WO2013125879A1 - Antibody to d-alanine-d-alanine ligase and detection of antibiotic-resistant bacteria using same - Google Patents

Antibody to d-alanine-d-alanine ligase and detection of antibiotic-resistant bacteria using same Download PDF

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Publication number
WO2013125879A1
WO2013125879A1 PCT/KR2013/001391 KR2013001391W WO2013125879A1 WO 2013125879 A1 WO2013125879 A1 WO 2013125879A1 KR 2013001391 W KR2013001391 W KR 2013001391W WO 2013125879 A1 WO2013125879 A1 WO 2013125879A1
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Prior art keywords
antibody
ligase
vana
vanb
alanine
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PCT/KR2013/001391
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French (fr)
Korean (ko)
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송형근
지길용
문유리
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다이노나(주)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring

Definitions

  • the present invention relates to an antibody against D-alanine-D-alanine ligase, a method for quantifying D-alanine-D-alanine ligase using the same, and detection of antibiotic resistant bacteria using the antibody.
  • Glycopeptide antibiotics are an important class of antibiotics that bind to peptidoglycan precursors and interfere with crosslinking of bacterial cell walls. It is often the last antibiotic of choice in treating life-threatening infections. Glycoated antibiotics include Vancomycin, Teicoplanin, Telavancin, Bleomycin Rarao lanin, and Decaplanin. Vancomycin and Televancin are also known to induce VanA ligase induction (Antimicrobial Agents and chemotherapy, Vol. 54, No. 7, 2010, p.2814-2818), and vancomycin-resistant enterococci are also known to be Teicoplanin resistant. .
  • Enterococci are classified into several phenotypes based on their resistance to and resistance to glycopeptide antibiotics such as teicoplanin or vancomycin. VanA type has induction resistance to vancomycin and teicoplanin, but VanB type has only induction resistance to vancomycin. Antibiotic resistance of the VanA type has been reported in a variety of enterococci, including E. faecalis, E. faecium, E. gal 1 inarm, E. cassel i flavus, E. durans, E. mundtii, E. raffinosus, and E.
  • Antibiotic resistance of the VanB type has been reported in E. faecalis and E. faecium.
  • Antibiotic resistance of the VanC type is low intrinsic to vancomycin. Characterized by resistance and reported in E. gallinarum, E. casseliflavus, and E. a esce2s. '
  • VanA, VanB and VanC Genes related to the types of VanA, VanB and VanC were found and named vanA, vanB, vanC-1, vanC - 2 and vanCS.
  • the vanA and vanB gene clusters can be propagated to other strains in the form of transposons. Since VanA and VanB type antibiotic resistance is obtained, it can be transmitted to Staphylococcus aureus and organisms in addition to enterococci, for example Vancotnyc in-resistant Staphylococcus aureus (VRSA). On the other hand, VanC type resistance is inherent in nature and therefore has no clinical significance.
  • VanA and VanB types need to be detected and typed because of their relatively high separation frequency, strong antibiotic resistance, and propagation capability.
  • Bacteria having resistance to glycopeptide antibiotics include enterococci, which are resistant to teicoplanin or vancomycin, and staphylococci resistant to vancomycin.
  • enterococci which are resistant to teicoplanin or vancomycin
  • staphylococci resistant to vancomycin For example, For vancomycin-resistant enterococci, fecal swabs or rectal swabs are used as detection samples for the prevention of transmission and early diagnosis. Recently, new diagnostic methods such as chromogenic agar have been introduced, and improved detection performance using fecal or rectal swab samples has been reported.
  • ChromID VRE agar (bioMerieux, Marcy l'Etoile, France) is a solid medium containing vancomycin at 8 ug / mL. It has advantages However, a diagnostic method based on cultures such as ChromID VRE agar has the disadvantage that it does not provide direct phenotypic information on the type of VanA or VanB antibiotic resistance.
  • the present invention solves the disadvantages of the high cost of the conventional PCR method and the difficulty of early diagnosis of the culture method and does not provide information on the phenotype of antibiotic resistant bacteria, glycopeptide system that can obtain the resistance gene type information in a short time
  • An antibody, a detection method, and a detection kit for detecting bacteria resistant to antibiotics are provided.
  • the present invention provides an antibody or antigen-binding fragment thereof that specifically recognizes D-alanine-D-alanine ligase of bacteria resistant to glycopeptide antibiotics, and uses the antibody for glycopeptide antibiotics.
  • An object of the present invention is to provide a detection kit and a detection method capable of quickly detecting resistant bacteria.
  • the present invention provides a D-alanine-D-alanine ligase present in a sample using an antibody or antigen-binding fragment thereof that specifically recognizes D-alanine-D-alanine ligase of bacteria resistant to glycopeptide-based antibiotics. It relates to a method for quantifying agent.
  • One embodiment of the present invention is produced using D-alanine -D-alanine ligase, preferably VanA ligase or VanB ligase protein as an antigen, specific for the D-alanine -D-alanine ligase To an antibody or antigen-binding fragment thereof that is recognized automatically.
  • the antibody may be a monoclonal antibody or a polyclonal antibody, and may also be a chimeric antibody or a humanized antibody.
  • Another embodiment of the present invention is a D-alanine-D-alanine ligase, preferably VanA ligase comprising an antibody or antigen-binding fragment thereof that specifically recognizes the D-alanine -D-alanine ligase A method for quantifying VanB ligase protein.
  • a further embodiment of the invention comprises a D-alanine-D-alanine ligase, preferably VanA ligase, comprising an antibody or antigen-binding fragment thereof that specifically recognizes the D-alanine -D-alanine ligase
  • the present invention relates to a detection kit for detecting bacteria that expresses VanB protein and is resistant to glycopeptide antibiotics.
  • the glycopeptide antibiotic may be at least one selected from the group consisting of Vancomycin, Teicoplanin, Telavancin, Bleomycin, Ramoplanin, and Decaplanin.
  • the bacterium may be a strain of the genus Enterococcus or a strain of the genus Staphylococcus.
  • a further embodiment of the present invention relates to a method for detecting bacteria having resistance to glycopeptide antibiotics by immunoassay using an antibody or antigen-binding fragment thereof that specifically recognizes the D-alanine-D-alanine ligase.
  • the detection method is an antigen-antibody with the antibody using VanA ligase or VanB ligase protein present in the sample as an antigen.
  • the immune response can be analyzed to detect bacteria that are resistant to glycopeptide antibiotics expressing VanA ligase or VanB ligase in the sample in the case of a positive immune response.
  • the detection kit according to the present invention and a method for detecting resistant bacteria can not only detect bacteria having resistance to glycopeptide antibiotics contained in a sample, but also distinguish between phenotypes of VanA ligase or VanB ligase type. Can be.
  • One embodiment of the present invention is produced by using D-alanine-D-alanine ligase, preferably VanA ligase or VanB ligase as an antigen, an antibody or a antibody that specifically recognizes the VanA ligase or VanB protein. It relates to an antigen binding fragment.
  • VanA ligase or VanB ligase protein as an antigen has been reported a variety of proteins that can use the antigen as D-alanine-D-alanine ligase (EC 6.3.2.4.).
  • the amino acid sequence of the VanA ligase protein may preferably be a sequence that includes the amino acid sequence of SEQ ID NO: 1 or is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 2.
  • Enterococcus faecium-derived protein sequences GenBank accession number X56895.1, Uniprot accession number Q7B608, D4RFM1, D4QQA6, E3USG8, and J.BBH6 and S.
  • aureus-derived protein sequences Uniprot accession number Q7BWD7, I3GWM9, and I3EUH6.
  • amino acid sequence of the VanA ligase protein is not 100% identical to the amino acid sequence of SEQ ID NO: 1, for example, an amino acid sequence identity of 90% or more, and acts as an antigen to prepare an antibody that specifically recognizes VanA ligase It is intended to include all proteins that exhibit the same antigenicity as possible.
  • the amino acid sequence of VanB ligase protein is preferably a sequence that includes the amino acid sequence of SEQ ID NO: 3, or is encoded by a gene containing a nucleotide sequence of SEQ ID NO: 4.
  • Enterococcus facalis-derived protein sequence GenBank accession number U00456.1, Uniprot accession numbers Q06893, C7WYV3, E6ZHJ4, Q7BHZ7, and the like.
  • Enter ococcus faeciu-derived protein sequences include Uniprot accession numbers C4MZP6, Q58F99, J7QUG3, Q84CN3, Q5MPQ4, P97205, Q9R3, Q5R3, Q5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5
  • the amino acid sequence of the VanB ligase protein is not 100% identical to the amino acid sequence of SEQ ID NO: 1, if the amino acid sequence identity is greater than 90% and acts as an antigen, an antibody that specifically recognizes VanB ligase may be prepared. It is intended to be included.
  • the term "specifically binds" or “specifically recognized” is the same as commonly known to those skilled in the art, and the antigen and the antibody specifically interact with each other to perform immunological reactions. Means that.
  • the term "antigen-binding fragment" of an antibody is a fragment thereof for the entire structure of an immunoglobulin and refers to a portion of a polypeptide comprising a portion to which an antigen can bind.
  • scFv scFv
  • Fab Fab 'or F (ab') 2.
  • the Fab in the antigen-binding fragment is a variable region of the light and heavy chains, the constant region of the light chain and the first constant region of the heavy chain (C Has one antigen binding site in a structure having H1 )
  • the antigen binding fragment can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with pine yields Fab and cleavage with pepsin). F (ab ') 2 fragments can be obtained), it can be produced through genetic recombination technology.
  • the antibody or fragment thereof may be monoclonal and polyclonal antibodies.
  • the antibody or fragment thereof may be an animal-derived antibody, for example, a rodent antibody such as a mouse, and may be a chimeric antibody or a humanized antibody.
  • the antibody that specifically recognizes the VanA ligase is a heavy chain of the antibody comprises three CDRs obtained from the heavy chain of the antibody produced by hybridoma having accession number KCLRF-BP-00277
  • the light chain of the antibody comprises three CDRs obtained from the light chain of the antibody produced by hybridoma having accession number KCLRF-BP-00277. It may be to include.
  • the antibody that specifically recognizes the VanA ligase specifically recognizes the VanA ligase protein, and may be one produced by a hybridoma having accession number KCLRF-BP-00277.
  • the heavy chain of the antibody comprises three CDRs obtained from the heavy chain of the antibody produced by hybridoma having accession number KCLRF-BP-00278 and the light chain of the antibody May comprise three CDRs obtained from the light chain of an antibody produced by hybridoma having accession number KCLRF-BP-00278.
  • the antibody that specifically recognizes the VanB ligase specifically recognizes the VanB ligase protein and may be one produced by a hybridoma having accession number KCLRF ⁇ BP-00278.
  • CDR complementarity determining region
  • the CDRs may provide a major contact residue for the antibody to bind antigen or epitope.
  • the antibody according to the present invention can be obtained by in vitro culture or by administering a cell producing the antibody to an animal.
  • the antibody can be obtained from ascites of animals in which the cells producing the antibody have been administered into the peritoneum.
  • the antibody can be purified from the culture supernatant or ascites by ion exchange chromatography or affinity column chromatography.
  • Monoclonal antibodies (mAbs) may be of animal (eg, mouse, rat, hamster or chicken) origin, or may be genetically produced.
  • Rodent monoclonal antibodies can be prepared by standard methods well known in the art and can be obtained by multiple immunization of vanAmycin-resistant enterococci VanA or VanB ligase into the intraperitoneal, intravenous, or footpad with appropriate adjuvant. Then spleen or lymph node cells are extracted and fused with an appropriate immortal cell line, Selecting hybridomas that produce antibodies that bind VanA or VanB ligase.
  • the present invention provides a cell producing the antibody or fragment thereof.
  • a method of producing an antibody or fragment thereof may comprise (a) immunizing the animal with D—alanine-D-alanine ligase, preferably VanA ligase, VanB mogase, or fragment thereof, (b) immunized animal Extracting splenocytes from the cells; (c) fusing the spleen cells of the animal with a myeloma cell line; and (d) screening the hybridoma cells and resistant bacteria to glycopeptide antibiotics. Selecting hybridoma cells that produce an antibody that specifically recognizes VanA ligase or VanB ligase.
  • the present invention also includes hybridoma cells obtained by the above method and antibodies produced by the hybridoma cells.
  • the present invention was established as a hybridoma producing monoclonal antibodies, eight anti-VanA monoclonal antibodies and three anti-VanB monoclonal antibodies were obtained, and clone number 3G11 cells were designated as VanA 3G11-63-22.
  • the clone number 7A12 cells were named VanB 7A12-12-4 and deposited with the Korean Cell Line Research Foundat ion (KCLRF) on February 20, 2012 and deposited with KCLRF-BP-00278. Received.
  • Another embodiment of the invention comprises an antibody or antigen-binding fragment thereof that specifically recognizes the VanA ligase or VanB ligase protein
  • a quantitative kit for quantifying VanA ligase or VanB ligase The presence or absence of a bacterium resistant to glycopeptide antibiotics expressing the VanA ligase or VanB ligase by quantifying the VanA ligase or VanB ligase present in the sample using the antibody or fragment thereof Ligase type can be detected by distinguishing phenotypes of resistant bacteria.
  • a further embodiment of the present invention relates to a detection kit for bacteria resistant to glycopeptide antibiotics comprising an antibody or antigen-binding fragment thereof that specifically recognizes the VanA ligase or VanB ligase protein.
  • the detection kit not only detects the presence of bacteria resistant to glycopeptide antibiotics in the sample, but can also detect the phenotype of resistant bacteria by VanA ligase or VanB ligase type.
  • the glycopeptide antibiotic may be at least one selected from the group consisting of Vancomycin, Teicoplanin, Te 1 avanc i n, Bleomycin, amo lanin, and Decaplanin, preferably Vancomycin, Teicoplanin or Telavancin.
  • the bacteria may be Enter ococcus strains (E. coli) or Staphylococcus strains, and the vanA type antibiotic resistant enterococci are E. faecalis, E. faecium, E. gal 1 inarum, E. casseliflavus, E. durans, E. mundtii, E. raffinosus, and E. avium, and the antibiotic resistant bacteria of type VanB may be E. faecal is and E. faec m.
  • the Staphylococcus genus strain may be Staphylococcus aureus (Staphylococcus aureus), preferably vancomycin resistant Staphylococcus aureus 0 ⁇ .
  • the detection kit may preferably further include a labeling substance for detecting an immune response between the VanA ligase or VanB ligase protein and the antibody.
  • the labeling substance may be a colorase, a colorant, a radioactive substance, or a fluorescent substance.
  • the detection kit according to the present invention may be an enzyme immunoassay kit (ELISA), a blotting kit, an immunoprecipitation kit, an immunofluorescence kit, or an immunochromatography kit of the antibody, preferably an enzyme immunoassay kit (ELISA) Or immunochromatography kits.
  • ELISA enzyme immunoassay kit
  • blotting kit an immunoprecipitation kit
  • immunofluorescence kit an immunofluorescence kit
  • immunochromatography kit of the antibody preferably an enzyme immunoassay kit (ELISA) Or immunochromatography kits.
  • the ELISA is intended to include both a general method using one antibody and a sandwich method using both capture and detection antibodies.
  • one antibody may be used in the immune strip, it is common to use two antibodies, usually a capture antibody and a detection antibody.
  • an antibody against VanA includes 1H9 and 3G11 (a monoclonal antibody produced by a hybridoma having accession number KCLRF-BP-00277) as a capture antibody.
  • Antibody pairs including monoclonal antibodies).
  • ELSIA enzyme-associated immunoassay
  • the enzyme-labeled antibody solution may include 50 to 150 ⁇ per plate well at an appropriate concentration with goat anti-mouse Ig-HRP, and the coloring solution may be selected from tetramethylbenzidine (TMB) and the reaction blocking solution. May be selected from the group consisting of IN HC1 or IN H 2 S0 4 .
  • the immunochromatography kit includes a general immunostrip kit, and the strip kit is a dipstick type used by immersing the sample solution vertically, horizontally moving the strip and placing the sample solution horizontally, or the dipstick. It may be a cassette type for fixing the mold or horizontal movable type to the cassette, but is not particularly limited.
  • An immune strip kit includes a support; Conjugate pads containing a conjugate of a detection antibody and a labeling substance; Signal detection pads to which capture antibodies are immobilized; And at least one analysis strip having a width of a short side and a length of a long side including an absorbent pad absorbing the sample developing solution.
  • the detection antibody and capture antibody may be an antibody or fragment thereof that specifically recognizes VanA ligase or VanB ligase according to the present invention.
  • the immunostrip kit may be used in combination with one or more strips to which VanA ligase or VanB ligase-recognizing antibodies are immobilized, respectively. It may be a double VanA / VanB detection kit combining a strip fixed with an antibody that specifically recognizes.
  • the chromophore is HRPQorseradish peroxidase or alkaline dephosphatase
  • the chromophore is colloid gold
  • the fluorescent molecules are poly L-lysine-f luorescein isothiocyanate (FITC) and RITC (rhodam) i ne-B- i sot hi ocyanat e).
  • the signal detection pad may be any one selected from the group consisting of nitrosarose, salloose, polyethylene, polyethersulfone and nylon.
  • the absorbent pad may include a porous support, and an absorbent dispersed in a cavity of the porous support or adsorbed or coated on the fiber yarn of the porous support.
  • the immune strip kit reacts with a detection antibody in which an antigen in a sample is bound to colloidal gold particles, and then fines the nitrocellulose membrane by capillary action. While moving through the micropore, it is possible to visually distinguish positive and negative by combining with a capture antibody fixed to the inner surface of the micropore to form a color band.
  • glycopeptide antibiotics comprising the step of detecting by immunoassay at least one protein selected from the group consisting of VanA ligase and VanB ligase contained in the sample It relates to a method for detecting bacteria resistant to.
  • the detection method is characterized in that the bacteria resistant to glycopeptide antibiotics in the sample when VanA ligase and VanB ligase are present in the sample. Determining what is present may be further included.
  • the detection method can detect the presence of bacteria resistant to glycopeptide antibiotics in the sample, and can also distinguish the phenotype of resistant bacteria by VanA ligase or VanB ligase type.
  • the sample Before performing the detection step, the sample may be inoculated and cultured in a medium to which glycopeptide antibiotics are added, cells are collected, and VanA ligase and VanB ligase are prepared by eluting.
  • the detection step may be performed by extracting a sample from the sample and eluting VanA ligase and VanB ligase, but the sensitivity of the detection method is increased by increasing the expression level of VanA ligase and VanB ligase and increasing the number of bacteria expressing the protein. It is preferable to carry out the culturing step so as to increase.
  • the culture medium may be a medium commonly used as a culture medium of bacteria resistant to glycopeptide antibiotics, for example, Enterococcus strain (E. coli) or Staphylococcus strain.
  • the concentration of glycopeptide antibiotics added during the culture may be any concentration of glycopeptide antibiotics added during the culture.
  • Any concentration that can increase the expression level of VanA ligase and VanB ligase may be appropriately selected and used, for example, 2 to 8 ug / mL, and preferably 4 to 6 ug / mL.
  • the cells can be analyzed by lysing the cells by chemical lysis or physical disruption, eluting the cells extracellularly, for example, by treating urea, VanA ligase and VanB ligase.
  • the agent can be water-soluble.
  • the immunoassay may be selected from the group consisting of radioimmunoassay, immunoassay (ELISA), sandwich immunoassay, and immunochromatography assay, preferably ELISA assay and immunochromatography assay.
  • ELISA immunoassay
  • sandwich immunoassay sandwich immunoassay
  • immunochromatography assay preferably ELISA assay and immunochromatography assay.
  • the sample sample is contacted with a monoclonal antibody of the present invention coated with a solid support, such as a microtiter plate, a membrane, a test strip, or an antibody specific for the VanA / B ligase.
  • a well of a microtiter plate is coated with a monoclonal antibody of the present invention or an antibody specific for the VanA / B ligase, and the nonoccupied binding site is blocked with, for example, BSA, and then coated with a plate.
  • Wells may be incubated with the sample sample, followed by determination of the presence of the antigen-antibody complex.
  • the antibody may be bound to a label as described above.
  • the antibody pair when using sandwich immunoassay and lateral flow immunographic assay, the antibody pair may be prepared using the monoclonal antibody of the present invention and the antibiotic resistant bacteria may be detected using the same.
  • the immunochromatography kit developed in the present invention detects VanA / B ligase proteins, it is preferable to secure proteins beyond the detection limit.
  • Detectable protein can be secured by increasing the number of bacteria and inducing VanA / B through the enrichment process. The enrichment process takes about a day and this becomes a limiting factor that impedes the speed of detection.
  • the VRE antibiotic resistance type can be identified without additional culture after enrichment, the detection time is shorter than that of the culture method.
  • the present invention provides an antibody that specifically recognizes alanine-D-alanine ligase, and a kit and a method for detecting bacteria resistant to glycopeptide-based antibiotics using the antibody.
  • Fig. La to lc are diagrams showing the purification results of the recombinant VanA and VanB recombinant proteins
  • Fig. La is the cloning result of Cloning of vanA and vanB ligase gene
  • Fig. Lb is the refolding and It is a photograph showing the purification result
  • Figure lc is a photograph showing the purification result of the water-soluble recombinant VanA / VanB ligase protein.
  • FIGS. 2A to 2C are diagrams showing the results of selecting an optimal anti-VanA antibody pair
  • FIG. 2A is a detection result of evaluating antibody performance by a conventional ELISA method
  • FIG. 2B shows capture antibody performance by a sandwich ELISA method
  • 2C shows VanA. Is a graph showing 1H9 / 3G11 or 1B7 / 3G11 (capture / detection) as the optimal antibody pair for.
  • 3A to 3C are diagrams showing the results of selection of an optimal anti-VanB antibody pair.
  • FIG. 3A is a detection result of evaluating antibody performance by a general ELISA method.
  • FIG. 3B is a capture ELISA method.
  • 3C is a graph showing 7E8 / 14B2 or 7A12 / 14B2 (capture / detection) as an optimal antibody pair against VanB.
  • 4 shows a signal-to-noise ratio (signal / noise ratio) and a three-dimensional schematic diagram for noise of a VanA antibody pair, wherein the X-axis represents the dilution factor of the detection antibody, Y-. The concentration of the contracted capture antibody is shown.
  • FIG. 5 shows the signal-to-noise ratio (signal / noise ratio) of noise of the VanB antibody pair, and antibody pair 7E8 / 7A12 (capture / detection) showed a high S / N ratio.
  • Optimal concentration of capture antibody The antibody pair was determined to be 2.5 ug / mL based on the 7E8 / 7A12 (capture / detection) S / N ratio and the optimal dilution range of the detected antibody was 800-12,800.
  • FIG. 8 is a diagram illustrating an immune strip of a dip-stick type according to an embodiment of the present invention.
  • FIG. 9 is a schematic diagram illustrating a process for detecting vancomycin-tolerant enterococci by immunochromatography (ICA) using an antibody against VanA / VanB ligase according to one embodiment of the present invention.
  • ICA immunochromatography
  • FIG. 10 is a photograph showing a schematic diagram and an analysis result of a VanA VanA immunochromatography kit according to an embodiment of the present invention.
  • FIG. 11 is a diagram showing the result of analyzing the correlation between VanA immunochromatography and ELISA.
  • FIG. 12 is a graph showing the results of analyzing the correlation between VanB immunochromatography and ELISA.
  • FIG. 13 is a diagram showing the results of analyzing vancomycin addition requirements to the culture medium to increase the expression level of VanA or VanB.
  • 14 is a view showing the performance of the dual VanA / B immunochromatography kit.
  • Example 1 Expression and Purification of Recombinant VanA and VanB Proteins 1100 base pairs of vanA and vanB genes were cloned from vancomycin-resistant enterococci, respectively, and purified to about 40 kDa protein through induction, water solubilization and elution.
  • vanA ligase gene was cloned in vancomycin resistant enterococci VRE 1506 in Table 7 below, and the vanB ligse gene in V583 VE, respectively (FIG. la).
  • vanA and vanB ligase gene sequences were obtained from the National Center for Biotechnology Information (NCBI) gene bank (va, GenBank: X56895.1, vanB, GenBank: U00456.1).
  • the vanA ligase gene was amplified using VRE 1141 and the vanB ligase gene from V583 VRE using the primer sequences shown below.
  • the gene was obtained using the 'move BL21 Star E. coli transformed the pETlOl / D-TOPO vectorClnvitrogen, Carlsbad , CA, USA).
  • IPTG 0.5 mM IPTG was added to the E. coli culture transformed with the gene prepared in 1-2 to induce histidine-attached recombinant protein. After further incubation for 4 hours, the culture medium was centrifuged to form a precipitate, and suspended in 2.0 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100 (pH 8.0) solution to characterize the recombinant protein. Analysis shows that most recombination
  • VanA and VanB proteins are induced in insoluble form.
  • the suspension is centrifuged again to form a precipitate, 20 mM Tris-HCl, 0.5 M NaCl, After resuspending in 5 mM imidazole, 8 M urea, 1 mM 2-mercaptoethanol (pH 8.0), the phases were stirred for 1 hour at silver. The supernatant was collected by centrifugation and filtered with a 0.22 urn filter.
  • the recombinant protein Since the recombinant protein has histidine tag, it was solubilized in 8.0 M urea solution and injected into Ni-column. The prepared filtered sample was injected into a 1.0 raL HisTrap (GE healthcare, Little Chalfont, UK) column. The protein bound to the column induced water solubilization by lowering urea concentration from 8.0 M to 3 ⁇ 4 (0.5 mL / min, 80 mL). Soluble recombinant protein was eluted under 20 mM Tris-HCl, 0.5 M NaCl, 150 mM imidazole, 1 mM 2-mercaptoethanol (pH 8.0).
  • VanA and VanB recombinant proteins purified in Example 1 were immunized four times at intervals of 100 ug ⁇ 3 weeks in the abdominal cavity of Balb / c mice. Three days after 4 immunizations, mouse spleens were extracted, 1 ⁇ 10 8 cells were taken, and X63-Ag8.653 (ATCC, Virginia, USA) mouse myeloma cancer cells were fused and induced. Fused hybridomi "was selected by incubation for 10 days in HAT (hypoxanthine, aminopterin and thymidine containing media) medium, and coated with 100ng of VanA and VanB recombinant protein per well for monoclonal antibody selection.
  • HAT hyperxanthine, aminopterin and thymidine containing media
  • KCLRF Korean Cel l Line Research Foundat, whose clone number 3G11 cells were named VanA 3G11-63-22 and were addressed at the Cancer Research Institute, Seolleh University School of Medicine, 28, Yeongun-dong, Seoul, Korea. ion was deposited on February 20, 2012 and received accession number KCLRF-BP-00277, and clone number 7A12 cells were named VanB 7A12-12-4, and the Korean Cell Line Research Foundation (KCLRF). ion was deposited on February 20, 2012 and received accession number KCLRF-BP- 00278. 2-3 : Screening of anti—VanA monoclonal antibodies
  • Antibody Preparation Antibody characteristics were analyzed for the selection of monoclonal antibodies suitable for the development of EL ISA and immunochromatography kits. Detection antibodies were coated with VanA or VanB recombinant protein as antigen and selected for evaluation of reactivity to the antigen. Capture antibodies were coated with the antibody to be evaluated and evaluated using HRP conjugated antibody pool (Pooled-mono) as the detection antibody.
  • HRP (Amresco, OH, USA) was dialyzed on 50 mM sodium phosphate (pH 7.2) complete solution for the production of detection antibodies.
  • Prepare 0.1 M SMCC (Succinimidyl 4- (TV-ma 1 eimi doraet hy 1) eye 1 ohexane- 1-car boxy 1 at e) and add 1/10 (v / v) to HRP solution for 2 hours at room temperature Stirred.
  • Monoclonal antibody was prepared by dialysis into a 50 mM sodium phosphate (pH 7.2) complete layer solution, and 14 mM Traut's reagent (2-Iminothiolane-HCl) was added 46 ul to 1.0 mL of the antibody solution, followed by stirring for 2 hours.
  • SMCC and Traut's reagent that did not participate in the reaction were removed by a sephadex G25 desalting column, and then HRP and antibody solutions were mixed and stirred for another 2 hours.
  • HRP conjugated antibody was separated by stirring for 30 minutes by adding the same amount of saturated ammonium sulfate solution (pH 7.4), centrifugation, and then dialyzed in phosphate buffered saline (pH 7.2).
  • Microplate (Nunc, Denmark) was coated with anti VanA or VanB antibody (1.0-2.5 ug / mU) and blocked using an aqueous solution of skim milk powder. Antigen (recombinant protein VanA / B or VRE lysate) was applied to each well. The reaction was repeated for 1 hour and washed twice at 37 ° C. The detection antibody (detection antibody) with HRP was added and the reaction was repeated for 1 hour and washed twice at 37 ° C. Then, the reaction was performed by adding TMB substrate solution. The reaction was terminated by addition of 1.0 N 3 ⁇ 4SO 4 .
  • VanA or VanB 2-fold series of recombinant protein
  • Standard antigen dilution buffer was used as VRE lysis complete solution, and standard antigen concentration was calculated by BCA assay.
  • the standard antigen dilution buffer was used as a VanA type VRE lysis buffer containing 1.5 M urea and 3% Tween 20 in Popculture reagent (Novagen, WI, USA) .
  • the standard antigen dilution buffer was 0.8 in VanB ELISA.
  • Murea, 3% Tween 20, 0.2% Polyvinyl alcohol, 0.5% skim milk, 10 mM phosphate buffer (pH 7.4) was used. The results are shown graphically in FIG. 2B.
  • FIGS. 2A to 2C are diagrams showing the results of selecting an optimal anti-VanA antibody pair
  • FIG. 2A is a detection result of evaluating antibody performance by a conventional ELISA method
  • FIG. 2B is a capture antibody performance by a sandwich ELISA method
  • 2C is a graph showing 1H9 / 3G11 or 1B7 / 3G11 (capture / detection) as an optimal antibody pair against VanA.
  • hybridoma cultures were obtained by culturing for 2 weeks in Cell-bag (Xcellerex, Marlborough, MA, USA) and filtered with a 0.22 urn filter. Prepared samples are injected into Hitrap Protein G 5 mL column (GE healthcare, Little Chalfont, UK), and 20 mM sodium phosphate (pH 7.2) solution is poured into 10 column volume (50 mL) to remove impurities and 0.1 M glycine-HCl (pH 3.0) eluted with 5 column volumes and purified. The purified antibody was dialyzed with phosphate buffered saline (pH 7.4) and used for various purposes.
  • Example 3 VanA and VanB ELISA Implementation
  • the signal-to-noise ratio was checked to confirm the antibody concentration appropriate for the ELISA implementation.
  • the capture antibody 1H9 was serially diluted from 10 ug / mL and coated at various concentrations, and the detection antibody 3G11 was also sequenced and implemented at various concentrations to confirm reactivity.
  • Signal was assessed by 50 ng / mL VanA recombinant protein and noise by reactivity with diluent. VanA semisexual results are expressed in signal / noise ratio and three-dimensional schematic (Table 4 and FIG. 4).
  • the X-axis represents the concentration of capture antibody
  • the Y-axis represents the dilution factor of the detection antibody
  • the signal was measured with 50 ng / mL VanA
  • the noise was determined with the dilution buffer.
  • the optimal capture antibody concentration was selected to be 2.5 ug / mL with high signal / noise ratio
  • the optimal dilution factor range of the detection antibody was selected from 800 to 6,400. Detected antibody dilution was reevaluated using VRE lysate as antigen.
  • FIG. 5 shows the signal-to-noise ratio (signal / noise ratio) against noise of the VanB antibody pair, wherein antibody pair 7E8 / 7A12 (capture / detection) showed a high S / N ratio.
  • the optimal concentration of capture antibody was determined to be 2.5 ug / mL using antibody pair 7E8 / 7A12 (capture / detection), and the optimal dilution range of the detected antibody was 800-12,800.
  • the capture antibody 7E8 was coated on a microtiter plate at a concentration of 2.5 ug / mL, and a detection antibody 7A12 diluted 500-fold was applied. Recombinant VanB protein was used as standard and QC samples. The detection range is 7.8 to 250 ng / mL. The standard curve is represented by a quadratic model fit.
  • VanA and VanB Immunochromatography Kits are constructed with 1A9 / 3G11 (capture / detection) and VanB Immunochromatography Kit with 7A12 / 7E8 (capture / detection) monoclonal antibody combinations. It was.
  • the capture antibody was coated on the membrane and the detection antibody was subjected to gold conjugation to dry and assembled into the device.
  • gold conjugated antibody 0.5 M sodium bicarbonate solution was added to colloidal gold (Dinona, Iksan, Korea) and titrated to pH 7.4.
  • Antibodies were prepared at a concentration of 0.1 mg / mL in 2 mM borax (pH 7.4).
  • Nitrocell was attached to a plastic pad with a rose film (HiflowPlus, millipore, MC, USA).
  • the capture antibody for VanA ICA used monoclonal antibody 1H9 1.0 mg / mL.
  • the antibody solution was sprayed in a straight line on the surface of the nitrous membrane using a BioDot machine (BioDot, California, USA) and dried.
  • the control line was a 0.3 mg / mL solution of goat ant i-mouse IgG (Dinona, Iksan, Korea) at 6 cm / sec to each membrane to form a control line and a detection site, respectively.
  • the capture antibody for VanB ICA used a 2.0 mg / mL solution of 7A12 monoclonal antibody.
  • An absorbent pad was attached to the top of the finished nitrocell film to design a flow rate, and the strip was cut at intervals of 5 mm.
  • the completed strips were used in dip-stick form by inserting one by one into a microplate containing a gold conjugated antibody solution (FIG. 8).
  • VanA type VREs (1141, 1142, 1506) were isolated from Chung-Buk National University Hospital (CBNUH) and VanB type VREs (V583, NJ3) were sold by ATCC (Virginia, USA). VanA type VRE isolated from the clinic was genotyped by ⁇ 4 ligase gene amplification.
  • VRE strains were cultured in solid medium for one night in BHI (BD bioscience, MD, USA) medium to which 0-8 ug / mL vancomycin was added. The cultured 1.0 mL culture was precipitated by centrifugation and used as a sample for ICA or ELISA analysis.
  • BHI BD bioscience, MD, USA
  • VRE precipitate was prepared by suspending with 30 ⁇ l of 8.0 M urea, 20 mM sodium phosphate buffer (pH 7.4) for 10 minutes and adding 3% Tween 20 in Popculture reagent 120.
  • VRE precipitate was suspended in 30 mL of 4.0 M urea, 20 mM phosphate buffer (pH 7.4) for 10 minutes, 3% Tween 20, 0.2% Polyvinyl alcohol, 0.5% skim mi lk, 10 mM phosphate buffer (pH 7.4) was added in an amount of 120 ul. Each prepared VRE lysate was centrifuged to remove precipitates and used as ICA or ELISA assay samples.
  • Microplate (Nunc, Denmark) was coated with 2.5 ug / mL anti VanA 1H9 or 7E82.5 ug / mL anti VanB antibody, and mocking was performed using an aqueous solution of skim milk powder.
  • Antigen recombinant protein VanA / B or VRE lysate
  • VanA detection antibody 3G11-HRP was 800 times
  • VanB detection antibody 7A12-HRP was prepared 500 times the diluent was added and reacted for 1 hour at 37 ° C and washed twice.
  • TMB since Substrate solution was added to induce a color reaction, and 1.0 NH 2 SO 4 was added to terminate reaction.
  • VanA / B sample concentrations were calculated using each quantitative curve format (Quadratic Fit).
  • Antibiotic resistant strains were cultured in solid medium for one night in BHI (BD bioscience, MD, USA) medium containing 0 or 4 ug / mL vancomycin and precipitated by centrifugation of 1.0 mL culture. Du lysate was made and analyzed.
  • antibiotic-resistant strain ELISA analysis antibiotic-resistant strain precipitates were suspended in 8.0 M urea, 20 mM sodium phosphate buffer (pH 7.4) 30 ul, left for 10 minutes and prepared by adding 120 ul of 3% Tween 20 in Popculture reagent. It was.
  • antibiotic-resistant strain precipitates were suspended in 4.0 M urea, 20 mM phosphate buffer (pH 7.4) 30 ul for 10 minutes, 3% Tween 20, 0.2% Polyvinyl alcohol, 0.5% skim milk, 10 mM phosphate buffer (pH 7.4) was added in an amount of 120 ul. Each prepared antibiotic resistant strain lysate was centrifuged to remove the precipitate and used as an ELISA assay sample.
  • Table 8 shows the results of detecting VanA ligase expressed in antibiotic-resistant strains using the above-described VanA ligase detection sandwich ELISA
  • Table 9 shows the results of quantifying VanB ligase by VanB ligase detection sandwich ELISA.
  • Immunoassay (ICR) 9 is a schematic diagram showing the use of the dual VanA / B immunochromatography kit.
  • Figure 10 is a photograph showing the schematic and analysis results of the VanA VanA immunochromatography kit according to an embodiment of the present invention. VanA VRE was observed to amplify the concentration of VanA ligase by about 10 times in 4 ug / mL of vancomycin (Fig. 11).
  • VanA ligase The concentration of VanA ligase was different according to the species, but even at the lowest concentration of 1142, VanA concentration was higher than 200 ng / mL under vancomycin-containing conditions, and was well detected by immunochromatography kit.
  • VanB VRE VanB ligase amplification was observed in vancomycin-containing medium, but the concentration of VanB ligase was found to be very low than 1/10 of VanA ligase (FIG. 12). However, the immunochromatography kit analysis showed positive results, although not dark contrast.
  • VanA and VanB ligase are both induced by vancomycin, but the use of excess vancomycin may inhibit V E growth.
  • VanB VRE has low resistance to vancomycin, so it is necessary to check the concentration of vancomycin in detail. ⁇
  • VanA / B ligase was quantified at various vancomycin concentrations in order to amplify VanA / B ligase well and confirm the vancomycin concentration that does not inhibit VRE growth. Specifically, the VanA ligase was quantitatively set to 0, 2, 6, and 8 ug / mL, and the VanB ligase was set to 0, 2, 4, 6 and 8 ug / mL. In the case of VanA 1142 species, the vanA ligase concentration was continuously increased to 264 ng / mL when the vancomycin concentration was increased to 8 ug / mL (FIG. 13).
  • VanB VRE In VanB VRE, no significant vanB ligase amplification was observed once the vancomycin concentration exceeded 2 ug / mL. This phenomenon may be attributed to the slowing of growth of VanB VRE, which has a relatively low resistance to vancomycin, as the number of bacteria in the enrichment medium decreased. Since the highest concentration of VanB ligase (10.97 ng / mL) was observed at vancomycin 4 — 6 ug / mL, the concentration of vancomycin in the enrichment medium was in the range of 4-6 ug / mL.
  • Example 6 Dual VanA / B Immunochromatography Kits 6-1 : Detection and Identification of Dual VanA / B Immunochromatography Kits
  • VRE was incubated for one day in vancomycin-free or containing (4 ug / mL) liquid medium and analyzed by dual VanA / B immunochromatography kits.
  • the assay was read using three VanA type (VRE 1141, VRE 1142, VRE1506) and two VanB type VRE VRE NJ3, VRE V583.
  • VanA type VRE was correctly detected and identified even in the absence of vancomycin, but VanB type was not detected.
  • ligase was induced by adding 4 ug / mL of vancomycin, the correct antibiotic resistance type was identified in all 5 strains (FIG. 14).
  • VanA / B immunochromatography kit was used by connecting the immunostrips prepared in Example 4-1 in parallel with VanA and VanB, respectively.
  • VanA 1142 and VanB V583 strains were inoculated in the vancomycin-containing solid medium and cultured for one day in order to confirm the minimum number of VRE bacteria or protein amount detected by the immunochromatography kit. After all the colonies were removed and transferred to PBS solution, 0D 600 was confirmed. Strain samples were aliquoted as described in FIG. 15 and analyzed by immunochromatography kit, respectively.
  • VanA 1142 strain was detectable at analytical volume of 100 ul or more, and VanB V583 was detected as detectable at analyte of 50 ul or more.
  • VanA or VanB protein was quantified using the same amount of sample determined as the limit of detection analyte, and the colony-forming unit (CFU) was isolated by sequencing the strains and culturing them in solid medium containing 2 ug / mL of vancomycin to confirm the number of bacteria. Confirmed.
  • the detection limits of VanA were 115 ng / mL and 6.78 X 10 8 CFU, and the detection limits of VanB were 7.2 ng / mL and 2.94 X 10 8 , respectively.

Abstract

The present invention relates to an antibody to VanA or VanB ligase and to a quantification method for VanA or VanB ligase using the antibody, and also relates to a detection kit and a detection method for bacteria resistant to glycopeptide antibiotics.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
D-알라닌 -D-알라닌 리가제에 대한 항체 및 이를 이용한 항생제 내성 세균의 탐지  Antibodies to D-alanine-D-alanine ligase and detection of antibiotic resistant bacteria using the same
【기술분야】 Technical Field
본 발명은 D-알라닌 -D-알라닌 리가제에 대한 항체 및 이를 이용한 D-알라닌 -D-알라닌 리가제의 정량방법， 및 상기 항체를 이용한 항생제 내성 세균의 검출에 관한 것이다.  The present invention relates to an antibody against D-alanine-D-alanine ligase, a method for quantifying D-alanine-D-alanine ligase using the same, and detection of antibiotic resistant bacteria using the antibody.
【발명의 배경이 되는 기술】 [Technique to become background of invention]
글리코펩티드계 항생제는 펩티도글리칸 전구체에 결합하여 세균 세포벽의 가교결합을 간섭하는 중요한 계통의 항생제이다. 이는 종종 생명을 위협하는 감염을 치료함에 있어 최후에 선택되는 항생제이다. 글리코템티드계 항생제는 Vancomycin, Teicoplanin, Telavancin, Bleomycin Rarao lanin, 및 Decaplanin등이 있다. 또한 Vancomycin과 Televancin등이 VanA ligase induction 을 유도하는 것을 알려져 있으며 (Antimicrobial Agents and chemotherapy, Vol . 54, No. 7, 2010， p.2814-2818) , 반코마이신 내성 장구균은 또한 Teicoplanin 내성을 가지는 것도 알려져 있다.  Glycopeptide antibiotics are an important class of antibiotics that bind to peptidoglycan precursors and interfere with crosslinking of bacterial cell walls. It is often the last antibiotic of choice in treating life-threatening infections. Glycoated antibiotics include Vancomycin, Teicoplanin, Telavancin, Bleomycin Rarao lanin, and Decaplanin. Vancomycin and Televancin are also known to induce VanA ligase induction (Antimicrobial Agents and chemotherapy, Vol. 54, No. 7, 2010, p.2814-2818), and vancomycin-resistant enterococci are also known to be Teicoplanin resistant. .
장구균은 테이코플라닌 또는 반코마이신과 같은 글리코핍티드계 항생제에 대한 저항성 및 저항성 유도 능력을 토대로 여러 종류의 표현형으로 분류 된다. VanA 유형의 경우 반코마이신과 테이코플라닌에 대한 유도 저항성을 가지나, VanB 유형은 반코마이신에 대한 유도 저항성만을 가지고 있다. VanA 유형의 항생제 내성은 장구균 중에서도 E. faecalis, E. faecium, E. gal 1 inarm, E. cassel i flavus, E. durans, E. mundtii, E. raffinosus, E. 등과 같은 다양한 균종에서 보고되었고， Enterococci are classified into several phenotypes based on their resistance to and resistance to glycopeptide antibiotics such as teicoplanin or vancomycin. VanA type has induction resistance to vancomycin and teicoplanin, but VanB type has only induction resistance to vancomycin. Antibiotic resistance of the VanA type has been reported in a variety of enterococci, including E. faecalis, E. faecium, E. gal 1 inarm, E. cassel i flavus, E. durans, E. mundtii, E. raffinosus, and E.
VanB 유형의 항생제 내성은 E. faecalis 과 E. faecium 에서 보고되었다. 반면 VanC 유형의 항생제 내성은 반코마이신에 대한 낮은 내재적 저항력으로 특징지을 수 있으며 , E. gallinarum, E. casseliflavus, E. a esce2s에서 보고되었다. ' Antibiotic resistance of the VanB type has been reported in E. faecalis and E. faecium. Antibiotic resistance of the VanC type, on the other hand, is low intrinsic to vancomycin. Characterized by resistance and reported in E. gallinarum, E. casseliflavus, and E. a esce2s. '
VanA, VanB 및 VanC 의 유형과 관련된 유전자들이 발견되었으며 vanA, vanB, vanC-1, vanC~2 및 vanCS 로 명명하게 되었다. vanA 및 vanB 유전자 클러스터는 트랜스포존 (transposon) 형태로 다른 균주로 전파될 수 있다. VanA 와 VanB 유형꾀 항생제 내성은 획득된 것이기 때문에 장구균이외에도 Staphylococcus aureus 과 유기체로 전달될 수 있으며， 예를 들면 반코마이신 내성 포도상구균 (Vancotnyc in-resistant Staphylococcus aureus, VRSA) 등일 수 있다. 반면 VanC 유형의 저항성은 내재적인 특성이기 때문에 임상적으로 중요한 의미는 없다. Genes related to the types of VanA, VanB and VanC were found and named vanA, vanB, vanC-1, vanC - 2 and vanCS. The vanA and vanB gene clusters can be propagated to other strains in the form of transposons. Since VanA and VanB type antibiotic resistance is obtained, it can be transmitted to Staphylococcus aureus and organisms in addition to enterococci, for example Vancotnyc in-resistant Staphylococcus aureus (VRSA). On the other hand, VanC type resistance is inherent in nature and therefore has no clinical significance.
상기 반코마이신 -내성 장구균의 분류를 하기 표 1 에 나타냈다 (Eurosurvellance Vol. 13. Issue 47. 20 November 2008) .  The classification of the vancomycin-resistant enterococci is shown in Table 1 (Eurosurvellance Vol. 13. Issue 47. 20 November 2008).
[표 1] 반코마이신 -내성 장구균의 분류  Table 1 Classification of Vancomycin-Resistant Enterococci
Figure imgf000004_0001
VanA 와 VanB 유형은 분리빈도가 상대적으로 높고， 항생제 저항성이 강하며， 전파 능력을 보유하고 있기 때문에 검출 및 유형분석이 필요하다. 글리코펩티드계 항생제에 대한 내성을 갖는 박테리아는 대표적으로 테이코플라닌 또는 반코마이신 등에 대한 내성을 갖는 장구균， 및 반코마이신에 내성을 갖는 포도상구균 등을 들 수 있다. 예를 들면， 반코마이신 내성 장구균은 전파 방지 및 조기 진단을 위하여 fecal swab 또는 rectal swab 이 검출 시료로 사용되고 있다. 최근에는 chromogenic agar 와 같은 새로운 진단법이 소개 되었으며， fecal 또는 rectal swab 시료를 사용하여 개선된 검출 성능이 보고되었다. ChromID VRE agar (bioMerieux, Marcy l'Etoile, France)는 반코마이신을 8 ug/mL 로 함유한 고체 배지로써， β-glucosidase 와 β -galactosidase 을 각각 검출하여 E. faecalis 와 E. faecium 동시에 구별할 수 있는 장점을 가지고 있다. 그러나 ChromID VRE agar 와 같은 배양을 기반으로 한 진단법은 VanA 또는 VanB 항생제 내성 유형에 대한 직접적인 표현형 정보를 제공하지 못한다는 단점이 있다.
Figure imgf000004_0001
VanA and VanB types need to be detected and typed because of their relatively high separation frequency, strong antibiotic resistance, and propagation capability. Bacteria having resistance to glycopeptide antibiotics include enterococci, which are resistant to teicoplanin or vancomycin, and staphylococci resistant to vancomycin. For example, For vancomycin-resistant enterococci, fecal swabs or rectal swabs are used as detection samples for the prevention of transmission and early diagnosis. Recently, new diagnostic methods such as chromogenic agar have been introduced, and improved detection performance using fecal or rectal swab samples has been reported. ChromID VRE agar (bioMerieux, Marcy l'Etoile, France) is a solid medium containing vancomycin at 8 ug / mL. It has advantages However, a diagnostic method based on cultures such as ChromID VRE agar has the disadvantage that it does not provide direct phenotypic information on the type of VanA or VanB antibiotic resistance.
현재까지 반코마이신 내성 장구균의 항생제 내성 유형을 동정할 수 있는 유일한 방법은 PCR (Polymerase Chain React ion)을 기반으로 한 분석법이다. 그러나 PCR 을 기반으로 한 분석법은 배양법에 비하여 신속하며 내성 유전자를 동정할 수 있는 장점이 있으나 관련 전문가의 부족 및 고비용의 한계가 있다.  To date, the only way to identify the antibiotic resistance type of vancomycin-resistant enterococci is by PCR (Polymerase Chain React ion). However, PCR-based assays are faster and more resistant to resistance genes than cultivation methods, but there is a shortage of related experts and high cost.
【발명의 내용】 [Content of invention]
【해결하고자 하는 과제】  Problem to be solved
본 발명은 종래 PCR 방법이 갖는 고비용의 단점과 배양법이 갖는 조기 진단이 어려우며 항생제 내성 균의 표현형에 대한 정보를 제공하지 못한다는 단점을 해소하여， 단시간내에 내성 유전자 유형 정보를 얻을 수 있는 글리코펩티드계 항생제에 대한 내성 세균의 검출에 필요한 항체， 검출방법 및 검출키트를 제공하고자 한다.  The present invention solves the disadvantages of the high cost of the conventional PCR method and the difficulty of early diagnosis of the culture method and does not provide information on the phenotype of antibiotic resistant bacteria, glycopeptide system that can obtain the resistance gene type information in a short time An antibody, a detection method, and a detection kit for detecting bacteria resistant to antibiotics are provided.
따라서， 본 발명은 글리코펩티드계 항생제에 대한 내성 세균의 D- 알라닌 -D-알라닌 리가제를 특이적으로 인식하는 항체 또는 이의 항원결합 단편을 제공하고， 상기 항체를 이용하여 글리코펩티드계 항생제에 대한 내성 세균을 직접 검출할 수 있는 신속하게 검출할 수 있는 검출키트 및 검출방법을 제공하고자 한다. 또한 본 발명은 글리코펩되드계 항생제에 대한 내성 세균의 D- 알라닌 -D-알라닌 리가제를 특이적으로 인식하는 항체 또는 이의 항원결합 단편을 이용하여 시료내에 존재하는 D-알라닌 -D-알라닌 리가제를 정량하는 방법에 관한 것이다. Accordingly, the present invention provides an antibody or antigen-binding fragment thereof that specifically recognizes D-alanine-D-alanine ligase of bacteria resistant to glycopeptide antibiotics, and uses the antibody for glycopeptide antibiotics. An object of the present invention is to provide a detection kit and a detection method capable of quickly detecting resistant bacteria. In addition, the present invention provides a D-alanine-D-alanine ligase present in a sample using an antibody or antigen-binding fragment thereof that specifically recognizes D-alanine-D-alanine ligase of bacteria resistant to glycopeptide-based antibiotics. It relates to a method for quantifying agent.
【과제 해결 수단】 [Task solution]
본 발명의 일구현예는 D-알라닌 -D-알라닌 리가제， 바람직하게는 VanA 리가제 (ligase) 또는 VanB 리가제 단백질을 항원으로 하여 생산되며， 상기 D-알라닌 -D-알라닌 리가제를 특이적으로 인식하는 항체 또는 이의 항원 결합 단편에 관한 것이다. 상기 항체는 단클론 항체 또는 다클론 항체일 수 있으며， 또한 키메라 항체 또는 인간화 항체일 수 있다.  One embodiment of the present invention is produced using D-alanine -D-alanine ligase, preferably VanA ligase or VanB ligase protein as an antigen, specific for the D-alanine -D-alanine ligase To an antibody or antigen-binding fragment thereof that is recognized automatically. The antibody may be a monoclonal antibody or a polyclonal antibody, and may also be a chimeric antibody or a humanized antibody.
본 발명의 또 다른 구현예는 상기 D-알라닌 -D-알라닌 리가제를 특이적으로 인식하는 항체 또는 이의 항원결합 단편을 포함하는 D-알라닌- D-알라닌 리가제， 바람직하게는 VanA 리가제 또는 VanB 리가제 단백질을 정량하는 방법에 관한 것이다.  Another embodiment of the present invention is a D-alanine-D-alanine ligase, preferably VanA ligase comprising an antibody or antigen-binding fragment thereof that specifically recognizes the D-alanine -D-alanine ligase A method for quantifying VanB ligase protein.
본 발명의 추가 구현예는 상기 D-알라닌 -D-알라닌 리가제를 특이적으로 인식하는 항체 또는 이의 항원결합 단편을 포함하는， D-알라닌- D-알라닌 리가제， 바람직하게는 VanA 리가제 또는 VanB 단백질을 발현하는， 글리코펩티드계 항생제에 대한 내성을 갖는 세균을 검출하는 탐지키트에 관한 것이다. 상기 글리코펩티드계 항생제는 Vancomycin, Teicoplanin, Telavancin, Bleomycin, Ramoplanin, 및 Decaplanin로 이루어지는 군에서 선택된 1종 이상일 수 있다. 또한 상기 세균은 Enterococcus 속 균주 또는 Staphylococcus속 균주일 수 있다.  A further embodiment of the invention comprises a D-alanine-D-alanine ligase, preferably VanA ligase, comprising an antibody or antigen-binding fragment thereof that specifically recognizes the D-alanine -D-alanine ligase The present invention relates to a detection kit for detecting bacteria that expresses VanB protein and is resistant to glycopeptide antibiotics. The glycopeptide antibiotic may be at least one selected from the group consisting of Vancomycin, Teicoplanin, Telavancin, Bleomycin, Ramoplanin, and Decaplanin. In addition, the bacterium may be a strain of the genus Enterococcus or a strain of the genus Staphylococcus.
본 발명의 추가 구현예는 상기 D-알라닌 -D-알라닌 리가제를 특이적으로 인식하는 항체 또는 이의 항원결합 단편을 이용한 면역분석법으로 글리코펩티드계 항생제에 대한 내성을 갖는 세균을 탐지하는 방법에 관한 것이다. 상기 탐지방법은 시료내 존재하는 VanA 리가제 또는 VanB 리가제 단백질을 항원으로 하여 상기 항체와의 항원 -항체 면역반응를 분석하여 양성 면역반응인 경우 시료에서 VanA 리가제 또는 VanB 리가제을 발현하는 글리코펩티드계 항생제에 대한 내성을 갖는 세균을 검출할 수 있다. A further embodiment of the present invention relates to a method for detecting bacteria having resistance to glycopeptide antibiotics by immunoassay using an antibody or antigen-binding fragment thereof that specifically recognizes the D-alanine-D-alanine ligase. will be. The detection method is an antigen-antibody with the antibody using VanA ligase or VanB ligase protein present in the sample as an antigen. The immune response can be analyzed to detect bacteria that are resistant to glycopeptide antibiotics expressing VanA ligase or VanB ligase in the sample in the case of a positive immune response.
본 발명에 따른 탐지키트 및 내성 세균의 탐지방법은 시료내에 포함된 글리코펩티드계 항생제에 대한 내성을 갖는 세균을 검출할 수 있을 뿐만 아니라， VanA 리가제 또는 VanB 리가제 유형의 표현형을 구분하여 검출할 수 있다.  The detection kit according to the present invention and a method for detecting resistant bacteria can not only detect bacteria having resistance to glycopeptide antibiotics contained in a sample, but also distinguish between phenotypes of VanA ligase or VanB ligase type. Can be.
이하, 본 발명을 더욱 자세히 설명하고자 한다.  Hereinafter, the present invention will be described in more detail.
본 발명의 일구현예는 D-알라닌 -D-알라닌 리가제， 바람직하게는 VanA 리가제 또는 VanB 리가제를 항원으로 하여 생산되며， 상기 VanA 리가제 또는 VanB 단백질을 특이적으로 인식하는 항체 또는 이의 항원 결합 단편에 관한 것이다.  One embodiment of the present invention is produced by using D-alanine-D-alanine ligase, preferably VanA ligase or VanB ligase as an antigen, an antibody or a antibody that specifically recognizes the VanA ligase or VanB protein. It relates to an antigen binding fragment.
본 발명에 따른 항원으로서 VanA 리가제 또는 VanB 리가제 단백질은 D-alanine-D-alanine ligase(EC 6.3.2.4.)로서 상기 항원을사용할 수 있는 단백질은 다양하게 보고되고 있다. 예를 들면， VanA 리가제 단백질의 아미노산 서열은 바람직하게는 서열번호 1 의 아미노산 서열을 포함하거나， 서열번호 2 의 염기서열을 포함하는 유전자에 의해서 암호화되는 서열일 수 있다. 구체적으로 Enterococcus faecium 유래 단백질 서열 GenBank accession number X56895.1 , Uniprot accession number Q7B608 , D4RFM1 , D4QQA6, E3USG8, 및 J7BBH6 등과 S. aureus 유래 단백질 서열 Uniprot accession number Q7BWD7, I3GWM9, 및 I3EUH6 등을 포함한다. 또한 상기 VanA 리가제 단백질의 아미노산 서열은 서열번호 1 의 아미노산 서열과 100% 일치하지 않더라도， 예를 들면 아미노산 서열 동일성이 90%이상이고 항원으로 작용하여 VanA 리가제를 특이적으로 인식하는 항체를 제조할 수 있는 동일한 항원원성을 나타내는 단백질을 모두 포함되는 의도이다.  VanA ligase or VanB ligase protein as an antigen according to the present invention has been reported a variety of proteins that can use the antigen as D-alanine-D-alanine ligase (EC 6.3.2.4.). For example, the amino acid sequence of the VanA ligase protein may preferably be a sequence that includes the amino acid sequence of SEQ ID NO: 1 or is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 2. Specifically, Enterococcus faecium-derived protein sequences GenBank accession number X56895.1, Uniprot accession number Q7B608, D4RFM1, D4QQA6, E3USG8, and J.BBH6 and S. aureus-derived protein sequences Uniprot accession number Q7BWD7, I3GWM9, and I3EUH6. In addition, even if the amino acid sequence of the VanA ligase protein is not 100% identical to the amino acid sequence of SEQ ID NO: 1, for example, an amino acid sequence identity of 90% or more, and acts as an antigen to prepare an antibody that specifically recognizes VanA ligase It is intended to include all proteins that exhibit the same antigenicity as possible.
또한， VanB 리가제 단백질의 아미노산 서열은 바람직하게는 서열번호 3 의 아미노산 서열을 포함하거나， 서열번호 4 의 염기서열을 포함하는 유전자에 의해서 암호화되는 서열일 수 있다. 구체적으로 Enterococcus facalis 유래 단백질 서열 GenBank accession number U00456.1, Uniprot accession number Q06893, C7WYV3, E6ZHJ4, Q7BHZ7 등이며， Enter ococcus faeciu 유래 단백질 서열을 Uniprot accession number C4MZP6, Q58F99, J7QUG3, Q84CN3, Q5MPQ4, P97205, Q9EY3, Q5MPQ2, J7CJR3 등을 포함한다. 또한 상기 VanB 리가제 단백질의 아미노산 서열은 서열번호 1 의 아미노산 서열과 100%일치하지 않더라도 아미노산 서열 동일성이 90%이상이고 항원으로 작용하여 VanB 리가제를 특이적으로 인식하는 항체를 제조할 수 있다면 모두 포함되는 의도이다. In addition, the amino acid sequence of VanB ligase protein is preferably a sequence that includes the amino acid sequence of SEQ ID NO: 3, or is encoded by a gene containing a nucleotide sequence of SEQ ID NO: 4. Specifically, Enterococcus facalis-derived protein sequence GenBank accession number U00456.1, Uniprot accession numbers Q06893, C7WYV3, E6ZHJ4, Q7BHZ7, and the like. Enter ococcus faeciu-derived protein sequences include Uniprot accession numbers C4MZP6, Q58F99, J7QUG3, Q84CN3, Q5MPQ4, P97205, Q9R3, Q5R3, Q5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J3, J5J, J. In addition, even if the amino acid sequence of the VanB ligase protein is not 100% identical to the amino acid sequence of SEQ ID NO: 1, if the amino acid sequence identity is greater than 90% and acts as an antigen, an antibody that specifically recognizes VanB ligase may be prepared. It is intended to be included.
한편, 본 명세서에 있어서， 용어， "특이적으로 결합" 또는 "특이적으로 인식"은 당업자에게 통상적으로 공지되어 있는 의미와 동일한 것으로서， 항원 및 항체가 특이적으로 상호작용하여 면역학적 반웅을 하는 것을 의미한다.  Meanwhile, in the present specification, the term "specifically binds" or "specifically recognized" is the same as commonly known to those skilled in the art, and the antigen and the antibody specifically interact with each other to perform immunological reactions. Means that.
본 명세서에서 용어， "항체의 항원 결합 단편' '은 면역글로불린 전체 구조에 대한 그의 단편으로, 항원이 결합할 수 있는 부분을 포함하는 폴리펩타이드의 일부를 의미한다. 예를 들어， scFv, (scFv)2, Fab, Fab' 또는 F(ab')2일 수 있으나, 이에 한정하지 않는다. 상기 항원 결합 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변 영역 및 중쇄의 첫 번째 불변 영역 (CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. 상기 항원 결합 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고 (예를 들어， 전체 항체를 파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다)， 유전자 재조합 기술을 통하여 제작할 수 있다. As used herein, the term "antigen-binding fragment" of an antibody is a fragment thereof for the entire structure of an immunoglobulin and refers to a portion of a polypeptide comprising a portion to which an antigen can bind. For example, scFv, (scFv ) 2 , Fab, Fab 'or F (ab') 2. The Fab in the antigen-binding fragment is a variable region of the light and heavy chains, the constant region of the light chain and the first constant region of the heavy chain (C Has one antigen binding site in a structure having H1 ) The antigen binding fragment can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with pine yields Fab and cleavage with pepsin). F (ab ') 2 fragments can be obtained), it can be produced through genetic recombination technology.
상기 항체 또는 이의 단편은 단클론 및 다클론 항체일 수 있다. 또한 상기 항체 또는 이의 단편은 동물 유래 항체， 예를 들면 마우스와 같은 설치류의 항체일 수 있으며， 키메라 항체 또는 인간화항체일 수 있다. 본 발명의 일구현예에서， 상기 VanA 리가제를 특이적으로 인식하는 항체는 상기 항체의 중쇄는 기탁번호 KCLRF-BP-00277를 갖는 하이브리도마에 의해 생산되는 항체의 중쇄로부터 얻어진 세 개의 CDR 을 포함하고 상기 항체의 경쇄는 기탁번호 KCLRF-BP-00277를 갖는 하이브리도마에 의해 생산되는 항체의 경쇄로부터 얻어진 세 개의 CDR 을 포함하는 것일 수 있다. 바람직하게는， 상기 VanA 리가제를 특이적으로 인식하는 항체는 VanA 리가제 단백질을 특이적으로 인식하며， 기탁번호 KCLRF-BP-00277을 갖는 하이브리도마에 의해 생산되는 것일 수 있다. The antibody or fragment thereof may be monoclonal and polyclonal antibodies. In addition, the antibody or fragment thereof may be an animal-derived antibody, for example, a rodent antibody such as a mouse, and may be a chimeric antibody or a humanized antibody. In one embodiment of the invention, the antibody that specifically recognizes the VanA ligase is a heavy chain of the antibody comprises three CDRs obtained from the heavy chain of the antibody produced by hybridoma having accession number KCLRF-BP-00277 And the light chain of the antibody comprises three CDRs obtained from the light chain of the antibody produced by hybridoma having accession number KCLRF-BP-00277. It may be to include. Preferably, the antibody that specifically recognizes the VanA ligase specifically recognizes the VanA ligase protein, and may be one produced by a hybridoma having accession number KCLRF-BP-00277.
또한, 상기 VanB 리가제를 특이적으로 인식하는 항체는 상기 항체의 중쇄는 기탁번호 KCLRF-BP-00278를 갖는 하이브리도마에 의해 생산되는 항체의 중쇄로부터 얻어진 세 개의 CDR 을 포함하고 상기 항체의 경쇄는 기탁번호 KCLRF-BP-00278를 갖는 하이브리도마에 의해 생산되는 항체의 경쇄로부터 얻어진 세 개의 CDR 을 포함하는 것일 수 있다. 바람직하게는, 상기 VanB 리가제를 특이적으로 인식하는 항체는 VanB 리가제 단백질을 특이적으로 인식하며， 기탁번호 KCLRFᅳ BP-00278을 갖는 하이브리도마에 의해 생산되는 것일 수 있다.  In addition, the antibody specifically recognizing the VanB ligase, the heavy chain of the antibody comprises three CDRs obtained from the heavy chain of the antibody produced by hybridoma having accession number KCLRF-BP-00278 and the light chain of the antibody May comprise three CDRs obtained from the light chain of an antibody produced by hybridoma having accession number KCLRF-BP-00278. Preferably, the antibody that specifically recognizes the VanB ligase specifically recognizes the VanB ligase protein and may be one produced by a hybridoma having accession number KCLRF ᅳ BP-00278.
본 명세서에서 사용된 용어， "CDR( complementarity determining region)' '은 면역글로불린의 중쇄 및 경쇄의 고가변 영역 (hypervar iable region)의 아미노산 서열을 의미한다. 중쇄 및 경쇄는 각각 3개의 CDR을 포함할 수 있다 (CDRHl, CDRH2, CDRH3 및 CDRLl, CDRL2, CDRL3). 상기 CDR은 항체가 항원 또는 에피토프에 결합하는 데 있어서 주요한 접촉 잔기를 제공할 수 있다.  As used herein, the term "complementarity determining region" (CDR) 'refers to the amino acid sequences of the hypervar iable regions of the heavy and light chains of immunoglobulins, each heavy and light chain comprising three CDRs. (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3) The CDRs may provide a major contact residue for the antibody to bind antigen or epitope.
본 발명에 따른 항체는 인 비트로 배양에 의하거나 또는 상기 항체를 생산하는 세포를 동물에 투여함으로써 얻을 수 있다. 상기 항체는 상기 항체를 생산하는 세포가 복막 내로 투여된 동물의 복수로부터 얻을 수 있다. 상기 항체는 이온 교환 크로마토그래피 또는 친화성 컬럼 크로마토그래피에 의하여 배양 상청액 또는 복수로부터 정제할 수 있다. 단클론 항체 (monoclonal antibody, mAb)는 동물 (예컨대， 마우스， 랫트， 햄스터 또는 닭) 기원일 수 있고, 또는 유전학적으로 제작될 수 있다. 설치류의 단클론 항체는 당업계에 잘 알려진 표준 방법에 의하여 제조될 수 있으며， 적절한 면역증강제와 함께 복강 내， 정맥 내， 또는 족척 (footpad) 내로 반토마이신 내성 장구균의 VanA또는 VanB 리가제를 다중 면역화한후 비장 또는 림프절 세포를 추출하고 적절한 불멸 세포주와 융합한 뒤， VanA 또는 VanB 리가제와 결합하는 항체를 생산하는 하이브리도마를 선별하는 것을 포함한다 . The antibody according to the present invention can be obtained by in vitro culture or by administering a cell producing the antibody to an animal. The antibody can be obtained from ascites of animals in which the cells producing the antibody have been administered into the peritoneum. The antibody can be purified from the culture supernatant or ascites by ion exchange chromatography or affinity column chromatography. Monoclonal antibodies (mAbs) may be of animal (eg, mouse, rat, hamster or chicken) origin, or may be genetically produced. Rodent monoclonal antibodies can be prepared by standard methods well known in the art and can be obtained by multiple immunization of vanAmycin-resistant enterococci VanA or VanB ligase into the intraperitoneal, intravenous, or footpad with appropriate adjuvant. Then spleen or lymph node cells are extracted and fused with an appropriate immortal cell line, Selecting hybridomas that produce antibodies that bind VanA or VanB ligase.
본 발명은 상기 항체 또는 이의 단편을 생산하는 세포를 제공한다 . 항체 또는 이의 단편을 생산하는 방법은 (a) D—알라닌 -D-알라닌 리가제， 바람직하게는 VanA 리가제， VanB 뫼가제 , 또는 이의 단편으로 동물을 면역화하는 단계， (b) 면역화된 동물로부터 비장 세포 (splenocyte)를 추출하는 단계， (c) 동물의 비장 세포를 골수종 (myeloma) 세포주와 융합시 키는 단계 및 (d) 하이브리도마 세포를 스크리닝하고， 글리코펩티드계 항생제에 대한 내성 세균의 VanA 리가제 또는 VanB 리가제를 특이 적으로 인식하는 항체를 생산하는 하이브리도마 세포를 선별하는 단계를 포함한다 .  The present invention provides a cell producing the antibody or fragment thereof. A method of producing an antibody or fragment thereof may comprise (a) immunizing the animal with D—alanine-D-alanine ligase, preferably VanA ligase, VanB mogase, or fragment thereof, (b) immunized animal Extracting splenocytes from the cells; (c) fusing the spleen cells of the animal with a myeloma cell line; and (d) screening the hybridoma cells and resistant bacteria to glycopeptide antibiotics. Selecting hybridoma cells that produce an antibody that specifically recognizes VanA ligase or VanB ligase.
상기 단클론 항체 또는 다른 항체들이 발현되면， 정밀여과 , 한외 여과， 단백질 A 또는 G 친화성 크로마토그래피， 크기 배제 크로마토그래피， 양이온 교환 크로마토그래피， 음이온 교환 크로마토그래피 및 /또는 유기 염료에 기초한 다른 형 태의 친화성 크로마토그래피 등과 같은 당업 계의 표준 기술에 따라 정 제할 수 있다 .  When the monoclonal antibody or other antibodies are expressed, microfiltration, ultrafiltration, protein A or G affinity chromatography, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography and / or other forms of parent based organic dyes Purification can be performed in accordance with standard techniques of the art such as chemical chromatography.
본 발명은 또한 상기 방법에 의해 얻어지는， 하이브리도마 세포 및 상기 하이브리도마 세포에 의해 생산되는 항체를 포함한다.  The present invention also includes hybridoma cells obtained by the above method and antibodies produced by the hybridoma cells.
구체적으로 본 발명은 단클론항체를 생산하는 하이브리도마로 확립하여， 항 -VanA 단클론 항체 8 종 , 항 -VanB 단클론 항체 3 종을 획득하였으며， 클론 번호 3G11 세포를 VanA 3G11-63-22 로 명 명하여 대한민국 서울시 종로구 연건동 28 번지 서울대학교 의과대학내 암연구소에 주소를 둔 한국세포주연구재단 (KCLRF , Korean Cel l Line Research Foundat ion) 에 2012 년 2 월 20 일자로 기 탁하여 기탁번호 KCLRF-BP- 00277 를 받았으며 , 클론 번호 7A12 세포를 VanB 7A12-12-4 로 명 명하여 한국세포주연구재단 (KCLRF , Korean Cel l Line Research Foundat ion) 에 2012 년 2 월 20 일자로 기 탁하여 기탁번호 KCLRF-BP-00278 를 받았다 .  Specifically, the present invention was established as a hybridoma producing monoclonal antibodies, eight anti-VanA monoclonal antibodies and three anti-VanB monoclonal antibodies were obtained, and clone number 3G11 cells were designated as VanA 3G11-63-22. Deposited KCLRF-BP-00277 with the Korean Cell Line Research Foundat ion (KCLRF), located at 28 Cancer Research Institute, Seoul National University College of Medicine, Jongno-gu, Seoul, Korea. The clone number 7A12 cells were named VanB 7A12-12-4 and deposited with the Korean Cell Line Research Foundat ion (KCLRF) on February 20, 2012 and deposited with KCLRF-BP-00278. Received.
본 발명 의 또 다른 구현예는 상기 VanA 리가제 또는 VanB 리가제 단백질을 특이 적으로 인식하는 항체 또는 이 의 항원결합 단편을 포함하는 VanA 리가제 또는 VanB 리가제의 정량하는 정량키트에 관한 것이다. 상기 항체 또는 이의 단편을 이용하여 시료내에 존재하는 VanA 리가제 또는 VanB 리가제의 정량함으로써， 상기 VanA 리가제 또는 VanB 리가제를 발현하는 글리코펩티드계 항생제에 대한 내성 세균의 존부 및 VanA 리가제 또는 VanB 리가제 유형으로 내성 세균의 표현형올 구분하여 검출할 수 있다. Another embodiment of the invention comprises an antibody or antigen-binding fragment thereof that specifically recognizes the VanA ligase or VanB ligase protein A quantitative kit for quantifying VanA ligase or VanB ligase. The presence or absence of a bacterium resistant to glycopeptide antibiotics expressing the VanA ligase or VanB ligase by quantifying the VanA ligase or VanB ligase present in the sample using the antibody or fragment thereof Ligase type can be detected by distinguishing phenotypes of resistant bacteria.
본 발명의 추가 구현예는 상기 VanA 리가제 또는 VanB 리가제 단백질을 특이적으로 인식하는 항체 또는 이의 항원결합 단편을 포함하는 글리코펩티드계 항생제에 대한 내성 세균의 검출키트에 관한 것이다. 상기 검출키트는 시료내에 글리코펩티드계 항생제에 대한 내성 세균의 존부를 검출할 뿐만 아니라， VanA 리가제 또는 VanB 리가제 유형으로 내성 세균의 표현형을 구분하여 검출할 수 있다.  A further embodiment of the present invention relates to a detection kit for bacteria resistant to glycopeptide antibiotics comprising an antibody or antigen-binding fragment thereof that specifically recognizes the VanA ligase or VanB ligase protein. The detection kit not only detects the presence of bacteria resistant to glycopeptide antibiotics in the sample, but can also detect the phenotype of resistant bacteria by VanA ligase or VanB ligase type.
상기 글리코펩티드계 항생제는 Vancomycin, Teicoplanin, Te 1 avanc i n , Bleomycin, amo lanin, 및 Decaplanin로 이루어지는 군에서 선택된 1종 이상일 수 있으며， 바람직하게는 Vancomycin, Teicoplanin또는 Telavancin일 수 있다.  The glycopeptide antibiotic may be at least one selected from the group consisting of Vancomycin, Teicoplanin, Te 1 avanc i n, Bleomycin, amo lanin, and Decaplanin, preferably Vancomycin, Teicoplanin or Telavancin.
상기 세균은 Enter ococcus 속 균주 (장구균) 또는 Staphylococcus 속 균주일 수 있으며， 상기 VanA유형의 항생제 내성 장구균은 E. faecalis, E. faecium, E. gal 1 inarum, E. casseliflavus, E. durans, E. mundtii , E. raffinosus, 및 E. avium 일 수 있고， VanB 유형의 항생제 내성 세균은 E. faecal is과 E. faec m일 수 있다. 상기 Staphylococcus 속 균주는 Staphylococcus aureus (포도상구균)일 수 있으며 바람직하게는 반코마이신 내성 Staphylococcus aureus0} . The bacteria may be Enter ococcus strains (E. coli) or Staphylococcus strains, and the vanA type antibiotic resistant enterococci are E. faecalis, E. faecium, E. gal 1 inarum, E. casseliflavus, E. durans, E. mundtii, E. raffinosus, and E. avium, and the antibiotic resistant bacteria of type VanB may be E. faecal is and E. faec m. The Staphylococcus genus strain may be Staphylococcus aureus (Staphylococcus aureus), preferably vancomycin resistant Staphylococcus aureus 0 }.
상기 검출키트는 바람직하게는 상기 VanA 리가제 또는 VanB 리가제 단백질과 상기 항체의 면역반응을 검출하는 표지 물질은 추가로 포함할 수 있다. 상기 표지 물질은 발색효소， 발색물질， 방사선 물질 또는 형광물질일 수 있다.  The detection kit may preferably further include a labeling substance for detecting an immune response between the VanA ligase or VanB ligase protein and the antibody. The labeling substance may be a colorase, a colorant, a radioactive substance, or a fluorescent substance.
본 발명에 따른 검출키트는 상기 항체의 효소면역 측정키트 (ELISA), 블롯팅 키트， 면역침전키트， 면역 형광검사 키트， 또는 면역 크로마토그래피 키트일 수 있으며， 바람직하게는 효소면역 측정키트 (ELISA) 또는 면역 크로마토그래피 키트일 수 있다. The detection kit according to the present invention may be an enzyme immunoassay kit (ELISA), a blotting kit, an immunoprecipitation kit, an immunofluorescence kit, or an immunochromatography kit of the antibody, preferably an enzyme immunoassay kit (ELISA) Or immunochromatography kits.
상기 ELISA는 하나의 항체를 이용하는 일반적인 방법과， 포획항체와 검출항체 두 가지 항체를 이용하는 샌드위치 방법을 모두 포함하는 의도이다. 상기 면역 스트립에 하나의 항체를 사용할 수도 있으나， 통상 포획항체와 검출항체 두 가지 항체를 이용하는 것이 일반적이다. 포획항체와 검출항체 두 가지 항체를 이용하는 경우에는, 예를 들면 VanA에 대한 항체는 포획항체로서 1H9와 검출항체 3G11 (기탁번호 KCLRF-BP-00277을 갖는 하이브리도마가 생산하는 단클론 항체)을 포함하는 항체쌍 또는 포획항체로서 1B7와 검출항체 3G11을 포함하는 항체쌍일 수 있고， 또는 VanB에 대한 항체는 포획항체로서 7E8와 검출항체 7A12(기탁번호 KCLRF-BP- 00278을 갖는 하이브리도마가 생산하는 단클론 항체)을 포함하는 항체쌍일 수도 있다.  The ELISA is intended to include both a general method using one antibody and a sandwich method using both capture and detection antibodies. Although one antibody may be used in the immune strip, it is common to use two antibodies, usually a capture antibody and a detection antibody. In the case of using both capture and detection antibodies, for example, an antibody against VanA includes 1H9 and 3G11 (a monoclonal antibody produced by a hybridoma having accession number KCLRF-BP-00277) as a capture antibody. May be an antibody pair or an antibody pair comprising 1B7 and a detection antibody 3G11 as a capture antibody, or an antibody against VanB is produced by a hybridoma having 7E8 and a detection antibody 7A12 (Accession Number KCLRF-BP- 00278) as a capture antibody. Antibody pairs), including monoclonal antibodies).
구체적으로， ELSIA 를 이용한 것이라면 고체상 지지체; 본 발명에 따른 단클론 항체; 및 항원과의 반응을 위한 효소 표지 항체액 및 효소반웅을 나타내는 발색액을 함유하는 효소 -연관 면역분석용 (ELSIA) 반웅액을 포함할 수 있다. 보다 구체적으로 효소 표지 항체액은 goat anti- mouse Ig-HRP 로 적정농도에서 플레이트 웰당 50 내지 150 ^가 포함될 수 있으며， 발색액은 테트라 메틸벤지딘 (Tetramethylbenzidine, TMB)을 선택될 수 있으며， 반응차단용액은 IN HC1 또는 IN H2S04 으로 이루어지는 군으로부터 선택될 수 있다. Specifically, if using ELSIA solid phase support; Monoclonal antibodies according to the invention; And an enzyme-associated immunoassay (ELSIA) reaction solution containing an enzyme-labeled antibody solution for reaction with an antigen and a chromophore showing an enzyme reaction. More specifically, the enzyme-labeled antibody solution may include 50 to 150 ^ per plate well at an appropriate concentration with goat anti-mouse Ig-HRP, and the coloring solution may be selected from tetramethylbenzidine (TMB) and the reaction blocking solution. May be selected from the group consisting of IN HC1 or IN H 2 S0 4 .
상기 면역 크로마토그래피 키트는 일반적인 면역 스트립 키트를 포함하며， 상기 스트립 키트는 시료용액에 수직으로 담구어 사용하는 딥스틱형， 스트립을 수평으로 놓고 시료용액을 점적시켜 사용하는 수평 이동형， 또는 상기 딥스틱형 또는 수평 이동형을 카세트에 끼워 고정하는 카세트형일 수 있으나 특별히 한정되지는 않는다.  The immunochromatography kit includes a general immunostrip kit, and the strip kit is a dipstick type used by immersing the sample solution vertically, horizontally moving the strip and placing the sample solution horizontally, or the dipstick. It may be a cassette type for fixing the mold or horizontal movable type to the cassette, but is not particularly limited.
본 발명의 일예에 따른 면역 스트립 키트는 지지체; 검출 항체와 표지 물질의 접합체 (con jugate)를 함유하는 컨쥬게이트 패드; 포획 항체가 고정된 신호 검출 패드; 및 검체 전개액을 흡수하는 흡수 패드를 포함하는 단변의 폭과 장변의 길이를 가지는 분석 스트립을 하나 이상 포함하며， 상기 검출 항체와 포획 항체는 본 발명에 따른 VanA 리가제 또는 VanB 리가제를 특이적으로 인식하는 항체 또는 이의 단편일 수 있다. An immune strip kit according to one embodiment of the present invention includes a support; Conjugate pads containing a conjugate of a detection antibody and a labeling substance; Signal detection pads to which capture antibodies are immobilized; And at least one analysis strip having a width of a short side and a length of a long side including an absorbent pad absorbing the sample developing solution. The detection antibody and capture antibody may be an antibody or fragment thereof that specifically recognizes VanA ligase or VanB ligase according to the present invention.
상기 면역 스트립 키트는 하나의 스트립에 VanA 뫼가제 또는 VanB 리가제을 인식하는 항체는 각각 고정시킨 하나 이상의 스트립을 조합하여 사용할 수도 있고， VanA 리가제를 인식하는 항체가 고정된 스트립과, VanB 리가제를 특이적으로 인식하는 항체가 고정된 스트립을 조합한 이중 VanA/VanB 검출키트일 수 있다.  The immunostrip kit may be used in combination with one or more strips to which VanA ligase or VanB ligase-recognizing antibodies are immobilized, respectively. It may be a double VanA / VanB detection kit combining a strip fixed with an antibody that specifically recognizes.
구체적으로， 상기 발색효소가 HRPQorseradish peroxidase) 또는 염기성 탈인산화효소 (alkaline phosphatase)이고， 발색물질은 콜로이드 골드 (coloid gold)이고， 형광분자는 FITC(poly L-lysine-f luorescein isothiocyanate) 및 RITC ( rhodam i ne-B- i sot h i ocyanat e ) 등일 수 있다. 또한 신호 검출 패드는 니트로샐를로오스， 샐를로오스， 플리에틸렌， 폴리에테르설폰 및 나일론으로 구성된 군으로부터 선택된 어느 하나일 수 있다. 상기 흡수 패드는 다공성 지지체， 및 상기 다공성 지지체의 공동 (pore)에 분산되거나 다공성 지지체의 섬유사에 흡착 또는 코팅되어 있는 흡수제를 포함하는 것일 수 있다.  Specifically, the chromophore is HRPQorseradish peroxidase or alkaline dephosphatase, the chromophore is colloid gold, and the fluorescent molecules are poly L-lysine-f luorescein isothiocyanate (FITC) and RITC (rhodam) i ne-B- i sot hi ocyanat e). In addition, the signal detection pad may be any one selected from the group consisting of nitrosarose, salloose, polyethylene, polyethersulfone and nylon. The absorbent pad may include a porous support, and an absorbent dispersed in a cavity of the porous support or adsorbed or coated on the fiber yarn of the porous support.
본 발명의 구체적인 예에서, 상기 면역 스트립 키트는 시료에 있는 항원이 콜로이드성 금 입자 (colloidal gold particle)에 결합된 검출 항체와 반응한 다음, 모세관 현상에 의해 니트로셀를로오스 (nitrocellulose) 멤브레인의 미세구멍 (micropore)을 통하여 이동하는 도중에 미세구멍의 내부 표면에 고정되어 있는 포획 항체 (capture antibody)와 결합하여 발색띠를 형성함으로써, 양성 및 음성을 육안으로 판별 가능하도록 할 수 있다.  In a specific example of the present invention, the immune strip kit reacts with a detection antibody in which an antigen in a sample is bound to colloidal gold particles, and then fines the nitrocellulose membrane by capillary action. While moving through the micropore, it is possible to visually distinguish positive and negative by combining with a capture antibody fixed to the inner surface of the micropore to form a color band.
본 발명의 또 다른 구체예는 상기 항체 또는 이의 단편을 이용하여， 시료에 함유된 VanA 리가제 및 VanB 리가제로 이루어지는 군에서 선택된 1종 이상의 단백질을 면역분석법으로 탐지하는 단계를 포함하는 글리코펩티드계 항생제에 대한 내성 세균의 검출방법에 관한 것이다. 상기 검출방법은 상기 탐지단계에서 VanA 리가제 및 VanB 리가제가 시료내에 존재하는 경우 글리코펩티드계 항생제에 대한 내성 세균이 시료내에 존재하는 것으로 결정하는 단계를 추가로 포함할 수 있다. 상기 검출방법은 시료내 글리코펩티드계 항생제에 대한 내성 세균의 존재를 검출할 수 있을 뿐만 아니라， VanA 리가제 또는 VanB 리가제 유형으로 내성 세균의 표현형을 구분할 수 있다. Another embodiment of the present invention using the antibody or fragments thereof, glycopeptide antibiotics comprising the step of detecting by immunoassay at least one protein selected from the group consisting of VanA ligase and VanB ligase contained in the sample It relates to a method for detecting bacteria resistant to. The detection method is characterized in that the bacteria resistant to glycopeptide antibiotics in the sample when VanA ligase and VanB ligase are present in the sample. Determining what is present may be further included. The detection method can detect the presence of bacteria resistant to glycopeptide antibiotics in the sample, and can also distinguish the phenotype of resistant bacteria by VanA ligase or VanB ligase type.
상기 탐지단계를 수행하기 전에， 상기 시료를， 글리코펩티드계 항생제가 첨가한 배지에서 접종하여 배양하고， 세포를 수집하고， VanA 리가제 및 VanB 리가제를 용출하여 준비하는 것일 수 있다. 검체에서 시료를 추출하여 VanA 리가제 및 VanB 리가제를 용출하여 탐지단계를 수행할 수도 있으나， VanA 리가제 및 VanB 리가제의 발현양을 증가시키고 상기 단백질을 발현하는 세균수를 늘려 검출방법의 민감도를 증가시칼 수 있도록 배양단계를 수행하는 것이 바람직하다. 상기 배양배지는 글리코펩티드계 항생제에 대한 내성 세균， 예를 들면 Enterococcus속 균주 (장구균) 또는 Staphylococcus 속 균주의 배양 배지로 통상 사용되는 배지를 사용할 수 있다.  Before performing the detection step, the sample may be inoculated and cultured in a medium to which glycopeptide antibiotics are added, cells are collected, and VanA ligase and VanB ligase are prepared by eluting. The detection step may be performed by extracting a sample from the sample and eluting VanA ligase and VanB ligase, but the sensitivity of the detection method is increased by increasing the expression level of VanA ligase and VanB ligase and increasing the number of bacteria expressing the protein. It is preferable to carry out the culturing step so as to increase. The culture medium may be a medium commonly used as a culture medium of bacteria resistant to glycopeptide antibiotics, for example, Enterococcus strain (E. coli) or Staphylococcus strain.
상기 배양시 첨가되는 글리코펩티드계 항생제의 농도는 내성 세균의 The concentration of glycopeptide antibiotics added during the culture may be
VanA 리가제 및 VanB 리가제의 발현량을 증가시킬 수 있는 농도이면 적절히 선택하여 사용할 수 있으며, 예를 들면 2 내지 8 ug/mL일 수 있으며 바람직하게는 4 내지 6 ug/mL 일 수 있다. Any concentration that can increase the expression level of VanA ligase and VanB ligase may be appropriately selected and used, for example, 2 to 8 ug / mL, and preferably 4 to 6 ug / mL.
상기 VanA 리가제 및 VanB 리가제는 수용성 단백질이 아니므로 세포를 화학적 용해 또는 물리적 파쇄 등으로 세포를 용해하고， 세포외로 용출시켜 분석할 수 있으며， 예를 들면 요소를 처리하여 VanA 리가제 및 VanB 리가제를 수용성화 시킬 수 있다.  Since the VanA ligase and VanB ligase are not water soluble proteins, the cells can be analyzed by lysing the cells by chemical lysis or physical disruption, eluting the cells extracellularly, for example, by treating urea, VanA ligase and VanB ligase. The agent can be water-soluble.
상기 면역 분석법은 방사성 면역분석법， 면역효소분석법 (ELISA), 샌드위치 면역분석법 및 면역 크로마토그래피 분석법 (immunographic assay)으로 이루어진 군에서 선택된 것일 수 있으며， 바람직하게는 ELISA 분석 및 면역크로마토그래피 분석법이며 이들 방법은 상기 검출키트에서 설명한 바와 같다. 구체적으로 항원-항체의 검출은 직접적으로나 간접적으로 표지된 항체의 사용을 수반하며， 사용 가능한 표지물질은 앞서 기술한 바와 같으며 상기 분석법의 구체적인 내용 및 방법은 당업계에 공지된 바에 의한다 . The immunoassay may be selected from the group consisting of radioimmunoassay, immunoassay (ELISA), sandwich immunoassay, and immunochromatography assay, preferably ELISA assay and immunochromatography assay. As described above in the detection kit. Specifically, the detection of antigen-antibody involves the use of directly or indirectly labeled antibodies, the available labeling materials are as described above and the details and methods of the assays are known in the art. As is known.
일례로 ELISA 검출 방법에 의하는 경우 시료 샘플은 고체 지지체， 예를 들어 마이크로타이터 플레이트， 멤브레인， 테스트 스트립 등에 코팅된 본 발명의 단클론 항체 또는 상기 VanA/B ligase 에 특이적인 항체와 접촉된다. 구체적 일례로서， 마이크로타이터 플레이트의 웰을 본 발명의 단클론 항체 또는 상기 VanA/B ligase 에 특이적인 항체로 코팅하고 점유되지 않은 (nonoccupied) 결합 부위를 예를 들어 BSA 로 차단한 후， 코팅된 플레이트의 웰을 시료 샘플과 인큐베이션하고， 이어서 항원—항체 복합체의 존재를 결정할 수 있다. 상기 항체는 앞서 기술한 바와 같이 표지물질이 결합되어 있을 수 있다.  For example, in the case of ELISA detection, the sample sample is contacted with a monoclonal antibody of the present invention coated with a solid support, such as a microtiter plate, a membrane, a test strip, or an antibody specific for the VanA / B ligase. As a specific example, a well of a microtiter plate is coated with a monoclonal antibody of the present invention or an antibody specific for the VanA / B ligase, and the nonoccupied binding site is blocked with, for example, BSA, and then coated with a plate. Wells may be incubated with the sample sample, followed by determination of the presence of the antigen-antibody complex. The antibody may be bound to a label as described above.
또한， 샌드위치 면역분석법 및 측방 유동 면역 크로마토그래피 분석법 (lateral flow immunographic assay)을 사용하는 경우 본 발명의 단클론 항체를 이용하여 항체 쌍을 제조하고 이를 이용하여 항생제 내성 세균을 탐지할 수 있다.  In addition, when using sandwich immunoassay and lateral flow immunographic assay, the antibody pair may be prepared using the monoclonal antibody of the present invention and the antibiotic resistant bacteria may be detected using the same.
본 발명에서 개발한 면역크로마토그래피 키트는 VanA/B ligase 단백질을 검출하는 것이기 때문에 검출 한계 이상의 단백질 확보가 필요하는 것이 바람직하다. 검출가능한 단백질 확보는 증균 과정을 통하여 균수의 증가 및 VanA/B 의 유도로서 확보될 수 있는데， 증균 과정은 하루 정도의 시간을 요하고 이는 검출의 신속성을 저해하는 한계요소가 되었다. 그러나 증균 이후 추가적인 배양 없이 VRE 항생제 내성 유형을 동정할 수 있기 때문에， 배양법 검출에 비하여 하루에서 이를 정도의 검출시기 단축효과가 있다.  Since the immunochromatography kit developed in the present invention detects VanA / B ligase proteins, it is preferable to secure proteins beyond the detection limit. Detectable protein can be secured by increasing the number of bacteria and inducing VanA / B through the enrichment process. The enrichment process takes about a day and this becomes a limiting factor that impedes the speed of detection. However, since the VRE antibiotic resistance type can be identified without additional culture after enrichment, the detection time is shorter than that of the culture method.
【발명의 효과】 【Effects of the Invention】
본 발명은 으알라닌 -D-알라닌 리가제를 특이적으로 인식하는 항체， 및 상기 항체를 이용한 글리코펩티드계 항생제에 대한 내성 세균의 검출키트 및 검출방법을 제공하여， 종래 PCR 방법이 갖는 고비용 및 전문 인력이 필요하다는 단점과 배양법이 갖는 조기 진단이 어려우며 항생제 내성 균의 표현형에 대한 정보를 제공하지 못한다는 단점을 해소하여, 배양법보다 빠른 진단이 가능하고， 내성 유전자 유형 정보를 함께 제공할 수 있다는 점에서 배양법 또는 PCR 진단법과 차별화가 가능하며， 글리코펩티드계 항생제에 대한 내성 세균의 검출의 새로운 대안을 제시하고 있다. The present invention provides an antibody that specifically recognizes alanine-D-alanine ligase, and a kit and a method for detecting bacteria resistant to glycopeptide-based antibiotics using the antibody. By eliminating the disadvantage of the need for manpower and the early diagnosis of culture methods and the inability to provide information on the phenotype of antibiotic resistant bacteria, It can be diagnosed faster than the culture method, and can be differentiated from the culture method or the PCR diagnosis method by providing the resistance gene type information together, and suggests a new alternative to the detection of resistant bacteria against glycopeptide antibiotics.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 la 내지 도 lc 는 은 재조합 VanA 및 VanB 재조합 단백질의 정제결과를 나타내는 도면으로서， 도 la 는 Cloning of vanA and vanB 리가제 유전자의 클로닝 결과이고， 도 lb 는 재조합 VanA 단백질의 재접힙 (Refolding) 및 정제 결과를 나타내는 사진이고， 도 lc 는 수용성 재조합 VanA/VanB 리가제 단백질의 정제 결과를 나타내는 사진이다.  Fig. La to lc are diagrams showing the purification results of the recombinant VanA and VanB recombinant proteins, Fig. La is the cloning result of Cloning of vanA and vanB ligase gene, and Fig. Lb is the refolding and It is a photograph showing the purification result, Figure lc is a photograph showing the purification result of the water-soluble recombinant VanA / VanB ligase protein.
도 2a 내지 도 2c 는 최적 항— VanA 항체쌍을 선택 결과를 나타내는 도면으로서， 도 2a 는 일반적인 ELISA 방법 (conventional ELISA)으로 항체 성능을 평가한 검출 결과이고， 도 2b 는 샌드위치 ELISA 방법으로 포획 항체 성능을 평가한 검출 결과이고, 도 2c 는 VanA. 에 대한 최적 항체쌍으로서 1H9/3G11 또는 1B7/3G11 (포획 /검출)을 나타내는 그래프이다. 도 3a 내지 도 3c 는 최적 항 -VanB 항체쌍의 선택 결과를 나타내는 도면으로서， 도 3a 는 일반적인 ELISA 방법 (convent ional ELISA)으로 항체 성능을 평가한 검출 결과이고， 도 3b 는 샌드위치 ELISA 방법으로 포획 항체 성능올 평가한 검출 결과이고， 도 3c 는 VanB 에 대한 최적 항체쌍으로서 7E8/14B2 or 7A12/14B2 (포획 /검출)을 나타내는 그래프이다. 도 4 는 VanA 항체쌍의 노이즈에 대한 시그널 비율 (signal-to—noise ratio)(signal/noise 비율) 및 3 차원 모식도를 보여주는 도면으로서， X- 축은 검출 항체의 회석인자 (dilution factor), Y-축^ 포획 항체의 농도를 나타낸다.  2A to 2C are diagrams showing the results of selecting an optimal anti-VanA antibody pair, and FIG. 2A is a detection result of evaluating antibody performance by a conventional ELISA method, and FIG. 2B shows capture antibody performance by a sandwich ELISA method. 2C shows VanA. Is a graph showing 1H9 / 3G11 or 1B7 / 3G11 (capture / detection) as the optimal antibody pair for. 3A to 3C are diagrams showing the results of selection of an optimal anti-VanB antibody pair. FIG. 3A is a detection result of evaluating antibody performance by a general ELISA method. FIG. 3B is a capture ELISA method. 3C is a graph showing 7E8 / 14B2 or 7A12 / 14B2 (capture / detection) as an optimal antibody pair against VanB. 4 shows a signal-to-noise ratio (signal / noise ratio) and a three-dimensional schematic diagram for noise of a VanA antibody pair, wherein the X-axis represents the dilution factor of the detection antibody, Y-. The concentration of the contracted capture antibody is shown.
도 5 는 VanB 항체쌍의 노이즈에 대한 시그널 비율 (signal-to-noise ratio)(signal/noise 비율)을 보여주는 도면으로서， 항체쌍 7E8/7A12 (포획 /검출)은 높은 S/N 비율을 나타냈으며, 포획 항체의 최적 농도는 항체쌍 7E8/7A12 (포획 /검출) S/N 비율을 바탕으로 2.5 ug/mL 로 결정하였고， 검출항체의 최적 희석 범위는 800내지 12,800 이다. FIG. 5 shows the signal-to-noise ratio (signal / noise ratio) of noise of the VanB antibody pair, and antibody pair 7E8 / 7A12 (capture / detection) showed a high S / N ratio. Optimal concentration of capture antibody The antibody pair was determined to be 2.5 ug / mL based on the 7E8 / 7A12 (capture / detection) S / N ratio and the optimal dilution range of the detected antibody was 800-12,800.
도 6 은 본 발명의 일실시예에 따른 VanA EL ISA 분석을 위한 표준 곡선이다.  6 is a standard curve for VanA EL ISA analysis according to an embodiment of the present invention.
도 7 은 본 발명의 일실시예에 따른 VanB ELISA 분석을 위한 표준 곡선이다.  7 is a standard curve for VanB ELISA analysis according to an embodiment of the present invention.
도 8은 본 발명의 일실시예에 따른 딥스틱 타입 (dip-stick type)의 면역 스트립을 나타내는 도면아다.  FIG. 8 is a diagram illustrating an immune strip of a dip-stick type according to an embodiment of the present invention.
도 9 은 본 발명의 일실시예에 따라 VanA/VanB 리가제에 대한 항체를 이용하여 면역크로마토그래피 (ICA)로 반코마이신 -내성 장구균를 검출하는 과정을 나타내는 모식도이다.  9 is a schematic diagram illustrating a process for detecting vancomycin-tolerant enterococci by immunochromatography (ICA) using an antibody against VanA / VanB ligase according to one embodiment of the present invention.
도 10 은 본 발명의 일예에 따라 VanA VanA 면역크로마토그래피 키트의 개략도와 분석 결과를 나타내는 사진이다.  10 is a photograph showing a schematic diagram and an analysis result of a VanA VanA immunochromatography kit according to an embodiment of the present invention.
도 11 는 VanA 면역크로마토그래피와 ELISA 의 상관관계를 분석한 결과를 나타내는 도면이다.  11 is a diagram showing the result of analyzing the correlation between VanA immunochromatography and ELISA.
도 12 은 VanB 면역크로마토그래피와 ELISA 의 상관관계를 분석한 결과를 나타내는 도면이다.  12 is a graph showing the results of analyzing the correlation between VanB immunochromatography and ELISA.
도 13 은 VanA 또는 VanB 의 발현 수준을 증가시키기 위해서 배양배지에 반코마이신 추가요건을 분석한 결과를 나타내는 도면이다. 도 14 는 이중 VanA/B 면역크로마토그래피 키트의 수행결과를 나타내는 도면이다.  FIG. 13 is a diagram showing the results of analyzing vancomycin addition requirements to the culture medium to increase the expression level of VanA or VanB. 14 is a view showing the performance of the dual VanA / B immunochromatography kit.
도 15 은 이중 VanA/B 면역크로마토그래피 키트를 이용한 검출방법에서， 반코마이신 내성 균주의 탐지량을 평가한 결과이다. 【발명을 실시하기 위한 구체적인 내용】  15 is a result of evaluating the detection amount of vancomycin resistant strains in the detection method using a double VanA / B immunochromatography kit. [Specific contents to carry out invention]
이하， 실시예 및 비교예를 통하여 본 발명을 더욱 상세히 설명한다. 다만， 하기 실시예는 본 발명을 예시하기 위한 것이며， 이들에 의하여 본 발명의 범위가 한정되는 것은 아니다. 실시예 1: 재조합 VanA 및 VanB단백질의 발현 및 정제 반코마이신 내성 장구균으로부터 1100 염기쌍의 vanA 및 vanB 유전자를 각각 클로닝 하였고， 유도， 수용성화 및 용출 과정을 거쳐 약 40 kDa에 달하는 단백질로 정제하였다. Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the following examples are provided to illustrate the present invention, and the scope of the present invention is not limited thereto. Example 1 Expression and Purification of Recombinant VanA and VanB Proteins 1100 base pairs of vanA and vanB genes were cloned from vancomycin-resistant enterococci, respectively, and purified to about 40 kDa protein through induction, water solubilization and elution.
1-1： vanA및 vanB유전자클로닝  1-1 ： vanA and vanB gene cloning
vanA ligase 유전자는 하기 표 7 의 반코마이신 내성 장구균 VRE 1506에서， vanB ligse 유전자는 V583 V E에서 각각 클로닝 되었다 (도 la). 구체적으로， vanA 및 vanB ligase 유전자 서열은 NCBI (National Center for Biotechnology Information) 유전자 은행에서 획득 하였다 (va , GenBank: X56895.1, vanB, GenBank: U00456.1). vanA ligase 유전자는 VRE 1141， vanB ligase 유전자는 V583 VRE 에서 각각 아래에 표시된 primer 서열을 사용하여 증폭하였다. 확보된 유전자는 pETlOl/D-TOPO vectorClnvitrogen, Carlsbad, CA, USA)에' 옮겨 BL21 Star E. coli 형질 전환에 사용되었다. The vanA ligase gene was cloned in vancomycin resistant enterococci VRE 1506 in Table 7 below, and the vanB ligse gene in V583 VE, respectively (FIG. la). Specifically, vanA and vanB ligase gene sequences were obtained from the National Center for Biotechnology Information (NCBI) gene bank (va, GenBank: X56895.1, vanB, GenBank: U00456.1). The vanA ligase gene was amplified using VRE 1141 and the vanB ligase gene from V583 VRE using the primer sequences shown below. The gene was obtained using the 'move BL21 Star E. coli transformed the pETlOl / D-TOPO vectorClnvitrogen, Carlsbad , CA, USA).
[표 2] 프라이머 서열  TABLE 2 Primer Sequences
Figure imgf000018_0001
Figure imgf000018_0001
1-2: VanA/VanB 재조합 단백질의 개발 1-2: Development of VanA / VanB Recombinant Protein
상기 1-2 에서 준비된 유전자로 형질전환 한 E. coli 배양액에 0.5 mM IPTG 를 첨가하여 histidine 이 부착된 재조합 단백질을 유도하였다. 4 시간 추가 배양을 실시 한 뒤 배양액을 원심분리하여 침전물을 만들고， 2.0 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100 (pH 8.0) 용액에 현탁하여 재조합 단백질의 특성을 분석한 결과 대부분의 재조합 0.5 mM IPTG was added to the E. coli culture transformed with the gene prepared in 1-2 to induce histidine-attached recombinant protein. After further incubation for 4 hours, the culture medium was centrifuged to form a precipitate, and suspended in 2.0 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100 (pH 8.0) solution to characterize the recombinant protein. Analysis shows that most recombination
VanA 및 VanB 단백질이 불용성 형태로 유도되고 있음이 확인되었다. 현탁액은 다시 원심분리하여 침전물을 만들고 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 8 M urea, 1 mM 2-mercaptoethanol (pH 8.0)에 재현탁한 후 상은에서 1 시간 동안 교반하였다. 원심분리하여 상청액을 취하고 0.22 urn filter로 여과하였다. It was confirmed that VanA and VanB proteins are induced in insoluble form. The suspension is centrifuged again to form a precipitate, 20 mM Tris-HCl, 0.5 M NaCl, After resuspending in 5 mM imidazole, 8 M urea, 1 mM 2-mercaptoethanol (pH 8.0), the phases were stirred for 1 hour at silver. The supernatant was collected by centrifugation and filtered with a 0.22 urn filter.
제작된 재조합 단백질은 histidine tag 을 가지고 있기 때문에 8.0 M urea 용액에서 수용성화하고 Ni-컬럼에 주입하였다. 준비된 여과 시료는 1.0 raL HisTrap (GE healthcare, Little Chalfont, UK) 컬럼에 주입하였다. 컬럼에 결합된 단백질은 요소 농도를 8.0 M 부터 서서 ¾ᅵ (0.5 mL/min, 80 mL) 낮추면서 수용성화를 유도 하였다. 수용화된 재조합 단백질은 20 mM Tris-HCl , 0.5 M NaCl , 150 mM imidazole, 1 mM 2-mercaptoethanol (pH 8.0) 조건에서 용출하였다.  Since the recombinant protein has histidine tag, it was solubilized in 8.0 M urea solution and injected into Ni-column. The prepared filtered sample was injected into a 1.0 raL HisTrap (GE healthcare, Little Chalfont, UK) column. The protein bound to the column induced water solubilization by lowering urea concentration from 8.0 M to ¾ (0.5 mL / min, 80 mL). Soluble recombinant protein was eluted under 20 mM Tris-HCl, 0.5 M NaCl, 150 mM imidazole, 1 mM 2-mercaptoethanol (pH 8.0).
Ni-컬럼에 결합된 단백질 refolding 을 유도하기 위해 urea 의 농도를 서서히 낮추어 urea 을 완전히 제거하였다 (도 lb). 재조합 단백질은 150 mM imidazole 용출 완층액을 사용하여 용출하였고, 분자량은 약 40 kDa 정도였다 (도 lc). 재조합 단백질 VanA 및 VanB 모두 동일한 방법으로 수용성화， refolding 및 용출이 진행되었다.  In order to induce protein refolding bound to the Ni-column, the concentration of urea was slowly lowered to completely remove urea (FIG. Lb). Recombinant protein was eluted using 150 mM imidazole elution complete solution, the molecular weight was about 40 kDa (Fig. Lc). Both recombinant proteins VanA and VanB were solubilized, refolded and eluted in the same manner.
상기 모든 과정은 VanA 및 VanB 재조합 단백질 유도， 수용성화 및 용출에 동일하게 사용되었다. 실시예 2: 단클론 항체 제조  All of these procedures were used identically for induction, water solubilization and elution of VanA and VanB recombinant proteins. Example 2: Monoclonal Antibody Preparation
2-1： 하이브리도마 제조  2-1 ： Hybridoma production
상기 실시예 1 에서 정제된 VanA 및 VanB 재조합 단백질을 Balb/c 마우스 복강에 100 ug^ 3주 간격으로 4회 면역하였다. 4회 면역 후 3일 경과 시점에 마우스 비장을 적출하여 1 X 108 세포를 취하고, X63-Ag8.653 (ATCC, Virginia, USA) 마우스 골수종 암세포와 융합올 유도하였다. 융합된 하이브리도미"는 HAT (hypoxanthine, aminopterin and thymidine containing media) 배지에서 10 일간 배양하여 선별하고， 단클론 항체 선별을 위하여 VanA 및 VanB 재조합 단백질을 웰당 100ng 씩 넣어 코팅한 후， 배양액 100 ul를 취하여 Goat 항-마우스 Ig-HRP를 넣어 ELISA 방법으로 재조합 단백질 VanA 또는 VanB 에 대한 반웅성을 확인하였다. 재조합 단백질과 반웅성을 나타내는 하이브리도마는 2 - 3 회 l imit ing di lut ion 법을 통하여 계대배양하여 단클론 하이브리도마로 확립하였다 . 최종적으로 항 -VanA 단클론 항체 8 종， 항 -VanB 단클론 항체 3 종을 획득하였다 (표 3) . VanA and VanB recombinant proteins purified in Example 1 were immunized four times at intervals of 100 ug ^ 3 weeks in the abdominal cavity of Balb / c mice. Three days after 4 immunizations, mouse spleens were extracted, 1 × 10 8 cells were taken, and X63-Ag8.653 (ATCC, Virginia, USA) mouse myeloma cancer cells were fused and induced. Fused hybridomi "was selected by incubation for 10 days in HAT (hypoxanthine, aminopterin and thymidine containing media) medium, and coated with 100ng of VanA and VanB recombinant protein per well for monoclonal antibody selection. Reaction of the recombinant protein VanA or VanB was confirmed by ELISA using Goat anti-mouse Ig-HRP. Indicative hybridomas were subcultured 2-3 times by l imiting di lut ion method to establish monoclonal hybridomas. Finally, eight anti-VanA monoclonal antibodies and three anti-VanB monoclonal antibodies were obtained (Table 3).
[표 3] 마우스의 항 -VanA 또는 항 -VanB 단클론 항체  Table 3 Anti-VanA or Anti-VanB Monoclonal Antibodies in Mice
Figure imgf000020_0001
상기 하이브리도마 세포 중 클론 번호 3G11 세포를 VanA 3G11-63- 22 로 명 명하여 대한민국 서울시 종로구 연건동 28 번지 서을대학교 의과대학내 암연구소에 주소를 둔 한국세포주연구재단 (KCLRF, Korean Cel l Line Research Foundat ion) 에 2012 년 2 월 20 일자로 기탁하여 기 탁번호 KCLRF-BP-00277 를 받았으며， 클론 번호 7A12 세포를 VanB 7A12-12-4 로 명 명하여 한국세포주연구재단 (KCLRF, Korean Cel l Line Research Foundat ion) 에 2012 년 2 월 20 일자로 기탁하여 기탁번호 KCLRF-BP- 00278 를 받았다 . 2-3： 항— VanA 단클론 항체의 선별
Figure imgf000020_0001
KCLRF, Korean Cel l Line Research Foundat, whose clone number 3G11 cells were named VanA 3G11-63-22 and were addressed at the Cancer Research Institute, Seolleh University School of Medicine, 28, Yeongun-dong, Seoul, Korea. ion was deposited on February 20, 2012 and received accession number KCLRF-BP-00277, and clone number 7A12 cells were named VanB 7A12-12-4, and the Korean Cell Line Research Foundation (KCLRF). ion was deposited on February 20, 2012 and received accession number KCLRF-BP- 00278. 2-3 ： Screening of anti—VanA monoclonal antibodies
(1) 항체준비 EL ISA 및 면역크로마토그래피 키트 개발에 적절한 단클론 항체 선별을 위하여 항체 특성을 분석하였다. 검출 항체 (detection antibody)는 항원으로서 VanA 또는 VanB 재조합 단백질을 코팅하고 항원에 대한 반응성 평가로 선정하였다. 포획 항체는 평가할 항체를 코팅하고 HRP 접합 항체 집합용액 (Pooled-mono)을 검출 항체로 사용하여 평가하였다. (1) Antibody Preparation Antibody characteristics were analyzed for the selection of monoclonal antibodies suitable for the development of EL ISA and immunochromatography kits. Detection antibodies were coated with VanA or VanB recombinant protein as antigen and selected for evaluation of reactivity to the antigen. Capture antibodies were coated with the antibody to be evaluated and evaluated using HRP conjugated antibody pool (Pooled-mono) as the detection antibody.
구체적으로， 검출 항체의 제조를 위하여 HRP (Amresco, OH, USA)를 50 mM sodium phosphate (pH 7.2) 완층액에 투석 하였다. 0.1 M SMCC (Succinimidyl 4- (TV-ma 1 e i m i doraet hy 1 ) eye 1 ohexane- 1-car boxy 1 at e ) 준비하여 HRP 용액 대비 1/10 (v/v)을 첨가하고 상온에서 2 시간 동안 교반하였다. 단클론 항체를 50 mM sodium phosphate (pH 7.2) 완층액에 투석하여 준비하고， 14 mM Traut' s reagent (2-Iminothiolane-HCl )를 항체 용액 1.0 mL 에 46 ul 첨가하여 2 시간 동안 교반하였다. 반웅에 참여하지 않은 SMCC 및 Traut ' s reagent 를 sephadex G25 desalting column 으로 제거한 뒤 HRP 용액과 항체 용액을 섞어 다시 2 시간 동안 교반하였다. HRP 접합 항체는 포화 ammonium sulfate 용액 (pH 7.4)을 동량 첨가하여 30 분 교반하고 원심분리로 침전하여 분리하였고， 이후 phosphate buffered saline (pH 7.2)에 투석하였다.  Specifically, HRP (Amresco, OH, USA) was dialyzed on 50 mM sodium phosphate (pH 7.2) complete solution for the production of detection antibodies. Prepare 0.1 M SMCC (Succinimidyl 4- (TV-ma 1 eimi doraet hy 1) eye 1 ohexane- 1-car boxy 1 at e) and add 1/10 (v / v) to HRP solution for 2 hours at room temperature Stirred. Monoclonal antibody was prepared by dialysis into a 50 mM sodium phosphate (pH 7.2) complete layer solution, and 14 mM Traut's reagent (2-Iminothiolane-HCl) was added 46 ul to 1.0 mL of the antibody solution, followed by stirring for 2 hours. SMCC and Traut's reagent that did not participate in the reaction were removed by a sephadex G25 desalting column, and then HRP and antibody solutions were mixed and stirred for another 2 hours. HRP conjugated antibody was separated by stirring for 30 minutes by adding the same amount of saturated ammonium sulfate solution (pH 7.4), centrifugation, and then dialyzed in phosphate buffered saline (pH 7.2).
(2)ELISA분석 (2) ELISA analysis
최적 항 -VanA 단클론 항체쌍올 선택하고자， 일반적인 ELISA 방법 (conventional ELISA) 및 샌드위치 ELISA 방법으로 항체 성능을 평가하였으며， 그 결과 최적 항 -VanA 항체쌍으로서 포획항체 1B7또는 1H9, 검출항체 3G11이 선택되었다 (도 2a 내지 도 2c).  To select the optimal anti-VanA monoclonal antibody pair, antibody performance was evaluated by conventional ELISA method and sandwich ELISA method. As a result, capture antibody 1B7 or 1H9 and detection antibody 3G11 were selected as the optimal anti-VanA antibody pair ( 2A-2C).
일반적인 ELISA (Conventional ELISA)  Conventional ELISA
Microplate (Nunc, Denmark)에 재조합 단백질 VanA 또는 VanB 수용액 1 ug/mL (phosphate buffered saline, pH 7.4) 100 ul 을 넣고 37°C에서 1 시간 방치하여 단백질을 코팅하였다. 이후 2% 탈지 분유 수용액을 200 ul 첨가하여 blocking 한 뒤， 일차 항체 (하이브리도마 배양액 또는 단클론 항체)를 첨가하여 1 시간 동안 37°C 에서 반응하였다. Phosphate buffer saline 으로 2 회 수세한 뒤 HRP 접합 Goat ant i -Mouse IgG (Dinona, Iksan, Korea)을 첨가하여 30 분 동안 추가 반웅을 실시하였다. TMB 기질 용액을 첨가하여 색깔 반응을 유도하고， 1.0 N H2S04 을 첨가하여 반응을 종료하였다. 이후 600 nm 파장을 참조하여 450 nra 파장에서 반웅성을 평가하였다. 그 결과를 도 2a에 그래프로 나타냈다. Into a microplate (Nunc, Denmark) 100 ul of recombinant protein VanA or VanB aqueous solution 1 ug / mL (phosphate buffered saline, pH 7.4) and left at 37 ° C for 1 hour to coat the protein. After blocking by adding 200 ul of 2% skim milk powder aqueous solution, a primary antibody (hybridoma culture solution or monoclonal antibody) was added and reacted at 37 ° C. for 1 hour. After washing twice with Phosphate buffer saline, HRP conjugated Goat ant i-Mouse IgG (Dinona, Iksan, Korea) was added and additional reaction was performed for 30 minutes. TMB substrate solution was added to induce a color reaction, and 1.0 NH 2 SO 4 was added to terminate the reaction. The reaction was then evaluated at 450 nra wavelength with reference to 600 nm wavelength. The results are shown graphically in FIG. 2A.
Sandwich ELISA  Sandwich ELISA
Microplate (Nunc, Denmark)을 항 VanA 또는 VanB 항체 (1.0 ~ 2.5 ug/mU로 코팅하고， 탈지 분유 수용액을 사용하여 blocking을 실시하였다. 항원 (재조합 단백질 VanA/B 또는 VRE lysate)을 각각의 well 에 첨가하여 37°C에서 1 시간 반웅하고 2 회 수세하였다. HRP 가 부착된 검출항체 (검출 antibody)를 첨가하여 37°C에서 1 시간 반웅하고 2 회 수세하였다. 이후 TMB 기질 용액을 첨가하여 색깔 반웅을 유도하고， 1.0 N ¾S04 을 첨가하여 반웅을 종료하였다. Microplate (Nunc, Denmark) was coated with anti VanA or VanB antibody (1.0-2.5 ug / mU) and blocked using an aqueous solution of skim milk powder. Antigen (recombinant protein VanA / B or VRE lysate) was applied to each well. The reaction was repeated for 1 hour and washed twice at 37 ° C. The detection antibody (detection antibody) with HRP was added and the reaction was repeated for 1 hour and washed twice at 37 ° C. Then, the reaction was performed by adding TMB substrate solution. The reaction was terminated by addition of 1.0 N ¾SO 4 .
정량곡선을 위해 재조합 단백질 (VanA 또는 VanB)을 2 배씩 계열회석한 6 개의 다른 농도 시료를 표준항원으로 사용하였다. 표준항원 희석 완충액은 VRE 용해 완층액으로 사용하였고, 표준항원 농도는 BCA assay 를 통하여 계산하였다. VanA ELISA 에서 표준항원 희석 완층액은 Popculture reagent (Novagen, WI， USA)에 1.5 M urea, 3% Tween 20 이 첨가된 VanA type VRE 용해 완층액을 사용하였고, VanB ELISA 에서 표준항원 희석 완층액은 0.8 M urea, 3% Tween 20， 0.2% Polyvinyl alcohol, 0.5% skim milk, 10 mM phosphate buffer (pH 7.4)를 사용하였다. 그 결과를 도 2b에 그래프로 나타냈다.  For the quantitative curves, six different concentration samples of 2-fold series of recombinant protein (VanA or VanB) were used as standard antigens. Standard antigen dilution buffer was used as VRE lysis complete solution, and standard antigen concentration was calculated by BCA assay. In the VanA ELISA, the standard antigen dilution buffer was used as a VanA type VRE lysis buffer containing 1.5 M urea and 3% Tween 20 in Popculture reagent (Novagen, WI, USA) .The standard antigen dilution buffer was 0.8 in VanB ELISA. Murea, 3% Tween 20, 0.2% Polyvinyl alcohol, 0.5% skim milk, 10 mM phosphate buffer (pH 7.4) was used. The results are shown graphically in FIG. 2B.
도 2a 내지 도 2c 는 최적 항 -VanA 항체쌍을 선택 결과를 나타내는 도면으로서， 도 2a 는 일반적인 ELISA 방법 (conventional ELISA)으로 항체 성능을 평가한 검출 결과이고， 도 2b 는 샌드위치 ELISA 방법으로 포획 항체 성능을 평가한 검출 결과이고， 도 2c 는 VanA 에 대한 최적 항체쌍으로서 1H9/3G11 또는 1B7/3G11 (포획 /검출)을 나타내는 그래프이다.  2A to 2C are diagrams showing the results of selecting an optimal anti-VanA antibody pair, FIG. 2A is a detection result of evaluating antibody performance by a conventional ELISA method, and FIG. 2B is a capture antibody performance by a sandwich ELISA method. 2C is a graph showing 1H9 / 3G11 or 1B7 / 3G11 (capture / detection) as an optimal antibody pair against VanA.
2-4： 항 -VanB단클론항체의 선별 사용한 클론이 상이한 것을 제외하고는 실질적으로 상기 2-3 과 동일한 방법으로 항체 성능을 평가하였고， 그 결과 항 -VanB 단클론 항체로는 포획항체 7E8 및 검출항체 14B2 가 선정되었다 (도 3a 내지 도 3c) · 2-5： 단클론 항체의 정제 2-4 ： Screening of anti-VanB monoclonal antibody Antibody performance was evaluated in the same manner as in the above 2-3 except that the clones used were different, and as a result, capture antibody 7E8 and detection antibody 14B2 were selected as anti-VanB monoclonal antibodies (FIGS. 3A to 3C). 2-5: Purification of monoclonal antibodies
항체 정제를 위해서, 하이브리도마 배양액은 Cell-bag (Xcellerex, Marlborough, MA, USA)에서 2 주간 배양하여 획득하고， 0.22 urn filter 로 여과하였다. 준비된 시료는 Hitrap Protein G 5 mL column (GE healthcare, Little Chalfont, UK)에 주입하고， 20 mM sodium phosphate (pH 7.2) 용액을 10 column volume (50 mL) 홀려주어 불순물을 제거한 뒤 0.1 M glycine-HCl (pH 3.0) 5 column volume 으로 용출하여 정제하였다. 정제된 항체는 phosphate buffered saline (pH 7.4)으로 투석한 뒤 다양한 목적으로 사용하였다. 실시예 3: VanA및 VanB ELISA구현  For antibody purification, hybridoma cultures were obtained by culturing for 2 weeks in Cell-bag (Xcellerex, Marlborough, MA, USA) and filtered with a 0.22 urn filter. Prepared samples are injected into Hitrap Protein G 5 mL column (GE healthcare, Little Chalfont, UK), and 20 mM sodium phosphate (pH 7.2) solution is poured into 10 column volume (50 mL) to remove impurities and 0.1 M glycine-HCl (pH 3.0) eluted with 5 column volumes and purified. The purified antibody was dialyzed with phosphate buffered saline (pH 7.4) and used for various purposes. Example 3: VanA and VanB ELISA Implementation
ELISA 구현에 적절한 항체 사용농도를 확인하기 위하여 signal-to- noise 비율을 확인하였다. 포획 항체 1H9 을 10 ug/mL 부터 계열 희석하여 다양한 농도로 코팅하고， 검출 항체 3G11또한 계열 회석하여 다양한 농도로 구현하여 반응성을 확인하였다. Signal 은 50 ng/mL VanA 재조합 단백질, noise 는 희석 액에 대한 반응성으로 각각 평가하였다. VanA 반웅성 결과는 signal/noise 비율 및 3차원 모식도로 표현하였다 (표 4 및 도 4).  The signal-to-noise ratio was checked to confirm the antibody concentration appropriate for the ELISA implementation. The capture antibody 1H9 was serially diluted from 10 ug / mL and coated at various concentrations, and the detection antibody 3G11 was also sequenced and implemented at various concentrations to confirm reactivity. Signal was assessed by 50 ng / mL VanA recombinant protein and noise by reactivity with diluent. VanA semisexual results are expressed in signal / noise ratio and three-dimensional schematic (Table 4 and FIG. 4).
[표 4]  TABLE 4
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000023_0001
Figure imgf000024_0001
X-축은 포획 항체의 농도를 나타내고， Y-축은 검출 항체의 희석인자 (dilution factor)을 나타내며， 신호는 50 ng/mL VanA 로 측정하였고， 노이즈는 희석 버퍼로 결정하였다. 최적 포획 항체의 농도는 signal/noise 비율이 높게 구현되는 2.5 ug/mL 로 선정하였고， 검출 항체의 최적 희석 배수 범위는 800 에서 6,400 범위를 선정하였다. 검출항체 회석 배수는 VRE 용해물 (lysate)를 항원으로 사용하여 재평가하였다. The X-axis represents the concentration of capture antibody, the Y-axis represents the dilution factor of the detection antibody, the signal was measured with 50 ng / mL VanA, and the noise was determined with the dilution buffer. The optimal capture antibody concentration was selected to be 2.5 ug / mL with high signal / noise ratio, and the optimal dilution factor range of the detection antibody was selected from 800 to 6,400. Detected antibody dilution was reevaluated using VRE lysate as antigen.
VanB 의 경우 포획 항체 농도를 2.5 ug/mL 로 고정하여 각각 signal/noise 비율을 계산하였다. 7E8을 포획항체으로， 7A12을 검출항체로 사용한 경우 변별력 있는 signal/noise 비율이 관찰되었다. 도 5 는 VanB 항체쌍의 노이즈에 대한 시그널 비율 (signal-to-noise ratio)(signal/noise 비율)을 보여주는 도면으로서， 항체쌍 7E8/7A12 (포획 /검출)은 높은 S/N 비율을 나타냈으나， 포획 항체의 최적 농도는 항체쌍 7E8/7A12 (포획 /검출)를 사용하여 2.5 ug/mL 로 결정하였으며， 검출항체의 최적 회석 범위는 800 내지 12，800으로 이었다. (도 5)  In the case of VanB, the capture / concentration ratio was fixed at 2.5 ug / mL to calculate the signal / noise ratio, respectively. When 7E8 was used as a capture antibody and 7A12 as a detection antibody, discriminative signal / noise ratios were observed. FIG. 5 shows the signal-to-noise ratio (signal / noise ratio) against noise of the VanB antibody pair, wherein antibody pair 7E8 / 7A12 (capture / detection) showed a high S / N ratio. However, the optimal concentration of capture antibody was determined to be 2.5 ug / mL using antibody pair 7E8 / 7A12 (capture / detection), and the optimal dilution range of the detected antibody was 800-12,800. (Figure 5)
VanA EL ISA 는 재조합 단백질 VanA 500 ng/mL 을 2 배씩 계열희석하여 6 종의 표준용액으로 구현하였다 (도 6). 성능 검증을 위하여 별도의 QC 시료 3 종을 준비하였고 구현된 샌드위치 ELISA 로 정량하여 표 5에 기록하였다 (표 5). VanA ELISA의 정밀도는 각기 다른 QC 시료에서 2.2， 2.2， 1.3 CV 로 우수하게 평가되었으며， 선형성은 정량값과 이론값을 별도의 선형 회귀함수로 표현하여 평가하였다. 선형성은 y=0.8977x, R2=0.9999로 우수하였다. VanA EL ISA was implemented in six standard solutions by serial dilution of 500 ng / mL of the recombinant protein VanA (Fig. 6). Three separate QC samples were prepared for performance verification and quantified by the implemented sandwich ELISA and reported in Table 5 (Table 5). The accuracy of VanA ELISA was excellent in 2.2, 2.2 and 1.3 CV in different QC samples, and linearity was evaluated by expressing quantitative and theoretical values as separate linear regression functions. Linearity was excellent with y = 0.8977x, R 2 = 0.9999.
[표 5]  TABLE 5
Figure imgf000024_0002
Figure imgf000025_0001
Figure imgf000024_0002
Figure imgf000025_0001
VanB 샌드위치 ELISA 는 7.8 - 250 ng/mL 범위의 검출 구간으로 구현하였고 (도 7)， 3종의 QC 시료로 선형성을 검증하였다. VanB ELISA또한 y=0.9213x, R2=0.9946 의 우수한 선형성으로 계산되었다. 성능 검증을 위하여 별도의 QC 시료 3 종을 준비하였고 구현된 샌드위치 ELISA 로 정량하여 표 6에 기록하였다 (표 6). VanB sandwich ELISA was implemented with a detection interval ranging from 7.8-250 ng / mL (FIG. 7), and linearity was verified with three QC samples. VanB ELISA was also calculated with good linearity of y = 0.9213x, R 2 = 0.9946. Three separate QC samples were prepared for performance verification and quantified by the implemented sandwich ELISA and reported in Table 6 (Table 6).
[표 6]  TABLE 6
Figure imgf000025_0002
상기 포획 항체 7E8 를 마이크로티터 (microtiter) 플레이트에 2.5 ug/mL 농도로 코팅하고， 500 배로 희석한 검출 항체 7A12 를 적용하였다. 재조합 VanB 단백질을 표준 및 QC 시료로 사용하였다. 검출범위는 7.8 내지 250 ng/mL 이다. 표준 곡선은 2 차 방정식 (quadratic model fit)으로 표현하였다.
Figure imgf000025_0002
The capture antibody 7E8 was coated on a microtiter plate at a concentration of 2.5 ug / mL, and a detection antibody 7A12 diluted 500-fold was applied. Recombinant VanB protein was used as standard and QC samples. The detection range is 7.8 to 250 ng / mL. The standard curve is represented by a quadratic model fit.
y=A+Bx + Cx2 y = A + Bx + Cx 2
A :0.0995， B: 0.0106， C： -0.104e-05 , R2=0.999 A: 0.0995, B: 0.0106, C: -0.104e-05, R 2 = 0.999
결정농도와 이론 농도와의 상관관계로 선형성 (Linearity)을 결정하였다. 선형성은 y=0.9213x 및 R2=0.9946로 나타냈다. 실시예 4: VanA및 VanB면역크로마토그래피 키트 평가 Linearity was determined by the correlation between crystal concentration and theoretical concentration. Linearity is represented by y = 0.9213x and R 2 = 0.9946. Example 4 VanA and VanB Immunochromatography Kit Evaluation
4-1： 면역크로마토그래피 (ICA) 스트립 제조  4-1 ： Immunochromatography (ICA) Strip Preparation
VanA 및 VanB 면역크로마토그래피 키트는 재료 및 방법에 기술한 바와 같이 VanA 면역크로마토그래피 키트는 1H9/3G11 (포획 /검출)， VanB 면역크로마토그래피 키트는 7A12/7E8 (포획 /검출) 단클론 항체 조합으로 구현하였다. 포획 항체는 membrane 에 코팅하고 검출 항체는 골드 접합을 수행하여 건조 후 디바이스에 조립하였다 골드 접합항체를 제조하기 위하여， 콜로이드 골드에 (Dinona, Iksan, Korea)에 0.5 M sodium bicarbonate 용액을 첨가하여 pH 7.4 로 적정하였다. 항체는 2 mM borax (pH 7.4)에 0.1 mg/mL 농도로 준비하였다. 콜로이드 골드 용액 50 mL 에 준비된 항체 용액 1.25 mL 을 한 방울씩 첨가하여 30 분간 상온에서 교반하였다. 이후 10% BSA, 2 mM borax (pH 7.6) 5 mL 을 한 방울씩 첨가하여 추가적으로 1 시간 동안 교반하였다. 골드 접합 항체는 원심분리 (8000 rpm, 1 시간)하여 침전을 유도한 뒤 2% BSA in 2 mM borax (pH 7.4) 5 mL을 첨가하고 현탁하였다. As described in Materials and Methods, VanA and VanB Immunochromatography Kits are constructed with 1A9 / 3G11 (capture / detection) and VanB Immunochromatography Kit with 7A12 / 7E8 (capture / detection) monoclonal antibody combinations. It was. The capture antibody was coated on the membrane and the detection antibody was subjected to gold conjugation to dry and assembled into the device. To prepare a gold conjugated antibody, 0.5 M sodium bicarbonate solution was added to colloidal gold (Dinona, Iksan, Korea) and titrated to pH 7.4. Antibodies were prepared at a concentration of 0.1 mg / mL in 2 mM borax (pH 7.4). 1.25 mL of the prepared antibody solution was added dropwise to 50 mL of the colloidal gold solution, followed by stirring at room temperature for 30 minutes. Then 5 mL of 10% BSA, 2 mM borax (pH 7.6) was added dropwise and stirred for an additional 1 hour. Gold conjugated antibodies were centrifuged (8000 rpm, 1 hour) to induce precipitation and then suspended and added 5 mL of 2% BSA in 2 mM borax (pH 7.4).
플라스틱 패드에 니트로셀를로오스막 (HiflowPlus, millipore, MC, USA)m을 붙여 사용하였다. VanA ICA를 위한 포획 항체는 단클론 항체 1H9 1.0 mg/mL 을 사용하였다. 항체용액을 BioDot machine (BioDot, California, USA)을 사용하여 니트로샐를로오스막 표면에 일직선으로 뿌려주고 건조하였다. 대조선 (control line)은 goat ant i -mouse IgG (Dinona, Iksan, Korea) 0.3 mg/mL 용액을 각각 6 cm/sec 속도로 멤브레인에 선을 그어 각각 탐지선 (detection site)과 대조선을 형성하였다. VanB ICA 를 위한 포획 항체는 7A12 단클론 항체 2.0 mg/mL 용액을 사용하였다. 완성된 니트로셀를로오스막 상단에는 흡수 패드를 부착하여 유량의 흐름이 발생하도록 설계하였고， 5 mm 간격으로 잘라 스트립을 완성하였다. 완성된 스트립은 골드 접합항체 용액이 담겨진 마이크로폴레이트 (microplate)에 하나씩 꽂아 dip-stick 형태로 사용하였다 (도 8)  Nitrocell was attached to a plastic pad with a rose film (HiflowPlus, millipore, MC, USA). The capture antibody for VanA ICA used monoclonal antibody 1H9 1.0 mg / mL. The antibody solution was sprayed in a straight line on the surface of the nitrous membrane using a BioDot machine (BioDot, California, USA) and dried. The control line was a 0.3 mg / mL solution of goat ant i-mouse IgG (Dinona, Iksan, Korea) at 6 cm / sec to each membrane to form a control line and a detection site, respectively. The capture antibody for VanB ICA used a 2.0 mg / mL solution of 7A12 monoclonal antibody. An absorbent pad was attached to the top of the finished nitrocell film to design a flow rate, and the strip was cut at intervals of 5 mm. The completed strips were used in dip-stick form by inserting one by one into a microplate containing a gold conjugated antibody solution (FIG. 8).
4-2： cell Lysate준비 4-2 ： Cell Lysate Preparation
VanA 유형 VRE (1141, 1142, 1506)은 충북대학교 병원 (Chung-Buk National University Hospital, CBNUH)에서 분리되었으며， VanB 유형의 VRE (V583, NJ3)는 ATCC (Virginia, USA)에서 분양 받았다. 임상에서 분리된 VanA유형 VRE는 腿 4 ligase 유전자 증폭으로 유전형을 확인하였다.  VanA type VREs (1141, 1142, 1506) were isolated from Chung-Buk National University Hospital (CBNUH) and VanB type VREs (V583, NJ3) were sold by ATCC (Virginia, USA). VanA type VRE isolated from the clinic was genotyped by 腿 4 ligase gene amplification.
[표 7] 반코마이신 내성 장구균 특성
Figure imgf000026_0001
Figure imgf000027_0001
TABLE 7 Vancomycin Resistant Enterococci Properties
Figure imgf000026_0001
Figure imgf000027_0001
VRE 균종은 고체배지에서 배양한 균락 (colony)을 0 내지 8 ug/mL 반코마이신이 첨가된 BHI (BD bioscience, MD, USA) 배지에서 하루 밤 동안 배양하였다. 배양된 1.0 mL 배양액을 원심분리하여 침전하였고， ICA 또는 ELISA분석을 위한 시료로 사용하였다. VRE strains were cultured in solid medium for one night in BHI (BD bioscience, MD, USA) medium to which 0-8 ug / mL vancomycin was added. The cultured 1.0 mL culture was precipitated by centrifugation and used as a sample for ICA or ELISA analysis.
VanA ICA 또는 ELISA 분석의 경우， VRE 침전물을 8.0 M urea, 20 mM sodium phosphate buffer (pH 7.4) 30 ul로 현탁하여 10 분 동안 방치하고 3% Tween 20 in Popculture reagent 120 를 첨가하여 준비하였다.  For VanA ICA or ELISA assays, the VRE precipitate was prepared by suspending with 30 μl of 8.0 M urea, 20 mM sodium phosphate buffer (pH 7.4) for 10 minutes and adding 3% Tween 20 in Popculture reagent 120.
반면 VanB ICA 또는 ELISA 분석의 경우， VRE 침전물을 4.0 M urea, 20 mM phosphate buffer (pH 7.4) 30 ul 에 현탁하여 10 분 방치하고， 3% Tween 20, 0.2% Polyvinyl alcohol , 0.5% skim mi lk, 10 mM phosphate buffer (pH 7.4)을 120 ul 양으로 첨가하여 준비하였다. 준비된 각각의 VRE lysate 는 원심분리하여 침전물을 제거하고 ICA 또는 ELISA 분석 시료로 사용하였다.  In the case of VanB ICA or ELISA analysis, the VRE precipitate was suspended in 30 mL of 4.0 M urea, 20 mM phosphate buffer (pH 7.4) for 10 minutes, 3% Tween 20, 0.2% Polyvinyl alcohol, 0.5% skim mi lk, 10 mM phosphate buffer (pH 7.4) was added in an amount of 120 ul. Each prepared VRE lysate was centrifuged to remove precipitates and used as ICA or ELISA assay samples.
4-3： 면역분석 (샌드위치 ELISA) 4-3 ： Immunoassay (Sandwich ELISA)
Microplate (Nunc, Denmark)을 항 VanA 1H9 2.5 ug/mL 또는 항 VanB 항체 7E82.5 ug/mL로 코팅하고， 탈지 분유 수용액을 사용하여 Mocking을 실시하였다. 항원 (재조합 단백질 VanA/B 또는 VRE lysate)을 각각의 well 에 첨가하여 37°C에서 1 시간 반웅하고 2 회 수세하였다. VanA 검출항체 3G11-HRP 는 800 배， VanB 검출항체 7A12-HRP 는 500 배 회석액을 제조하여 첨가하고 37°C에서 1 시간 반응한 뒤 2 회 수세하였다. 이후 TMB 기질 용액을 첨가하여 색깔 반응을 유도하고， 1.0 N H2S04 을 첨가하여 반웅을 종료하였다. Microplate (Nunc, Denmark) was coated with 2.5 ug / mL anti VanA 1H9 or 7E82.5 ug / mL anti VanB antibody, and mocking was performed using an aqueous solution of skim milk powder. Antigen (recombinant protein VanA / B or VRE lysate) was added to each well and reacted at 37 ° C for 1 hour and washed twice. VanA detection antibody 3G11-HRP was 800 times, VanB detection antibody 7A12-HRP was prepared 500 times the diluent was added and reacted for 1 hour at 37 ° C and washed twice. TMB since Substrate solution was added to induce a color reaction, and 1.0 NH 2 SO 4 was added to terminate reaction.
정량곡선을 위해 재조합 단백질 (VanA 또는 VanB)을 2 배씩 계열희석한 6 개의 다른 농도 시료를 표준항원으로 사용하였다. 표준항원 희석 완층액은 VRE 용해 완층액으로 사용하였고， 표준항원 농도는 BCA assay 를 통하여 계산하였다. VanA ELISA 에서 표준항원 회석 완충액은 Popculture reagent (Novagen, WI, USA)에 1.5 M urea, 3% Tween 20 이 첨가된 VanA type VRE 용해 완층액을 사용하였고, VanB ELISA 에서 표준항원 희석 완층액은 0.8 M urea, 3% Tween 20， 0.2% Polyvinyl alcohol 0.5% skim milk; 10 mM phosphate buffer (pH 7.4)를 사용하였다. VanA/B 시료 농도는 각각의 정량곡선 서식 (Quadratic Fit)을 이용하여 계산하였다. 항생제 내성 균주는 고체배지에서 배양한 균락 (colony)을 0 또는 4 ug/mL 반코마이신이 첨가된 BHI (BD bioscience, MD, USA) 배지에서 하루 밤 동안 배양하고, 1.0 mL 배양액을 원심분리하여 침전한 뒤 lysate 로 만들어 분석 하였다. 항생제 내성 균주 ELISA 분석의 경우， 항생제 내성 균주 침전물을 8.0 M urea, 20 mM sodium phosphate buffer (pH 7.4) 30 ul 로 현탁하여 10 분 동안 방치하고， 3% Tween 20 in Popculture reagent 120 ul를 첨가하여 준비하였다.  For the quantitative curves, six different concentration samples with 2-fold dilution of recombinant proteins (VanA or VanB) were used as standard antigens. The standard antigen diluted complete solution was used as the VRE lysis complete solution, and the standard antigen concentration was calculated by BCA assay. The standard antigen dilution buffer in VanA ELISA was a VanA type VRE lysis supernatant with 1.5 M urea, 3% Tween 20 added to Popculture reagent (Novagen, WI, USA). urea, 3% Tween 20, 0.2% Polyvinyl alcohol 0.5% skim milk; 10 mM phosphate buffer (pH 7.4) was used. VanA / B sample concentrations were calculated using each quantitative curve format (Quadratic Fit). Antibiotic resistant strains were cultured in solid medium for one night in BHI (BD bioscience, MD, USA) medium containing 0 or 4 ug / mL vancomycin and precipitated by centrifugation of 1.0 mL culture. Du lysate was made and analyzed. For antibiotic-resistant strain ELISA analysis, antibiotic-resistant strain precipitates were suspended in 8.0 M urea, 20 mM sodium phosphate buffer (pH 7.4) 30 ul, left for 10 minutes and prepared by adding 120 ul of 3% Tween 20 in Popculture reagent. It was.
반면 VanB ELISA 분석의 경우， 항생제 내성 균주 침전물을 4.0 M urea, 20 mM phosphate buffer (pH 7.4) 30 ul 에 현탁하여 10 분 방치하고， 3% Tween 20， 0.2% Polyvinyl alcohol, 0.5% skim milk, 10 mM phosphate buffer (pH 7.4)을 120 ul 양으로 첨가하여 준비하였다. 준비된 각각의 항생제 내성 균주 lysate 는 원심분리하여 침전물을 제거하고 ELISA 분석 시료로 사용하였다.  On the other hand, in the case of VanB ELISA, antibiotic-resistant strain precipitates were suspended in 4.0 M urea, 20 mM phosphate buffer (pH 7.4) 30 ul for 10 minutes, 3% Tween 20, 0.2% Polyvinyl alcohol, 0.5% skim milk, 10 mM phosphate buffer (pH 7.4) was added in an amount of 120 ul. Each prepared antibiotic resistant strain lysate was centrifuged to remove the precipitate and used as an ELISA assay sample.
표 8 은 상기 제시한 VanA 리가제 검출 샌드위치 ELISA 를 사용하여 항생제 내성 균주에서 발현되는 VanA 리가제를 검출한 결과이며， 표 9 는 VanB 리가제 검출 샌드위치 ELISA로 VanB 리가제를 정량한 결과이다.  Table 8 shows the results of detecting VanA ligase expressed in antibiotic-resistant strains using the above-described VanA ligase detection sandwich ELISA, and Table 9 shows the results of quantifying VanB ligase by VanB ligase detection sandwich ELISA.
4-4: 면역분석 (ICR) 하기 도 9 는 이중 VanA/B 면역크로마토그래피 키트 사용법을 나타내는 모식도이다. 도 10 은 본 발명의 일예에 따라 VanA VanA 면역크로마토그래피 키트의 개략도와 분석 결과를 나타내는사진.이다. VanA VRE 는 반코마이신 4 ug/mL 조건에서 VanA ligase 농도가 10 배 정도 증폭되는 현상이 관찰되었다 (도 11). 4-4: Immunoassay (ICR) 9 is a schematic diagram showing the use of the dual VanA / B immunochromatography kit. Figure 10 is a photograph showing the schematic and analysis results of the VanA VanA immunochromatography kit according to an embodiment of the present invention. VanA VRE was observed to amplify the concentration of VanA ligase by about 10 times in 4 ug / mL of vancomycin (Fig. 11).
[표 8]  TABLE 8
Figure imgf000029_0001
균종에 따라 VanA ligase 의 농도에 차이가 있었으나 가장 낮은 농도로 확인된 1142 균종에서도 VanA농도는 반코마이신 함유 조건에서 200 ng/mL을 상회하였으며， 면역크로마토그래피 키트로도 잘 검출되었다.
Figure imgf000029_0001
The concentration of VanA ligase was different according to the species, but even at the lowest concentration of 1142, VanA concentration was higher than 200 ng / mL under vancomycin-containing conditions, and was well detected by immunochromatography kit.
VanB VRE 의 경우에도 반코마이신 함유 배지에서 VanB ligase 증폭현상이 관찰되었으나, VanB ligase 농도는 VanA ligase 에 비해 1/10 이하의 매우 낮은 농도로 검출 되었다 (도 12). 그러나 면역크로마토그래피 키트 분석 결과에서 비록 진한 명암으로 대비되지는 않으나 양성 결과를 확인할 수 있었다.  In the case of VanB VRE, VanB ligase amplification was observed in vancomycin-containing medium, but the concentration of VanB ligase was found to be very low than 1/10 of VanA ligase (FIG. 12). However, the immunochromatography kit analysis showed positive results, although not dark contrast.
[표 9]
Figure imgf000029_0002
Figure imgf000030_0001
실시예 5: 증균 액체배지내 반코마이신 농도 결정 TABLE 9
Figure imgf000029_0002
Figure imgf000030_0001
Example 5: Determination of vancomycin concentration in enriched liquid medium
VanA 및 VanB ligase 모두 반코마이신에 의하여 유도되는 특성이 있으나 과량의 반코마이신을 사용하게 되면 V E 성장이 저해될 수 있다. 특히 VanB VRE 의 경우 반코마이신에 대한 저항성이 낮아 반코마이신 사용 농도를 세밀하게 확인하는 것이 필요하다. ᅳ  VanA and VanB ligase are both induced by vancomycin, but the use of excess vancomycin may inhibit V E growth. In particular, VanB VRE has low resistance to vancomycin, so it is necessary to check the concentration of vancomycin in detail. ᅳ
VanA/B ligase 를 잘 증폭하고 VRE 성장을 저해하지 않는 반코마이신 농도를 확인하기 위하여 다양한 반코마이신 농도에서 VanA/B ligase 를 정량하였다. 구체적으로, VanA ligase 의 경우 0， 2， 6， 및 8ug/mL 로 하고， VanB ligase 의 경우 0， 2， 4， 6 및 8ug/mL 로 설정하여 정량하였다. VanA 1142 균종의 경우 반코마이신 농도를 8 ug/mL 까지 증가시키면 VanA ligase 농도도 연속하여 264 ng/mL 까지 증가하였다 (도 13). 반면 VanB VRE 에서는 반코마이신 농도가 2 ug/mL 을 일단 초과하면 두드러진 VanB ligase 증폭 현상이 관찰되지 않았다. 이러한 현상은 반코마이신에 상대적으로 내성이 약한 특성을 가지는 VanB VRE 의 성장이 둔화되면서 증균배지내 균수가 감소했기 때문으로 판단된다. VanB ligase 최고 농도 (10.97 ng/mL)는 반코마이신 4 — 6 ug/mL 에서 관찰되었으므로， 증균배지내 반코마이신의 농도는 4 - 6 ug/mL 범위가 바람직하다고 판단되었다. 실시예 6: 이중 VanA/B 면역크로마토그래피 키트 6-1： 이중 VanA/B 면역크로마토그래피 키트의 검출 및 동정 성능 분석 VanA / B ligase was quantified at various vancomycin concentrations in order to amplify VanA / B ligase well and confirm the vancomycin concentration that does not inhibit VRE growth. Specifically, the VanA ligase was quantitatively set to 0, 2, 6, and 8 ug / mL, and the VanB ligase was set to 0, 2, 4, 6 and 8 ug / mL. In the case of VanA 1142 species, the vanA ligase concentration was continuously increased to 264 ng / mL when the vancomycin concentration was increased to 8 ug / mL (FIG. 13). In VanB VRE, no significant vanB ligase amplification was observed once the vancomycin concentration exceeded 2 ug / mL. This phenomenon may be attributed to the slowing of growth of VanB VRE, which has a relatively low resistance to vancomycin, as the number of bacteria in the enrichment medium decreased. Since the highest concentration of VanB ligase (10.97 ng / mL) was observed at vancomycin 4 — 6 ug / mL, the concentration of vancomycin in the enrichment medium was in the range of 4-6 ug / mL. Example 6: Dual VanA / B Immunochromatography Kits 6-1 ： Detection and Identification of Dual VanA / B Immunochromatography Kits
VRE 를 반코마이신 미함유 또는 함유 (4 ug/mL) 액체배지에서 하루 동안 배양하여 이중 VanA/B 면역크로마토그래피 키트로 분석하였다. 분석은 VanA유형 3 종 (VRE 1141, VRE 1142, VRE1506) 및 VanB유형 2 종 VRE VRE NJ3, VRE V583)를 사용하여 판독하였다.  VRE was incubated for one day in vancomycin-free or containing (4 ug / mL) liquid medium and analyzed by dual VanA / B immunochromatography kits. The assay was read using three VanA type (VRE 1141, VRE 1142, VRE1506) and two VanB type VRE VRE NJ3, VRE V583.
반코마이신 미함유 조건에서도 VanA 유형 VRE 는 정확하게 검출 및 동정 되었으나， VanB유형은 검출되지 않았다. 반면 반코마이신 4 ug/mL 을 첨가하여 ligase 를 유도한 경우 5 종의 균주 모두에서 정확한 항생제 내성 유형이 동정 되었다 (도 14).  VanA type VRE was correctly detected and identified even in the absence of vancomycin, but VanB type was not detected. On the other hand, when ligase was induced by adding 4 ug / mL of vancomycin, the correct antibiotic resistance type was identified in all 5 strains (FIG. 14).
6-2: 이중 VanA/B면역크로마토그래피 키트의 검출한계 분석 6-2: Detection Limit Analysis of Dual VanA / B Immunochromatography Kits
이중 VanA/B 면역크로마토그래피 키트는 상기 실시예 4-1 에서 제조한 면역 스트립을 VanA와 VanB각각을 병렬로 연결하여 사용하였다. 면역크로마토그래피 키트가 검출할 수 있는 최소 VRE 균수 또는 단백질 량을 확인하기 위하여 VanA 1142 균주와 VanB V583 균주를 반코마이신 함유 고체배지에 과량 접종하여 하루 동안 배양하였다. 균집락을 모두 취하여 PBS 용액으로 옮긴 ᅳ후 0D600 을 각각 확인하였다. 균주 시료는 도 15 에 기술한 만큼 분주하여 면역크로마토그래피 키트로 각각 분석하였다. The VanA / B immunochromatography kit was used by connecting the immunostrips prepared in Example 4-1 in parallel with VanA and VanB, respectively. VanA 1142 and VanB V583 strains were inoculated in the vancomycin-containing solid medium and cultured for one day in order to confirm the minimum number of VRE bacteria or protein amount detected by the immunochromatography kit. After all the colonies were removed and transferred to PBS solution, 0D 600 was confirmed. Strain samples were aliquoted as described in FIG. 15 and analyzed by immunochromatography kit, respectively.
VanA 1142 균주는 100 ul 이상의 분석량에서 검출이 가능했으며， VanB V583 은 50 ul 이상의 분석량에서 검출이 가능한 것으로 판독되었다. 검출 한계 분석량으로 판단된 시료 동량을 사용하여 VanA 또는 VanB 단백질을 정량하였고， 균수를 확인하기 위하여 균주를 계열회석하고 반코마이신 2 ug/mL 함유 고체배지에 배양하여 colony- forming unit (CFU) 을 각각 확인하였다. VanA 검출 한계는 VanA protein 115 ng/mL, 6.78 X 108 CFU 로 확인되었으며， VanB 검출 한계는 VanB protein 7.2 ng/mL, 2.94 X 108으로 각각 확인되었다. (번 역 문) 원기탁에 의한수탁 수 신: 송 형 근 대한민국 서을특별시 마포구 도화동 마포 트라팰리스 15층 1505 The VanA 1142 strain was detectable at analytical volume of 100 ul or more, and VanB V583 was detected as detectable at analyte of 50 ul or more. VanA or VanB protein was quantified using the same amount of sample determined as the limit of detection analyte, and the colony-forming unit (CFU) was isolated by sequencing the strains and culturing them in solid medium containing 2 ug / mL of vancomycin to confirm the number of bacteria. Confirmed. The detection limits of VanA were 115 ng / mL and 6.78 X 10 8 CFU, and the detection limits of VanB were 7.2 ng / mL and 2.94 X 10 8 , respectively. (Translation) Receipt by Won Deposit: Song Hyung-Geun 15F, 15F, Mapo Trapalace, Dohwa-dong, Mapo-gu, Seolle-gu, South Korea
I . 미생물의 명칭 기탁자에 의해 주어진 명칭 국제기탁기관이 부여한 수탁번호: VanA 3G11-63-11 KCLRF-BP-00277  I. Name of microorganism Name given by depositor Accession number assigned by international depositary authority: VanA 3G11-63-11 KCLRF-BP-00277
Π. 과학적 성질 및 /또는 분류학상 위치 상기 I에 표시된 미생물에 다음을 첨부하였다. Π. Scientific properties and / or taxonomic location The microorganisms indicated in I are attached below.
[ X ] 과학적 성질  [X] scientific properties
[ X ] 분류학상 위치 m.수령 및 수탁 본 국제기탁기관은 상기 Iᅳ에 표시된 미생물을 수탁받고 2012년 2월 14 수령하였다.  [X] Taxonomic location m. Receipt and deposit The International Depositary has received the microorganisms indicated in I ᅳ above and received it on February 14, 2012.
IV. 국제기탁기관 명칭: 한국세포주연구재단 IV. International Depository Name: Korea Cell Line Research Foundation
대표자 서명  Signature of Representative
주소: 대한민국 서을시 종로구 연건동 28  Address: 28, Yeongun-dong, Jongno-gu, Seolle-si, South Korea
서울대학교 의과대학내 암연구소  Cancer Research Institute, Seoul National University College of Medicine
2012년 02월 20일  February 20, 2012
(우) 110-744  (Right) 110-744
Form BP/4 ( CLRF form 17)  Form BP / 4 (CLRF form 17)
위 번역문 원본과 상위없음 (번 역 문) 원기탁에 의한수탁증 수 신: 송 형 근 대한민국 서울특별시 마포구 도화동 마포 트라팰리스 15층 1505 No translation above the original translation (Translation) Receipt of receipt by Won Deposit: 1505, 15F, Mapo Trapalace, Dohwa-dong, Mapo-gu, Seoul, Korea
I . 미생물의 명칭 기탁자에 의해 주어진 명칭 국제기탁기관이 부여한 수탁번호: VanB 7A12-12-4 KCLRF-BP-00278 I. Name of the microorganism Name given by the depositor Accession number assigned by the International Depositary: VanB 7A12-12-4 KCLRF-BP-00278
Π. 과학적 성질 및 /또는분류학상위치 상기 I에 표시된 미생물에 다음을 첨부하였다. Π. Scientific properties and / or taxonomic location The following microbes are attached to the microorganisms indicated above.
[ X ] 과학적 성질  [X] scientific properties
[ X 〕 분류학상 위치 m.수령 및 수탁 본 국제기탁기관은 상기 I .에 표시된 미생물을 수탁받고 2012년 2월 14  [X] Taxonomic location m. Receipt and entrustment The International Depositary has received the microorganisms indicated in I.
수령하였다.  Received.
IV. 국제기탁기관 명칭: 한국세포주연구재단 IV. International Depository Name: Korea Cell Line Research Foundation
대표자서명  Signature of Representative
주소: 대한민국 서을시 종로구 연건동 28 번지  Address: 28, Yeongun-dong, Jongno-gu, Seolle-si, South Korea
서울대학교 의과대학내 암연구소  Cancer Research Institute, Seoul National University College of Medicine
2012년 02월 20일  February 20, 2012
(우) 110-744  (Right) 110-744
Form BP/4 ( CLRF form 17) 위 번역문 원본과 상위없음  Form BP / 4 (CLRF form 17) No translations found above

Claims

【특허 청구범위】  [Patent Claims]
【청구항 11  [Claim 11
글리코펩티드계 항생제에 대한 내성 세균의 D-알라닌 -D-알라닌 리가제 (D-alanine— -D-alanine l igase) 단백질을 항원으로 하여 생산되며， 상기 D-알라닌 -D-알라닌 리가제를 특이 적으로 인식하는 항체 또는 이의 항원 결합 단편 .  D-alanine-D-alanine ligase (D-alanine—D-alanine ligase) protein of bacteria resistant to glycopeptide antibiotics is produced as an antigen, and the D-alanine -D-alanine ligase is specific Antibodies or antigen-binding fragments thereof that are recognized automatically.
【청구항 2]  [Claim 2]
제 1 항에 있어서， 상기 D-알라닌 -D-알라닌 리가제는 VanA 리가제 또는 VanB 리가제인 항체 또는 이의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the D-alanine-D-alanine ligase is VanA ligase or VanB ligase.
【청구항 3】  [Claim 3]
제 1 항에 있어서， 상기 항체는 단클론 또는 다클론 항체인 항체 또는 이의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a monoclonal or polyclonal antibody.
【청구항 4】  [Claim 4]
제 1 항에 있어서， 상기 항체는 VanA 리가제를 특이 적으로 인식하며， 상기 항체의 중쇄는 기탁번호 KCLRF— BP-00277를 갖는 하이브리도마에 의해 생산되는 항체의 중쇄로부터 얻어진 세 개의 CDR 을 포함하고 상기 항체의 경 쇄는 기탁번호 KCLRF-BP-00277를 갖는 하이브리도마에 의 해 생산되는 항체의 경 쇄로부터 얻어진 세 개의 CDR 을 포함하는 것인 항체 또는 이의 항원 결합 단편 .  The antibody of claim 1, wherein the antibody specifically recognizes VanA ligase and the heavy chain of the antibody comprises three CDRs obtained from the heavy chain of an antibody produced by hybridoma having accession number KCLRF—BP-00277. And the light chain of the antibody comprises three CDRs obtained from the light chain of an antibody produced by hybridoma having accession number KCLRF-BP-00277.
【청구항 5】  [Claim 5]
제 1 항에 있어서， 상기 항원 단백질은 서 열번호 1의 아미노산 서 열을 갖는 펩티드인 VanA 리가제 단백질인 항체 또는 이의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the antigenic protein is VanA ligase protein, which is a peptide having an amino acid sequence of SEQ ID NO: 1.
【청구항 6]  [Claim 6]
제 1 항에 있어서， 상기 항원 단백질은 서 열번호 2의 염기서 열에 의 해 암호화되는 VanA 리가제 단백질인 항체 또는 이의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the antigenic protein is a VanA ligase protein encoded by the nucleotide sequence of SEQ ID NO: 2.
【청구항 7】 [Claim 7]
제 1 항에 있어서， 상기 항원 단백질은 서 열번호 3의 아미노산 서 열을 갖는 펩티드인 VanB 리가제 단백질인 항체 또는 이의 항원 결합 단편 . The antibody or antigen-binding thereof according to claim 1, wherein the antigenic protein is VanB ligase protein, which is a peptide having an amino acid sequence of SEQ ID NO: 3 Short story.
【청구항 8]  [Claim 8]
제 1 항에 있어서， 상기 항원 단백질은 서 열번호 4의 염기서 열에 의해 암호화되는 VanB 리가제 단백질인 항체 또는 이 의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the antigenic protein is a VanB ligase protein encoded by the nucleotide sequence of SEQ ID NO: 4.
【청구항 9】 [Claim 9]
제 1 항에 있어서， 상기 항체는 VanB 리가제 단백질을 특이 적으로 인식하며， 상기 항체의 중쇄는 기 탁번호 KCLRF-BP-00278를 갖는 하이브리도마에 의해 생산되는 항체의 중쇄로부터 얻어진 세 개의 CDR 을 포함하고 상기 항체의 경 쇄는 기탁번호 KCLRF— BP-00278를 갖는 하이브리도마에 의해 생산되는 항체의 경 쇄로부터 얻어진 세 개의 CDR 을 포함하는 것 인 항체 또는 이의 항원 결합 단편 .  The antibody of claim 1, wherein the antibody specifically recognizes a VanB ligase protein and the heavy chain of the antibody comprises three CDRs obtained from the heavy chain of an antibody produced by a hybridoma having accession number KCLRF-BP-00278 And the light chain of the antibody comprises three CDRs obtained from the light chain of an antibody produced by a hybridoma having accession number KCLRF—BP-00278.
【청구항 10]  [Claim 10]
제 1 항에 있어서， 상기 항체는 키 메라 항체 또는 인간화 항체인 항체 또는 이의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a chimeric antibody or a humanized antibody.
【청구항 11】  [Claim 11]
제 1 항에 있어서， 상기 항체는 VanA 리가제 단백질을 특이 적으로 인식하며， 기탁번호 KCLRF-BP-00277을 갖는 하이브리도마에 의 해 생산되는 것 인 항체 또는 이의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody specifically recognizes a VanA ligase protein and is produced by a hybridoma having accession number KCLRF-BP-00277.
【청구항 12】  [Claim 12]
제 1 항에 있어서， 상기 항체는 VanB 리 가제 단백질을 특이 적으로 인식하며 , 기 탁번호 KCLRF-BP-00278을 갖는 하이브리도마에 의 해 생산되는 것인 항체 또는 이의 항원 결합 단편 .  The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody specifically recognizes a VanB ligase protein and is produced by a hybridoma having accession number KCLRF-BP-00278.
【청구항 13】  [Claim 13]
제 1항 내지 제 12항중 어느 한항에 따른， VanA 리가제 및 VanB 리가제로 이루어지는 군에서 선택된 1종 이상의 D-알라닌 -D-알라닌 리가제를 특이 적으로 인식하는 항체 또는 이의 항원결합 단편을 포함하는 글리코펩티드계 항생제에 대한 내성 세균의 검출키트 .  An antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which specifically recognizes at least one D-alanine-D-alanine ligase selected from the group consisting of VanA ligase and VanB ligase. Detection kit for bacteria resistant to glycopeptide antibiotics.
【청구항 14]  [Claim 14]
제 13 항에 있어서 , 상기 검출키트는 VanA 리 가제 또는 VanB 리가제 유형으로 내성 세균의 표현형을 구분하여 검출하는 검출키트.The method of claim 13, wherein the detection kit is VanA ligase or VanB ligase Detection kit that detects phenotypes of resistant bacteria by type.
【청구항 15】 [Claim 15]
제 13 항에 있어서， 상기 글리코펩티드계 항생제는 Vancomycin, Teicoplanin, Telavancin, Bleomycin, Ramo lanin, 및 Decaplanin로 이루어지는 군에서 선택된 1종 이상인 검출키트.  The detection kit according to claim 13, wherein the glycopeptide antibiotic is at least one selected from the group consisting of Vancomycin, Teicoplanin, Telavancin, Bleomycin, Ramo lanin, and Decaplanin.
【청구항 16】  [Claim 16]
제 13 항에 있어서ᅤ 상기 세균은 Enterococcus 속 균주 또는 Staphylococcus속 균주인 검출키트. 14. The method of claim 13 wherein the bacterium is Enterococcus sp yae or Staphylococcus spp the detection kit.
【청구항 17]  [Claim 17]
제 13 항에 있어서， 상기 검출키트는 상기 VanA 리가제 또는 VanB 리가제 단백질과 상기 항체의 면역반웅을 검출하는 표지 물질은 더욱 포함하는 검출키트.  The detection kit of claim 13, wherein the detection kit further comprises a labeling substance for detecting an immune response of the VanA ligase or VanB ligase protein and the antibody.
【청구항 18】  [Claim 18]
제 17 항에 있어서， 상기 표지 물질은 발색효소， 발색물질， 또는 형광물질인 검출키트.  18. The detection kit according to claim 17, wherein the labeling substance is a colorase, a colorant, or a fluorescent substance.
【청구항 19】  [Claim 19]
거 1 13 항에 있어서， 상기 키트는 상기 항체의 효소면역 측정키트， 블롯팅 키트， 면역침전키트， 면역 형광검사 키트， 또는 면역 스트립 키트인 검출키트.  The detection kit according to claim 1, wherein the kit is an enzyme immunoassay kit, a blotting kit, an immunoprecipitation kit, an immunofluorescence kit, or an immunostrip kit of the antibody.
【청구항 20】  [Claim 20]
제 19 항에 있어서， 상기 효소면역 측정키트는 VanA 리가제 또는 VanB 리가제 단백질을 특이적으로 인식하는 항체 또는 이의 항원결합 단편; 시료내 측정키트는 VanA 리가제 또는 VanB 리가제 단백질과 반웅을 위한 효소 표지 항체; 및 효소반응을 나타내는 발색물질을 포함하는 검출키트.  The method of claim 19, wherein the enzyme immunoassay kit comprises: an antibody or antigen-binding fragment thereof that specifically recognizes VanA ligase or VanB ligase protein; In-sample assay kits include VanA ligase or VanB ligase protein and enzyme labeled antibodies for reaction; And detection kit comprising a coloring material exhibiting an enzyme reaction.
【청구항 21】 [Claim 21]
제 19 항에 있어서， 상기 면역 스트립 키트는 지지체; 검출 항체와 표지 물질의 접합체 (conjugate)를 함유하는 컨쥬게이트 패드; 포획 항체가 고정된 반응 패드; 및 검체 전개액을 흡수하는 흡수 패드를 포함하는 단변의 폭과 장변의 길이를 가지는 분석 스트립을 하나 이상포함하며， 상기 검출 항체와 포획 항체가 제 1항 내지 제 9항중 어느 한항에 따른 항체 및 이의 항원결합 단편으로 이루어진 군에서 선택되는 것인 검출키트. 20. The kit of claim 19, wherein the immune strip kit comprises: a support; Conjugate pads containing a conjugate of a detection antibody and a labeling substance; Reaction pads to which capture antibodies are immobilized; And at least one analysis strip having a width of a short side and a length of a long side including an absorbent pad that absorbs the sample developing solution. The detection kit, wherein the detection antibody and the capture antibody is selected from the group consisting of the antibody according to any one of claims 1 to 9 and antigen-binding fragments thereof.
【청구항 22]  [Claim 22]
VanA 리가제 (ligase) 단백질을 특이적으로 인식하는 항체를 생산하는 기탁번호 KCLRF-BP-00277를 갖는 하이브리도마 세포.  A hybridoma cell with Accession No. KCLRF-BP-00277, which produces an antibody that specifically recognizes a VanA ligase protein.
【청구항 23】  [Claim 23]
VanB 리가제 단백질을 특이적으로 인식하는 항체를 생산하는 기탁번호 KCLRF-BP-00278를 갖는 하이브리도마 세포.  A hybridoma cell with Accession No. KCLRF-BP-00278, which produces an antibody that specifically recognizes a VanB ligase protein.
【청구항 24】  [Claim 24]
제 1항 내지 제 12항중 어느 한항에 따른 항체 또는 이의 단편을 이용하여， 시료에 함유된 VanA 리가제 및 VanB 리가제로 이루어지는 군에서 선택된 1종 이상의 D-알라닌 -D-알라닌 리가제를 면역분석법으로 탐지하는 단계 ; 및  Using at least one D-alanine -D-alanine ligase selected from the group consisting of VanA ligase and VanB ligase contained in a sample, using an antibody or a fragment thereof according to any one of claims 1 to 12 by immunoassay Detecting; And
상기 D-알라닌 -D-알라닌 라가제가 시료내에 존재하는 경우 글리코펩티드계 항생제에 대한 내성 세균이 시료내에 존재하는 것으로 결정하는 단계를 포함하는， 글리코펩티드계 항생제에 대한 내성 세균의 검출방법.  When the D-alanine -D-alanine ligase is present in the sample, comprising the step of determining that the bacteria resistant to the glycopeptide-based antibiotic in the sample, the detection method of bacteria resistant to glycopeptide-based antibiotics.
【청구항 25】  [Claim 25]
제 24 항에 있어서， 상기 VanA 리가제 또는 VanB 리가제 유형으로 내성 세균의 표현형을 구분하여 검출하는 검출방법 .  25. The detection method according to claim 24, wherein the vanA ligase or VanB ligase type detects and distinguishes the phenotype of resistant bacteria.
[청구항 26】  [Claim 26]
제 24 항에 있어서, 상기 시료는 글리코펩티드계 항생제가 첨가한 배지에서 접종하여 배양하고， 세포를 수집하고， VanA 리가제 및 VanB 리가제를 용출하여 준비하는 것인 검출방법.  25. The detection method according to claim 24, wherein the sample is inoculated and cultured in a medium to which glycopeptide antibiotics are added, cells are collected, and VanA ligase and VanB ligase are prepared by eluting.
【청구항 27】  [Claim 27]
제 24 항에 있어서, 상기 면역 분석법은 방사성 면역분석법， 면역효소분석법 (ELISA), 샌드위치 면역분석법 및 면역 크로마토그래피 분석법 (i讓 unographic assay)으로 이루어진 군에서 선택된 것인 검출방법.  The method of claim 24, wherein the immunoassay is selected from the group consisting of radioimmunoassay, immunoassay (ELISA), sandwich immunoassay, and immunochromatography (i 讓 unographic assay).
PCT/KR2013/001391 2012-02-21 2013-02-21 Antibody to d-alanine-d-alanine ligase and detection of antibiotic-resistant bacteria using same WO2013125879A1 (en)

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* Cited by examiner, † Cited by third party
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US5871910A (en) * 1990-10-31 1999-02-16 Institut Pasteur Probes for the detection of nucleotide sequences implicated in the expression of resistance to glycopeptides, in particular in gram-positive bacteria
US6087106A (en) * 1992-12-18 2000-07-11 Institut Pasteur Nucleic acids conferring and inducible resistance to glycopeptides particularly in gram-positive bacteria
US6054269A (en) * 1997-06-25 2000-04-25 Institut Pasteur Polynucleotides and their use for detecting enterococci and streptococci bacterial strains
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