarum is provided, comprising a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6.

[0018] Also provided is an antibody specific for the LDH1 protein of Lactobacillus plantarum, comprising a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 14.

[0019] According to another embodiment of the present invention, a polynucleotide encoding the antibody is provided.

[0020] According to another embodiment of the present invention, an expression vector comprising the polynucleotide is provided.

[0021] According to another embodiment of the present invention, a host cell comprising the expression vector is provided.

[0022] According to another embodiment of the present invention, a composition for detecting Lactobacillus plantarum comprising the antibody is provided.

[0023] According to another embodiment of the present invention, a kit for detecting Lactobacillus plantarum comprising the antibody is provided.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. In addition, when describing embodiments of the present invention, if a detailed description of a related known structure or function is judged to hinder the understanding of the embodiments of the present invention, a detailed description thereof will be omitted. In addition, although embodiments of the present invention will be described below, the technical idea of ​​the present invention is not limited or restricted thereto, and can be modified and implemented in various ways by those skilled in the art.

[0025] When a part in this specification is said to include a certain component, this does not exclude other components, unless otherwise specifically stated, but rather means that other components may be included. In this specification, the term "and / or" includes a combination of multiple related items or any one of multiple related items.

[0026]

[0027] According to an embodiment of the present invention, a novel antibody specific for the LDH1 protein of Lactobacillus plantarum is provided.

[0028] In the present invention, “antibody” means a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments.

[0029] In the present invention, "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.

[0030] For the purpose of the present invention, the antibody may be a monoclonal antibody that specifically binds to the LDH1 protein of Lactobacillus plantarum.

[0031] In the present invention, the immunoglobulin has a heavy chain and a light chain, and each of the heavy chain and the light chain includes a constant region and a variable region (these regions are known as domains). The variable regions of the light chain and the heavy chain include three variable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs mainly play a role in binding to epitopes of an antigen. The CDRs of each chain are sequentially called CDR1, CDR2, and CDR3 starting from the N-terminus, and are also identified by the chain on which the specific CDR is located.

[0032] According to one embodiment of the present invention, an antibody specific for the LDH1 protein of Lactobacillus plantarum is provided, which comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6, and is designated as 2H1.

[0033] The above antibody may include a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8.

[0034] According to another embodiment of the present invention, there is provided an antibody specific for the LDH1 protein of Lactobacillus plantarum, comprising a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 14, and this antibody is designated as 3D5.

[0035] The above 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.

[0036] The antibody of the present invention may include variants of the amino acid sequence set forth in the attached sequence listing, within the scope of being capable of specifically recognizing the LDH1 protein of Lactobacillus plantarum. For example, the amino acid sequence of the antibody may be changed to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals 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 these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0037] When introducing mutations, the hydrophobicity index of an amino acid can be considered. Each amino acid is assigned a hydrophobicity 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); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0038] The hydrophobic amino acid index is crucial for imparting interactive biological functions to proteins. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with hydrophobic indices differing by ±2, more preferably ±1, or even more preferably ±0.5.

[0039] Meanwhile, it is also well known that substitutions between amino acids with similar hydrophilicity values ​​result in proteins with equivalent biological activity, and the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+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).

[0040] When introducing a mutation with reference to a hydrophilicity value, substitution is preferably made between amino acids showing a difference in hydrophilicity value within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0041] Amino acid exchanges in proteins that do not alter the overall activity of the molecule are also known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between 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, Asp / Gly.

[0042] Therefore, the antibody according to the present invention may have 80 to 99% homology, 90 to 99% homology, or 95 to 99% homology with the amino acid sequence described above.

[0043] According to another embodiment of the present invention, a polynucleotide encoding the antibody is provided. The polynucleotide includes not only natural nucleotides but also analogues with modified sugar or base positions. The polynucleotide may be modified, and the modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0044] According to another embodiment of the present invention, an expression vector comprising the polynucleotide and a host cell comprising the expression vector are provided.

[0045] In the present invention, the term “vector” refers to a means for expressing a target gene in a host cell, including a plasmid vector; a cosmid vector; and viral vectors such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. In the vector of the present invention, a polynucleotide encoding an antibody may be operatively linked to a promoter. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the control sequence controls transcription and / or translation of the other nucleic acid sequence. The vector system of the present invention can be constructed by various methods known in the art, and can typically be constructed as a vector for cloning or a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0046] Meanwhile, the expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, and the antibiotic resistance gene may be at least one selected from among ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0047] In addition, in the present invention, the host cell may be a bacterial or animal cell. The cell transformed with the vector is a host cell capable of stably and continuously cloning and expressing the vector of the present invention, and any host cell known in the art may be used. For example, suitable eukaryotic host cells for the vector may include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK-293 cells.

[0048] According to another embodiment of the present invention, a composition or kit for detecting Lactobacillus plantarum comprising the antibody is provided. The composition or kit may be used for diagnosing skin conditions.

[0049] The composition or kit according to the present invention can detect Lactobacillus plantarum by detecting an antigen-antibody complex, wherein the antigen may include the LDH1 protein of Lactobacillus plantarum or the strain itself. The number of antibodies may be one or two or more depending on the detection method. For example, when there are two antibodies, one antibody may be a capture antibody and the other antibody may be a detection antibody. In one embodiment of the present invention, a rapid antigen diagnostic kit was manufactured using 2H1 antibody as a capture antibody and 3D5 antibody as a detection antibody.

[0050] In the present invention, the “antigen-antibody complex” refers to a combination of a corresponding protein antigen in a sample and an antibody that recognizes it. The above antigen-antibody complex can be detected using a method known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical and other methods, and specifically, can be detected by any method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment and a scintillation counting method, and can be detected by western blotting, an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay, a radioimmunodiffusion, an Ouchterlony immunodiffusion, a Rocket immunoelectrophoresis, tissue immunostaining, an immunoprecipitation assay, a complete fixation assay, Methods such as FACS and protein chips can be used, but are not limited thereto. In the present invention, various labels can be used to detect antigen-antibody complexes. Specific examples include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, radioactive isotopes, and the like, and include, but are not limited to, colloidal gold particles or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0051] In the present invention, the kit may be an ELISA (Enzyme-linked immunosorbent assay) kit, a sandwich ELISA kit, a protein chip kit, or a rapid kit, and specifically, may be a sandwich ELISA kit or a rapid kit using two antibodies, but is not limited thereto and may be applied to all kits using one or more antibodies.

[0052] In the present invention, "ELISA (Enzyme-linked immunosorbent assay)" is also called enzyme-linked immunosorbent assay, and is a method of quantifying using absorbance through a reaction between an enzyme and a substrate by forming an antigen-antibody complex by binding an enzyme to an antibody. The ELISA includes a direct ELISA using a labeled secondary antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibodies and antigens attached to the solid support, and an indirect sandwich ELISA using a labeled secondary antibody that recognizes an antibody after reacting with another antibody that recognizes an antigen in a complex of antibodies and antigens attached to the solid support.

[0053] In the present invention, the “rapid kit” is also called a rapid diagnostic test (RDT), rapid antigen test, or immunochromatography kit analysis. The rapid kit analysis is an analysis method using an immunochromatography strip including a first sample pad, a membrane, and an absorbent pad, which allows the user to simply detect an analyte from a biological or chemical sample in a short period of time without any special skills or equipment. The antibody according to the present invention has superior reactivity with an antigen compared to commercially available antibodies, and thus has excellent sensitivity and specificity, and is characterized by being applicable to a rapid kit that detects the presence of an antigen in a short period of time. The rapid kit may be capable of detecting an antigen within, for example, 60 minutes, specifically, 30 minutes, or even 15 minutes, but is not limited thereto.

[0054] The specimen (sample) used in the above kit may include all of tissue, whole blood, urine, saliva, etc., but may specifically be skin tissue depending on the purpose of the present invention. For example, the specimen may be prepared by scraping the surface of skin tissue with a stick and dissolving it in a buffer solution. Specifically, the kit according to the present invention can be used to quickly and easily self-diagnose whether Lactobacillus plantarum proliferates on the skin and its concentration by directly using the skin surface, i.e., the epidermal tissue. More specifically, by measuring the skin surface concentration of Lactobacillus plantarum, which is known to be involved in preventing skin aging and alleviating the symptoms of skin diseases including atopic dermatitis, the skin condition can be diagnosed, the possibility of occurrence and persistence of related diseases can be predicted, and it can be utilized as a rapid self-diagnosis kit to determine a subsequent treatment method.

[0055]

[0056] Hereinafter, examples are presented to more specifically explain the present invention, but the present invention is not limited thereto.

[0057]

[0058] Example 1. Preparation of a monoclonal antibody against Lactobacillus plantarum LDH1

[0059] 1-1. Immunization of mice

[0060] The primary immunization was performed by mixing PBS containing 150 μg of LDH1 protein with an equal volume of Freund's adjuvant (Incomplete, Sigma) to make a total of 600 μl and injecting 200 μl per mouse into 6-week-old female BALB / c mice. Two weeks later, the secondary immunization was performed by injecting 200 μl per mouse into the same mice that had received the primary immunization, dissolving 150 μg of LDH1 in PBS to make a total of 600 μl.

[0061]

[0062] 1-2. Cell fusion

[0063] Four days after the secondary immunization, the lymph nodes of the mice were aseptically removed and washed twice with DMEM medium. The washed lymph nodes were made into single cells using a cell strainer (Falcon), washed again with DMEM, and suspended in DMEM. Lymph node cells and SP2 / O cells were mixed at a ratio of 5:1, and 1 ml of PEG (Polyethylene Glycol 1500, Sigma) was slowly added for 2 minutes to induce cell fusion. DMEM medium was added, and the cells were incubated at 37°C for 15 minutes, centrifuged at 1200 rpm, and the supernatant was removed. 1x10 cells were incubated in DMEM supplemented with HAT (0.1 mM hypoxanthine, 0.4 M aminopterin, 16 μM thymidine, Sigma) and 20% FBS. 6 After suspending at a concentration of 10 cells / ml, 100 μl of this suspension was dispensed into a 96-well plate and cultured in a cell incubator for 2 weeks to select fused cells.

[0064]

[0065] 1-3. Confirmation of antibody formation in fused cells

[0066] The production of antibodies in fused cells was observed using an indirect ELISA method using the culture medium of the fused cells and the immune antigen. The immune antigen was diluted to a concentration of 0.5 μg / ml and 50 μl was added to each well. The wells were incubated overnight in the refrigerator to coat the antigen. Each well was washed three times with 0.05% PBST. 180 μl of 1% BSA / PBS was added to each well and blocked for 1 hour at room temperature. 50 μl of the culture medium of the fused cells was added to each well, incubated for 1 hour at room temperature, and then washed three times with 0.05% PBST. 50 μl of HRP-conjugated anti-mouse IgG antibody (Sigma) was dispensed into each well at a ratio of 1:10000, incubated for 30 minutes at room temperature, and then washed three times with 0.05% PBST. TMB substrate (Surmodics) was added to each well at 50 μl and reacted at room temperature for 15 minutes in a dark-shielded environment. The reaction was stopped by adding 50 μl of 1 N sulfuric acid to each well, and the optical density (OD) was measured using an ELISA reader.

[0067]

[0068] 1-4. Antibody purification

[0069] The antibody-producing fused cells were transferred to 25T flasks for mass cultivation, and antibodies were purified from the resulting cell supernatant using a Protein G affinity column. A total of eight antibodies were isolated, purified, loaded onto a gel, and subjected to SDS-PAGE. The results are shown in Figure 1.

[0070] As shown in Figure 1, it was confirmed that the product was well purified by confirming the expression of eight monoclonal antibodies (1G9, 2H1, 3D5, 2A3, 3B10, 3E3, 4F7, 3D10).

[0071]

[0072] 1-5. Selection of monoclonal antibody candidates

[0073] Antibodies were selected by examining antigen-antibody reactivity using the sandwich ELISA method. The sandwich ELISA was performed as follows. First, a total of eight monoclonal antibodies purified in Examples 1-4 were each diluted to a concentration of 1 μg / ml in carbonate buffer, 100 μl was added to each well, and the wells were left in the refrigerator overnight 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 wells were blocked for 1 hour at room temperature. The immune antigens were diluted to concentrations of 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml, and 100 μl were added each, and the wells were reacted for 1 hour at room temperature, and then washed three times with 0.05% PBST. Each biotinylated antibody was diluted to a concentration of 1 μg / ml, added to each well (100 μl), and incubated for 1 hour at room temperature. After washing three times with 0.05% PBST, SA-HRP (Sigma) was diluted 1:8000, added to each well (100 μl), and incubated for 30 minutes at room temperature. After washing as described above, TMB (Surmodics) was added to each well (100 μl), and incubated for 15 minutes at room temperature in a light-shielded environment. After stopping the reaction, 50 μl of 1 N sulfuric acid was added to each well, and the OD (optical density) was measured in an ELISA reader. Monoclonal antibody candidates were selected through the concentration reaction of antigen and antibody in the same manner as described above. The results are shown in Table 1.

[0074]

[0075] As shown in Table 1, two of the eight monoclonal antibodies were selected in pairs and sandwich ELISA was performed, and it was confirmed that they exhibited high reactivity to the antigen, and among them, 2H1, 3B10, 3D5, 3D10, and 4F7 exhibited high OD values.

[0076]

[0077] Example 2. Comparison of the affinity of selected monoclonal antibodies and commercialized antibodies for the LDH1 antigen.

[0078] 2-1. Comparison using the indirect ELISA method

[0079] LDH1 antigen was serially diluted in carbonate buffer to concentrations of 10, 5, 2.5, 1.25, 0.625, 0.313, and 0.156 ng / ml, added 100 μl to each well, and left to stand overnight in the refrigerator 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 reaction was blocked for 1 hour at room temperature. After washing all wells three times with 0.05% PBST in the same manner as above, the monoclonal antibodies selected in Example 1-5 and the purchased antibody from Biorbyt (orb847579) were diluted with 1% BSA / PBS to a concentration of 1 μg / ml, and then 100 μl was added to each well, reacted for 1 hour at room temperature, and then washed three times with 0.05% PBST. 100 μl of HRP-conjugated anti mouse IgG antibody (Sigma) was dispensed to each well at a ratio of 1:10000, reacted for 30 minutes at room temperature, and then washed three times with 0.05% PBST. 50 μl of TMB substrate (Surmodics) was added to each well, and reacted for 15 minutes at room temperature in a light-shielded manner. The reaction was stopped by adding 50 μl of 1N sulfuric acid to each well, and the OD (optical density) was measured using an ELISA reader. The results are shown in Figure 2.

[0080] As shown in Fig. 2, the monoclonal antibodies 2H1, 3B10, 3D5, 3D10, and 4F7 selected through the present invention showed better reactivity than commercialized antibodies, and in particular, 2H1 and 3D5 showed excellent results.

[0081]

[0082] 2-2. Comparison through Western blot

[0083] 2 μg of LDH1 antigen was added to SDS sample buffer and heated at 95-100℃ for 10 minutes, and 20 μl each of sample and marker were loaded onto a 12% SDS-PAGE gel and electrophoresed at 80-120 V for 2 hours. After completion, the gel was removed, washed in running water, and then placed on a plastic transfer pad in the order of 3M paper-PVDF-gel-3M paper. The pad was mounted on a transfer chamber and transferred at 185 mA for about 2 hours. After blocking with 5% skim milk, the monoclonal antibody selected in Example 1 and the antibody purchased from Biorbyt (orb847579) were reacted overnight in a refrigerator at a ratio of 1:1000. After washing three times with 1x TBST for 5 minutes each, the membrane was reacted with secondary antibody at a ratio of 1:4000 for 1 hour at room temperature, and washed three times with 1x TBST for 5 minutes each. ECL buffer was evenly distributed over the membrane, and detection was performed. The results are shown in Figure 3.

[0084] As shown in Fig. 3, it was confirmed that the monoclonal antibodies 3D5 and 2H1 selected through the present invention exhibited better reactivity than commercialized antibodies (Size 35.3 Kda).

[0085]

[0086] Example 3. Production of a rapid diagnostic kit using a novel monoclonal antibody.

[0087] 3-1. Antibody screening using rapid kit strips

[0088] A rapid kit containing the monoclonal antibodies selected in Example 2 was manufactured, and its detection ability for LDH1 was confirmed. More specifically, the monoclonal antibody candidates selected by the ELISA method were each diluted to a concentration of 1 mg / ml and spotted 1 μl onto a nitrocellulose membrane. In addition, the antibody-gold nanoparticle condensate was mixed with LDH1 (1 mg / ml) or a Lactobacillus strain, and the degree of color change at the spot was observed. The results are shown in Fig. 4.

[0089] As shown in Fig. 4, when the monoclonal antibodies according to the present invention were reacted in sets of two, it was confirmed that they did not bind to Blank, but reacted strongly to LDH1 and Lactobacillus strains, and among them, the set number 3, 2H1-3D5, showed the best results.

[0090]

[0091] 3-2. Confirmation of the detection ability of the final screening antibody on the rapid diagnostic kit

[0092] The detection ability of 2H1 and 3D5, the monoclonal antibodies finally selected in 3-1 above, against the LDH1 antigen was confirmed using a rapid kit. Specifically, the 2H1 antibody was used as a capture antibody, and the 3D5 antibody was used as a detection antibody. Strips measuring 27 x 300 mm were prepared by spotting the 3D5 antibody (test line) and anti-Nus A antibody (control line) on a nitrocellulose membrane. Next, the 2H1 antibody-gold nanoparticle condensate and the Nus A Protein-gold nanoparticle were dispensed onto a conjugate pad and dried to prepare them. The conjugate pad and sample pad were attached to the bottom of the nitrocellulose membrane, and the absorbent pad was attached to the top to manufacture a rapid kit. After that, samples prepared at various concentrations of LDH1 protein were added to the sample injection port, and the degree of color change of the test line was observed. The results are shown in Fig. 5.

[0093] As shown in Fig. 5, two monoclonal antibodies, 2H1 and 3D5, can be applied to a rapid diagnostic kit for detecting Lactobacillus plantarum in the skin, and in particular, it was confirmed that rapid and accurate detection was possible up to a level of about 0.032 μg / ml.

[0094] Next, an experiment was conducted to determine whether Lactobacillus plantarum could be detected using the rapid diagnostic kit developed above, using a sample prepared by scraping actual skin epidermis where Lactobacillus plantarum exists. The results are shown in Fig. 6.

[0095] As shown in Fig. 6, it was confirmed that rapid diagnosis of Lactobacillus plantarum on actual skin was possible using 2H1 and 3D5, which are monoclonal antibodies according to the present invention.

[0096]

[0097] Example 4. Sequence analysis of novel monoclonal antibodies 2H1 and 3D5.

[0098] Sequence analysis of monoclonal antibodies 2H1 and 3D5, whose effectiveness was confirmed in Example 3 above, was performed.

[0099] As a result, it was confirmed that the monoclonal antibody 2H1 is composed of a heavy chain of SEQ ID NO: 7 containing CDRs 1 to 3 of SEQ ID NOs: 1 to 3 and a light chain of SEQ ID NO: 8 containing CDRs 1 to 3 of SEQ ID NOs: 4 to 6. In addition, it was confirmed that the monoclonal antibody 3D5 is composed of a heavy chain of SEQ ID NO: 15 containing CDRs 1 to 3 of SEQ ID NOs: 9 to 11 and a light chain of SEQ ID NO: 16 containing CDRs 1 to 3 of SEQ ID NOs: 12 to 14.

[0100]

[0101] Through the above experimental results, it was confirmed that a rapid diagnostic kit capable of diagnosing skin conditions by directly determining the presence of Lactobacillus plantarum on the skin can be manufactured using novel monoclonal antibodies 2H1 and 3D5 that specifically bind to the LDH1 protein of Lactobacillus plantarum.

[0102]

[0103] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; An antibody specific for the LDH1 protein of Lactobacillus plantarum, comprising a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO:

6.

2. In paragraph 1, An antibody characterized in that the antibody comprises a heavy chain variable region represented by the amino acid sequence of sequence number 7.

3. In paragraph 1, An antibody characterized in that the antibody comprises a light chain variable region represented by the amino acid sequence of sequence number 8.

4. A heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11; An antibody specific for the LDH1 protein of Lactobacillus plantarum, comprising a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO:

14.

5. In paragraph 4, An antibody characterized in that the antibody comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO:

15.

6. In paragraph 4, An antibody characterized in that the antibody comprises a light chain variable region represented by the amino acid sequence of SEQ ID NO:

16.

7. A polynucleotide encoding an antibody according to any one of claims 1 to 6.

8. An expression vector comprising the polynucleotide of claim 7.

9. A host cell comprising the expression vector of clause 8.

10. A composition for detecting Lactobacillus plantarum comprising an antibody according to any one of claims 1 to 6.

11. A kit for detecting Lactobacillus plantarum comprising an antibody according to any one of claims 1 to 6.

12. In the 11th paragraph, the kit is an ELISA (Enzyme-linked immunosorbent assay) kit, a sandwich ELISA kit, a protein chip kit or a rapid kit.

13. A kit according to claim 11, wherein the kit comprises two types of antibodies, one type of antibody being a capture antibody and the other type of antibody being a detection antibody.

14. In clause 11, the kit is a kit for diagnosing skin conditions.

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