WO2013125879A1 - Anticorps dirigé contre la d-alanine-d-alanine ligase et détection de bactéries résistantes à des antibiotiques l'utilisant - Google Patents

Anticorps dirigé contre la d-alanine-d-alanine ligase et détection de bactéries résistantes à des antibiotiques l'utilisant Download PDF

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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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antibody
ligase
vana
vanb
alanine
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PCT/KR2013/001391
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English (en)
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.

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Abstract

La présente invention concerne un anticorps dirigé contre la VanA ligase ou VanB ligase et un procédé de quantification de la VanA ligase ou de la VanB ligase, en utilisant l'anticorps, et concerne également un kit de détection et un procédé de détection de bactéries résistantes aux antibiotiques glycopeptidiques.
PCT/KR2013/001391 2012-02-21 2013-02-21 Anticorps dirigé contre la d-alanine-d-alanine ligase et détection de bactéries résistantes à des antibiotiques l'utilisant WO2013125879A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US6054269A (en) * 1997-06-25 2000-04-25 Institut Pasteur Polynucleotides and their use for detecting enterococci and streptococci bacterial strains
US6087106A (en) * 1992-12-18 2000-07-11 Institut Pasteur Nucleic acids conferring and inducible resistance to glycopeptides particularly in gram-positive bacteria
WO2010149159A1 (fr) * 2009-06-22 2010-12-29 Statens Serum Institut Procédés basés sur l'adn pour l'identification spécifique de clone de staphylococcus aureus
US20110003306A1 (en) * 2008-02-01 2011-01-06 Miacom Diagnostics Gmbh Identification of antibiotic resistance using labelled antibiotics

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20110003306A1 (en) * 2008-02-01 2011-01-06 Miacom Diagnostics Gmbh Identification of antibiotic resistance using labelled antibiotics
WO2010149159A1 (fr) * 2009-06-22 2010-12-29 Statens Serum Institut Procédés basés sur l'adn pour l'identification spécifique de clone de staphylococcus aureus

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