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WO2011055968A2 - Nécessaire de détection d'une infection bactérienne comprenant des anticorps monoclonaux inédits - Google Patents

Nécessaire de détection d'une infection bactérienne comprenant des anticorps monoclonaux inédits Download PDF

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Publication number
WO2011055968A2
WO2011055968A2 PCT/KR2010/007710 KR2010007710W WO2011055968A2 WO 2011055968 A2 WO2011055968 A2 WO 2011055968A2 KR 2010007710 W KR2010007710 W KR 2010007710W WO 2011055968 A2 WO2011055968 A2 WO 2011055968A2
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Prior art keywords
pgrp
seq
chain variable
pgn
variable region
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WO2011055968A3 (fr
Inventor
Jong-Gyun Kim
Sang-Koo Park
Byung-Hyun Choi
Jae-Sun Lee
Soo-Kwang Kim
Yoon-Seon Jang
Won-Jae Kwag
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Yuhan Corp
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Yuhan Corp
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4722—Proteoglycans, e.g. aggreccan

Definitions

  • the present invention relates to a kit for detecting bacterial infection, more specifically a kit for detecting bacterial infection comprising novel monoclonal antibodies useful for detecting bacterial peptidoglycan in a sample.
  • Drosophila melanogaster peptidoglycan (PGN) recognition protein Drosophila PGRP-SA and Drosophila PGRP-SD activate the Toll pathway (Michel, T., Reichhart, J. M., Hoffmann, J. A. & Royet, J. (2001) Nature 414, 756-759; and Bischoff, V., Vignal, C., Boneca, I. G., Michel, T., Hoffmann, J. A. & Royet, J.
  • Drosophila PGRP-LC and Drosophila PGRP-LE are receptors for the Imd pathway (Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J. A., Ferrandon, D. & Royet, J. (2002) Nature 416, 640-644; Choe, K. M., Werner, T., Stoven, S., Hultmark, D. & Anderson, K. V.
  • pro-phenoloxidase (pro-PO) activation cascade which leads to melanization of invading microbes, is another major innate immune defense mechanism in invertebrates that is triggered by peptidoglycan (PGN) and ⁇ -1,3-glucan (Cerenius, L. & Soderhall, K. (2004) Immunol Rev 198, 116-126; and Kanost, M. R., Jiang, H. & Yu, X. Q. (2004) Immunol Rev 198, 97-105).
  • the pro-PO cascade like the vertebrate complement system, is a proteolytic cascade in blood plasma. Therefore, the pro-PO system is an ideal tool for biochemical studies of PGN and ⁇ -1,3-glucan recognition and subsequent signaling under a cell-free condition.
  • the present inventors performed various studies in order to develop a kit for simple, precise, and fast detection of bacterial infection, using the soluble linearized Lys-type peptidoglycan (SLPG) and the peptidoglycan-recognition protein (PGRP) reported by the present inventors.
  • SLPG soluble linearized Lys-type peptidoglycan
  • PGRP peptidoglycan-recognition protein
  • the present inventors carried out amino acid sequencing and nucleotide sequencing for antigen-binding sites of the antibodies (i.e., heavy and light chain variable regions); and determined each complementarity determining regions therefrom.
  • the present invention provides a kit for detecting bacterial infection comprising a monoclonal antibody specifically binding to the PGRP (anti-PGRP antibody) and a monoclonal antibody specifically binding to a complex of peptidoglycan and the PGRP (anti-PGN-PGRP-complex antibody).
  • the present invention also provides monoclonal antibodies useful for the kit for detecting bacterial infection, i.e., anti-PGRP antibody and anti-PGN-PGRP-complex antibody; amino acids of heavy and light chain variable regions thereof; and nucleotides encoding the same.
  • the present invention also provides recombinant vectors comprising a cDNA encoding the heavy or light chain variable region; and transformants transformed with the recombinant vectors.
  • kits for detecting bacterial infection comprising a peptidoglycan-recognition protein of SEQ ID NO: 21 (PGRP); a monoclonal antibody specifically binding to the PGRP (anti-PGRP antibody); and a monoclonal antibody specifically binding to a complex of peptidoglycan and the PGRP (anti-PGN-PGRP-complex antibody).
  • PGRP peptidoglycan-recognition protein of SEQ ID NO: 21
  • anti-PGRP antibody anti-PGRP antibody
  • a monoclonal antibody specifically binding to a complex of peptidoglycan and the PGRP anti-PGN-PGRP-complex antibody
  • the kit for detecting bacterial infection of the present invention may comprise a peptidoglycan-recognition protein of SEQ ID NO: 21 (PGRP); an anti-PGRP antibody wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 1, 2 and 3 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 6, 7 and 8 as a complementarity determining region thereof; and an anti-PGN-PGRP-complex antibody wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 11, 12 and 13 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 16, 17 and 18 as a complementarity determining region thereof.
  • PGRP peptidoglycan-recognition protein of SEQ ID NO: 21
  • an anti-PGRP antibody wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 1, 2
  • the anti-PGRP antibody has an amino acid sequence of SEQ ID NO: 4 as a heavy chain variable region and an amino acid sequence of SEQ ID NO: 9 as a light chain variable region.
  • the anti-PGN-PGRP-complex antibody has an amino acid sequence of SEQ ID NO: 14 as a heavy chain variable region and a amino acid sequence of SEQ ID NO: 19 as a light chain variable region.
  • peptidoglycan in a sample is detected by an immunochromatographic assay or an enzyme-linked immuno-sorbent assay (ELISA).
  • ELISA enzyme-linked immuno-sorbent assay
  • peptidoglycan in a sample is detected by an immunochromatographic assay; and the kit may be in a strip form.
  • a monoclonal antibody specifically binding to a peptidoglycan-recognition protein (anti-PGRP antibody), wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 1, 2 and 3 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 6, 7 and 8 as a complementarity determining region thereof.
  • the anti-PGRP antibody may have an amino acid sequence of SEQ ID NO: 4 as a heavy chain variable region and an amino acid sequence of SEQ ID NO: 9 as a light chain variable region.
  • the heavy chain variable region may be encoded by cDNA of SEQ ID NO: 5; and the light chain variable region may be encoded by cDNA of SEQ ID NO: 10.
  • a monoclonal antibody specifically binding to a complex of peptidoglycan and a peptidoglycan-recognition protein (anti-PGN-PGRP-complex antibody), wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 11, 12 and 13 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 16, 17 and 18 as a complementarity determining region thereof.
  • the anti-PGN-PGRP-complex antibody may have an amino acid sequence of SEQ ID NO: 14 as a heavy chain variable region and an amino acid sequence of SEQ ID NO: 19 as a light chain variable region.
  • the heavy chain variable region may be encoded by cDNA of SEQ ID NO: 15; and the light chain variable region may be encoded by cDNA of SEQ ID NO: 20.
  • a recombinant vector which is selected from the group consisting of a recombinant vector pC1120-VH comprising cDNA of SEQ ID NO: 5; a recombinant vector pC1120-VL comprising cDNA of SEQ ID NO: 10; a recombinant vector pC2172-VH comprising cDNA of SEQ ID NO: 15; and a recombinant vector pC2172-VL comprising cDNA of SEQ ID NO: 20.
  • a transformant transformed with the recombinant vector which is selected from the group consisting of a transformant E. coli DH5 ⁇ /pC1120-VH having the accession number KCTC 11572BP transformed with the recombinant vector pC1120-VH; a transformant E. coli DH5 ⁇ /pC1120-VL having the accession number KCTC 11571BP transformed with the recombinant vector pC1120-VL; a transformant E.
  • the antibodies according to the present invention i.e., anti-PGRP antibody and anti-PGN-PGRP-complex antibody, can specifically detect the respective PGRP and PGN-PGRP-complex with remarkably high specificity and binding affinity.
  • the antibodies can even detect 20 pg of PGN selectively. Therefore, when the antibodies are applied to an immunochromatographic assay or an enzyme-linked immuno-sorbent assay (ELISA), it is possible to detect bacterial infection even in a blood for transfusion, in which PGN is present in very low level.
  • ELISA enzyme-linked immuno-sorbent assay
  • FIG. 1 shows an example of detection kit for immunochromatographic assay; and detection mechanism thereof.
  • the antibody G represents a colored particle-bound anti-PGRP antibody
  • the white antibody represents an anti-PGN-PGRP-complex antibody
  • the gray antibody represents a goat anti-mouse immunoglobulin antibody.
  • FIG. 2 shows an example of detection kit for enzyme-linked immuno-sorbent assay (ELISA); and detection mechanism thereof.
  • the white antibody represents an anti-PGN-PGRP-complex antibody; and the black antibody represents a colored particle-bound anti-PGRP antibody.
  • FIG. 3 shows the detection result of bacterial infection in a sample including Staphylococcus epidermidis (one of the Gram positive bacteria) (A) or Pseudomonas aeruginosa (one of the Gram negative bacteria) (B), using the detection kit according to the present invention.
  • the present invention provides monoclonal antibodies useful for detecting bacterial infection, which recognizes a component of bacterial cell wall, i.e., PGN.
  • the present inventors prepared a lot of hybridized cell lines, using SLPG (WO2008/096994 published by the present inventors) and PGRP (WO2007/081067, also published by the present inventors) and isolated various monoclonal antibodies therefrom. Through screening the monoclonal antibodies isolated, we selected about 10 cell lines providing monoclonal antibodies, which have high reactivity to PGRP and PGN-PGRP complex. Among them, we obtain a hybridoma cell line producing a monoclonal antibody with remarkably high binding affinity and specificity to PGRP or PGN-PGRP complex. Total RNAs were extracted from the hybridoma cell lines and subject to cloning cDNAs of the heavy and light chains.
  • PCR Polymerase chain reactions
  • heavy and light chain variable regions of a monoclonal antibody specifically binding to PGRP, a gene encoding the same, and a monoclonal antibody comprising the same are provided.
  • the present invention provides a heavy chain variable region of a monoclonal antibody specifically binding to the PGRP, which comprises amino acid sequences of SEQ ID NOs: 1, 2 and 3 as a complementarity determining region.
  • the heavy chain variable region may have an amino acid sequence of SEQ ID NO: 4.
  • the present invention provides a cDNA encoding the heavy chain variable region, preferably a cDNA having a nucleotide sequence of SEQ ID NO: 5.
  • the present invention provides a light chain variable region of a monoclonal antibody specifically binding to the PGRP, which has amino acid sequences of SEQ ID NOs: 6, 7 and 8 as a complementarity determining region.
  • the light chain variable region may have an amino acid sequence of SEQ ID NO: 9.
  • the present invention provides a cDNA encoding the light chain variable region, preferably a cDNA having a nucleotide sequence of SEQ ID NO: 10.
  • the present invention provides a monoclonal antibody comprising the heavy and light chain variable regions.
  • the present invention provides a monoclonal antibody specifically binding to the PGRP (anti-PGRP antibody), wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 1, 2 and 3 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 6, 7 and 8 as a complementarity determining region thereof.
  • the anti-PGRP antibody may have an amino acid sequence of SEQ ID NO: 4 as a heavy chain variable region and an amino acid sequence of SEQ ID NO: 9 as a light chain variable region, respectively.
  • heavy and light chain variable regions of a monoclonal antibody specifically binding to PGN-PGRP complex, a gene encoding the same, and a monoclonal antibody comprising the same are provided.
  • the present invention provides a heavy chain variable region of a monoclonal antibody specifically binding to the PGN-PGRP complex, which has amino acid sequences of SEQ ID NOs: 11, 12 and 13 as a complementarity determining region.
  • the heavy chain variable region may have an amino acid sequence of SEQ ID NO: 14.
  • the present invention provides a cDNA encoding the heavy chain variable region, preferably a cDNA having a nucleotide sequence of SEQ ID NO: 15.
  • the present invention provides a light chain variable region of a monoclonal antibody specifically binding to the PGN-PGRP complex, which has amino acid sequences of SEQ ID NOs: 16, 17 and 18 as a complementarity determining region.
  • the light chain variable region may have an amino acid sequence of SEQ ID NO: 19.
  • the present invention provides a cDNA encoding the light chain variable region, preferably a cDNA having a nucleotide sequence of SEQ ID NO: 20.
  • the present invention provides a monoclonal antibody comprising the heavy and light chain variable regions.
  • the present invention provides a monoclonal antibody specifically binding to the PGN-PGRP complex (anti-PGN-PGRP-complex antibody), wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 11, 12 and 13 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 16, 17 and 18 as a complementarity determining region thereof.
  • the anti-PGN-PGRP-complex antibody may have an amino acid sequence of SEQ ID NO: 14 as a heavy chain variable region and an amino acid sequence of SEQ ID NO: 19 as a light chain variable region, respectively.
  • a recombinant vector comprising a cDNA gene encoding the heavy or light chain variable region of the monoclonal antibodies specifically binding to PGN or PGN-PGRP complex.
  • the recombinant vector may be prepared by inserting each gene into a known plasmid vector, e.g., pCR2.1-TOPO (Invitrogen, USA).
  • the recombinant vector may be selected from the group consisting of a recombinant vector pC1120-VH comprising cDNA of SEQ ID NO: 5; a recombinant vector pC1120-VL comprising cDNA of SEQ ID NO: 10; a recombinant vector pC2172-VH comprising cDNA of SEQ ID NO: 15; and a recombinant vector pC2172-VL comprising cDNA of SEQ ID NO: 20.
  • a transformant may be prepared by transforming a host cell, e.g., E. coli DH5 ⁇ T1 (Invitrogen, USA), using the recombinant vector. Therefore, the present invention provides a transformant transformed with each of recombinant vectors, which may be selected from the group consisting of a transformant E. coli DH5 ⁇ /pC1120-VH having the accession number KCTC 11572BP transformed with the recombinant vector pC1120-VH; a transformant E. coli DH5 ⁇ /pC1120-VL having the accession number KCTC 11571BP transformed with the recombinant vector pC1120-VL; a transformant E.
  • a transformant E. coli DH5 ⁇ /pC1120-VH having the accession number KCTC 11572BP transformed with the recombinant vector pC1120-VH
  • the above recombinant vectors may be recovered from each of the transformants, e.g., using Wizard plus SV minipreps DNA purification system (Promega, USA).
  • the above recombinant vectors may be also recovered from each of the transformants, using other conventional methods.
  • Complementarity determining regions of the monoclonal antibodies according to the present invention i.e., the complementarity determining regions of heavy and light chains of the monoclonal antibody specifically binding to PGRP; and the complementarity determining regions of heavy and light chains of the monoclonal antibody specifically binding to PGN-PGRP complex (for example, SEQ ID NOs: 1, 2, 3, 6, 7, and 8; or SEQ ID NOs: 11, 12, 13, 16, 17, and 18) may be fused with a humanized antibody (i.e., CDR grafting).
  • the framework region other than the complementarity determining region may be modified or substituted.
  • each of the variable regions may be fused with a gene encoding a human antibody or substituted for a variable region of a human antibody, so as to obtain humanized monoclonal antibodies. And also, if necessary, the cDNAs may be used as an antibody-fragment having each variable regions.
  • the anti-PGRP antibody and anti-PGN-PGRP-complex antibody may be used as a kit for detecting bacterial infection, in combination with an immunochromatographic assay or an enzyme-linked immuno-sorbent assay (ELISA). Therefore, the present invention provides a kit for detecting bacterial infection.
  • the kit may comprise a peptidoglycan-recognition protein of SEQ ID NO: 21 (PGRP); a monoclonal antibody specifically binding to the PGRP (anti-PGRP antibody); and a monoclonal antibody specifically binding to a complex of peptidoglycan and the PGRP (anti-PGN-PGRP-complex antibody).
  • the kit for detecting bacterial infection comprises a peptidoglycan-recognition protein of SEQ ID NO: 21 (PGRP); an anti-PGRP antibody wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 1, 2 and 3 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 6, 7 and 8 as a complementarity determining region thereof; and an anti-PGN-PGRP-complex antibody wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 11, 12 and 13 as a complementarity determining region thereof, and wherein a light chain variable region comprises amino acid sequences of SEQ ID NOs: 16, 17 and 18 as a complementarity determining region thereof.
  • PGRP peptidoglycan-recognition protein of SEQ ID NO: 21
  • an anti-PGRP antibody wherein a heavy chain variable region comprises amino acid sequences of SEQ ID NOs: 1, 2 and 3 as a
  • the anti-PGRP antibody has an amino acid sequence of SEQ ID NO: 4 as a heavy chain variable region and an amino acid sequence of SEQ ID NO: 9 as a light chain variable region.
  • the anti-PGN-PGRP-complex antibody has an amino acid sequence of SEQ ID NO: 14 as a heavy chain variable region and a amino acid sequence of SEQ ID NO: 19 as a light chain variable region.
  • the kit according to the present invention may further comprise an agent for pre-treatment, in order to improve binding affinity and/or specificity.
  • the kit according to the present invention may further comprise trichloroacetic acid and/or NaOH as an agent for pre-treatment, which helps exposing effective sites of the PGN in a sample and/or improving the binding of the monoclonal antibody with PGRP or PGN in a sample.
  • the kit of the present invention may be in a form of detection kit suitable for an immunochromatographic assay.
  • the kit may be a detection kit having a strip form, which comprises (i) a separate container having the peptidoglycan-recognition protein of SEQ ID NO: 21 in a form of solution or powder; or a sensitization pad having the peptidoglycan-recognition protein of SEQ ID NO: 21, (ii) a pad having an anti-PGRP antibody bound with a colored particle (i.e., colored particulate pad), and (iii) two antibodies-fixed membrane, one of which has an anti-PGN-PGRP-complex antibody.
  • FIG. 1 An example of the detection kit having a strip form is shown in FIG. 1. With reference to FIG. 1, the detection kit of the present invention is described as follows:
  • the kit comprises
  • a pad having an anti-PGRP antibody bound with a colored particle i.e., colored particulate pad
  • a portion of the sample pad is fixed by being overlapped over one end of the colored particulate pad
  • a supporting plate fixed with the colored particulate pad, two antibodies-fixed membrane, and a absorbing pad thereon, (a) wherein a bottom of the portion that is not overlapped with the sample pad in the colored particulate pad is fixed by being overlapped over one end of the supporting plate, (b) wherein the absorbing pad is fixed by being overlapped over the other end of the supporting plate, (c) wherein the two antibodies-fixed membrane are located between the colored particulate pad and the absorbing pad and (d) wherein the antibody-fixed membrane adjacent to the sample pad has an anti-PGN-PGRP-complex antibody.
  • the sample pad receives a test sample obtained by adding the PGRP to a sample.
  • the test sample may be applied to the test sample, e.g., by dropping it on the sample pad.
  • the sample pad may be prepared with e.g., glass fiber (Millipore, USA).
  • the colored particulate pad may be prepared with any colored particles as long as they do not impede antibody-reactivity and enable distinguishing with the naked eye.
  • the colored particulate pad may be prepared with colloidal gold-conjugated anti-PGRP antibody.
  • the supporting plate may be prepared with e.g., various polyethylene resins, in combination of an adhesive for binding the membranes and pads.
  • the absorbing pad On the supporting plate, the absorbing pad is fixed at the opposite end to the end fixed with the colored particulate pad.
  • the absorbing pad which induces absorbing the test sample through capillary action, may be made of e.g., celluloses (Pall, USA).
  • the first antibody line i.e., the membrane adjacent to the sample pad
  • the second antibody line comprises e.g., goat anti-mouse-immunoglobulin antibody so as to display an ending line (or control line) for determining completion of the detection test.
  • the sample treated with PGRP gives a PGN-PGRP complex. If the test sample having the PGN-PGRP complex is loaded on the sample pad, the PGN-PGRP complex is moved through capillary action along the colored particulate pad, so as to react with the colored particle-bound monoclonal antibody fixed in the pad. The antibody-bound mixture is continuously moved along the supporting pad and then bound, in sandwich form, with the anti-PGN-PGRP-complex antibody fixed on the first membrane, so as to give a result line (or detection line). When PGN is not present in a sample, the sample treated with PGRP does not give a PGN-PGRP complex.
  • test sample not having the PGN-PGRP complex passes through the first membrane without binding the colored particle-bound monoclonal antibody, so as to give only an ending line (or control line) at the membrane having e.g., goat anti-mouse-immunoglobulin antibody.
  • the kit of the present invention may be in a form of detection kit suitable for an enzyme-linked immuno-sorbent assay (ELISA).
  • ELISA enzyme-linked immuno-sorbent assay
  • the peptidoglycan-recognition protein of SEQ ID NO: 21 may be present in a form of solution or powder, in a separate container.
  • the anti-PGN-PGRP-complex antibody may be fixed on a detection plate.
  • FIG. 2 An example of the detection kit suitable for ELISA is shown in FIG. 2. With reference to FIG. 2, the detection kit of the present invention is described as follows:
  • An anti-PGN-PGRP-complex antibody is fixed on a conventional detection plate used in ELISA.
  • the sample treated with PGRP gives a PGN-PGRP complex.
  • the test sample having the PGN-PGRP complex is loaded on the detection plate, the PGN-PGRP complex is bound with the monoclonal antibody fixed on the plate.
  • the PGN in a sample may be detected through color-generation using a colorimetric substrate-bound antibody which has a binding-site other than the fixed antibody (for example, a colorimetric substrate-bound anti-PGRP antibody).
  • a colorimetric substrate-bound anti-PGRP antibody for example, a colorimetric substrate-bound anti-PGRP antibody
  • any conventional colorimetric substrates may be used in the kit of the present invention.
  • horseradish peroxidase HRP
  • kit suitable for ELISA may be also applied to quantitative analysis of PGN in a sample, using a calibration curve for PGN.
  • the sample may be a blood for transfusion; a mammalian blood, including human blood; food, such as vegetables, meats, fruits, or the like; cooked or non-cooked foods; water, such as tap water, underground water, rain water, or the like; sterile products; or the like.
  • the sample may be any sample that is required for microorganism detection.
  • the detection kit of the present invention is suitable for detecting bacterial infection in a blood for transfusion or a mammalian blood including human blood.
  • SLPG SLPG
  • PGRP PGRP of SEQ ID NO: 21
  • PBS phosphate buffered saline
  • the mixture was then emulsified by mixing with 150 uL of complete Freund's adjuvant (Sigma F5881).
  • the resulting emulsion was intraperitoneally injected into 7 week-old Balb/c mice (first injection).
  • mice were intraperitoneally injected with an emulsion containing PGN (100 ug), PGRP (100 ug), PBS (150 uL) and incomplete Freund's adjuvant (Sigma F5506) (150 uL) (second injection).
  • PGN 100 ug
  • PGRP 100 ug
  • PBS 150 uL
  • incomplete Freund's adjuvant Sigma F5506
  • the bloods were taken from each mouse for determining antibody formation with ELISA, as follows: 1 ug/mL of PGRP (100 uL) was added to each well of 96-well plate for ELISA (Nunc 469949) and then fixed at 4 °C for 15 hours or more. 0.5% Casein in PBS (350 uL) was added to each well and then incubated at 37 °C for 1.5 hours. The bloods taken from each mice were allowed to stand at 37 °C for 20 minutes and then centrifuged to recover each supernatant, i.e., serum. The recovered serums were diluted in 1,000 or 10,000 fold with PBS.
  • the diluted serums were added to each well of the ELISA plate and then incubated at 37 °C for 2 hours.
  • Goat anti-mouse Immunoglobulin G labeled with horseradish peroxidase (HRP) (goat anti-mouse IgG-HRP, Bio-rad 170-6516) was diluted in 1,000 fold with PBS.
  • the diluted goat anti-mouse IgG-HRP (100 uL) was added to each well in the above and then incubated at 37 °C for an hour.
  • 100 uL of HRP substrate solution was added to each well and allowed for color-generation at a room temperature for 3 minutes.
  • Reactivity of each mouse was determined by measuring each optical density (OD) using ELISA reader at 405 nm.
  • OD optical density
  • a mixture of PGN (100 ug) and PGRP (100 ug) in PBS (200 uL) was administered via tail veins of the mice showing reactivity in the above determination with ELISA.
  • the mice were sacrificed and then the spleens were extracted therefrom.
  • the obtained spleen cells were fused with Sp2/O cell (ATCC CRL-1581) and 50 % polyethylene glycol 1500 (Roche 783641).
  • the fused cells i.e., hybridoma cell lines
  • HAT 0.1 mM sodium hypoxanthine, 0.4 ⁇ M aminopterin, 16 ⁇ M thymidine, Invitrogen 31062-037
  • HAT 0.1 mM sodium hypoxanthine, 0.4 ⁇ M aminopterin, 16 ⁇ M thymidine, Invitrogen 31062-037
  • Reactivity against PGN-PGRP complex was determined for the fused cells screened from the spleen cells (2.5 x 10 8 ) obtained through immunization, as follows: 1 ug/mL of PGN (100 uL) was added to each well of 96-well plate for ELISA (Nunc 469949) and then fixed at 4 °C for 15 hours or more. 0.5% Casein in PBS (350 uL) was added to each well and then incubated at 37 °C for 1.5 hours. 1 ug/mL of PGRP (100 uL) was added to each PGN-fixed well and then incubated at 37 °C for 1 hour to prepare a PGN-PGRP complex-fixed plate.
  • the culture medium 100 uL was taken and then added to each well of the above ELISA plate.
  • the ELISA plate was incubated at 37 °C for 2 hours.
  • Goat anti-mouse Immunoglobulin G labeled with horseradish peroxidase (HRP) (goat anti-mouse IgG-HRP, Bio-rad 170-6516) was diluted in 1,000 fold with PBS.
  • the diluted goat anti-mouse IgG-HRP 100 uL was added to each well in the above and then incubated at 37 °C for an hour.
  • HRP substrate solution horseradish peroxidase substrate kit, Bio-rad 172-1064
  • OD optical density
  • Each culture medium having the cell line producing antibody was centrifuged at 6,000 x g to remove the precipitates and cells; and recover the supernatant. Each supernatant was purified with a column chromatography packed with Protein A sepharose 4 resin (Amersham 17-0974-02) to obtain each antibody.
  • Example 1 The antibodies obtained in Example 1 were analyzed for binding specificity and reactivity using ELISA.
  • PGN-fixed plate 1 ug/mL of PGN (100 uL) was added to each well of 96-well plate for ELISA (Nunc 469949) and then fixed at 4 °C for 15 hours or more. 0.5% Casein in PBS (300 uL) was added to each well and then incubated at 37 °C for 1.5 hours to prepare a PGN-fixed plate.
  • PGRP-fixed plate According to the same method as in (i), 0.001 to 1 ug/mL of PGRP (100 uL) was added to each well of 96-well plate for ELISA (Nunc 469949) and then fixed at 4 °C for 15 hours or more. 0.5% Casein in PBS (275 uL) was added to each well and then incubated at 37 °C for 1.5 hours to prepare a PGRP-fixed plate.
  • PGRP was added in a concentration of 0.001 to 1 ug/mL and then incubated at 37 °C for 1 hour.
  • the 10 antibodies which had been screened and purified in Example 1 were respectively added in a concentration of 1 ug/mL (100 uL) to each well of the plate prepared in the above and then incubated at 37 °C for 2 hours.
  • 100 uL of goat anti-mouse IgG-HRP (Bio-rad 170-6516) which had been diluted in 1,000 fold was added to each well, which was then incubated at 37 °C for an hour.
  • HRP substrate solution horserad 172-1064
  • HRP substrate solution horserad 172-1064
  • the 10 antibodies which had been screened and purified in Example 1 were respectively added in a concentration of 1 ug/mL (100 uL) to each well of the plate prepared in the above and then incubated at 37 °C for 2 hours.
  • 100 uL of goat anti-mouse IgG-HRP (Bio-rad 170-6516) which had been diluted in 1,000 fold was added to each well, which was then incubated at 37 °C for an hour.
  • 100 uL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each well and allowed for color-generation at a room temperature for 3 minutes.
  • Each optical density (OD) was measured using ELISA reader at 405 nm to screen anti-PGRP monoclonal antibodies having binding affinity to the PGRP.
  • the anti-PGN-PGRP-complex antibody showing the highest specificity was named as C2-172; and the anti-PGRP antibody showing the highest specificity was named as C1-120.
  • the results are shown in Table 1.
  • anti-PGN-PGRP-complex antibody (C2-172) obtained according to the present invention did not show any reactivity in the plate containing PGRP only, but showed reactivity in the plate pre-treated with PGN and followed by adding PGRP. From the results, it can be seen that the anti-PGN-PGRP-complex antibody (C2-172) of the present invention specifically binds to the PGN-PGRP complex.
  • the anti-PGN-PGRP-complex antibody (C2-172) and the anti-PGRP antibody (C1-120) did not bind with the PGN, and the anti-PGRP antibody represented binding specificity to both of the PGRP and the PGN-PGRP complex.
  • PGN sample of Gram-positive bacteria was prepared using Staphylococcus aureus , according to the disclosures in WO2008/096994.
  • each PGN 100 uL originated from Gram positive or negative bacteria was added to each well of 96-well plate for ELISA (Nunc 469949) and then fixed at 4 °C for 15 hours or more. 0.5% Casein in PBS (275 uL) was added to each well and then incubated at 37 °C for 1.5 hours to prepare a PGN-fixed plate. 1 ug/mL of PGRP (100 uL) was added to each PGN-fixed well and then incubated at 37 °C for 1 hour.
  • Example 2 100 uL (1 ug/mL) of anti-PGRP monoclonal antibody (C1-120) purified in Example 1 was added to each well of the plate and then incubated at 37 °C for 2 hours. 100 uL of goat anti-mouse IgG-HRP (Bio-rad 170-6516) which had been diluted in 1,000 fold was added to each well, which was then incubated at 37 °C for an hour. 100 uL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each well and allowed for color-generation at a room temperature for 3 minutes. Each optical density (OD) was measured using ELISA reader at 405 nm to verify reactivity of PGRP to the PGNs. The results are shown in Table 2.
  • the anti-PGN-PGRP-complex antibody (C2-172) obtained in Example 1 was added to each well of the ELISA plate (Nunc 469949) and then fixed at 4 °C for 15 hours or more. 0.5% Casein in PBS (300 uL) was added to each well and then incubated at 37 °C for 1.5 hours. 1 ug/mL of PGRP (100 uL) was added to each PGN-fixed well and then incubated at 37 °C for 1 hour.
  • HRP-labeling for the respective antibodies was performed with Peroxidase Labeling Kit (Dojindo, Japan) according to the manufacturer's manual. 100 uL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each well and allowed color-generation at a room temperature for 3 minutes. Each optical density (OD) was measured using ELISA reader at 405 nm.
  • a bacterial extract was prepared by culturing Gram positive bacteria (i.e., Staphylococcus aureus ) in a conventional medium for 12 to 24 hours, and then extracting the PGN of bacterial cell as follows.
  • the culture medium was centrifuged at 10,000 rpm for 15 minutes to obtain a precipitate, which was then suspended in sterilized water (50 uL).
  • 40 % of trichloroacetic acid (50 uL) was added to the resulting suspension, which was heated at 90 °C for 15 minutes and then centrifuged under the same condition to discard the supernatant.
  • Colloidal gold having a size of about 40 nm was used to prepare monoclonal antibody-bound colored particulates. While stirring a solution of colloidal gold (100 mL) having about 1 of maximum optical density (O.D.), the anti-PGRP antibody obtained in Example 1 was added in a concentration of 6 ug per 1 mL to the solution, which was then reacted for 30 minutes. Casein solution was added in a concentration of 0.2 % to the reaction mixture, which was then reacted for 1 hour. The resulting solution was centrifuged at 9,000 rpm for 15 minutes to discard the supernatant.
  • colloidal gold 100 mL
  • O.D. maximum optical density
  • the precipitate was then suspended in the stabilizing solution (1 % casein, 3 % sucrose, 50 mM Tris-buffer (pH 9), 50 mM borate buffer (pH 9), 0.5 % Triton X-100, and 0.5 % Tween 20).
  • the concentration of the suspension was adjusted to have 5 of maximum O.D. in a range of 400 to 800 nm.
  • a pad was immersed in the suspension and then lyophilized to prepare a colored particulate pad.
  • An antibody-fixed membrane was prepared in order to provide a result line capable of detecting the reaction with the naked eye.
  • a solution of anti-PGN-PGRP-complex antibody obtained in Example 1 having a concentration of 2 mg/mL was dropwisely added in 0.8 uL per cm pad onto a membrane for fixation thereof.
  • the membrane had been formed on a supporting plate.
  • the supporting plate was made of PVE/PE materials.
  • the solution of the anti-PGN-PGRP-complex antibody was prepared using PBS containing 0.5 % sucrose.
  • an ending line which displays completion of reaction is prepared on the other site of the supporting plate. For preparing the ending line, 2 mg/mL of goat anti-mouse immunoglobulin antibody was used, and the conditions for dropwise addition were the same as in preparing the result line.
  • the detection kit of the present invention comprised a sample pad receiving a sample and an absorbing pad inducing and maintaining absorption of a test sample.
  • the sample pad was prepared with glass fiber (Millipore, USA) and the absorbing pad was prepared with cellulose (Pall, USA).
  • a portion of the antibody-bound colored particulate pad prepared in (1) was fixed by being overlapped over one end of the supporting plate (in which antibody-bound membranes are fixed) prepared in (2).
  • a portion of the sample pad was also fixed by being overlapped over the other portion (i.e., the portion other than the fixed portion of the colored particulate pad and the supporting plate) of the colored particulate pad.
  • the absorbing pad was fixed by being overlapped over the other end of the supporting plate.
  • a purified PGN was detected using the PGN-detection kit prepared in Example 4.
  • 90 uL of PBS containing a certain amount of the purified PGN was mixed with a solution (10 uL) containing 100 ng of PGRP to prepare a sample.
  • the sample was applied on the sample pad of the detection kit.
  • As a result of the evaluation using 40 ng of the purified PGN there was occurred the result line which was detectable with the naked eye.
  • samples were prepared using Gram positive bacteria and Gram negative bacteria as a test microorganism. Culture media of various Gram positive and Gram negative bacteria were centrifuged at 13,400 rpm for 10 minutes to discard the supernatants. 40 % trichloroacetic acid (100 uL) was added to the obtained precipitates, which were then subject to heat treatment at 90 °C for 15 minutes. The mixtures were centrifuged under the same condition to discard the supernatants.
  • Example 4 0.1N NaOH (0.1 mL) was added to each resulting precipitate, which was heated at 90 °C for 15 minutes and then neutralized with 0.1 M sodium phosphate solution (10 uL). The obtained samples were loaded on the detection kit prepared in Example 4. As a result, it was found that the detection kit prepared in Example 4 could detect the PGNs originated from both of Gram positive and negative bacteria, even at a level of 10 5 cfu/mL or less.
  • reactivity of the detection kit was measured, using a sample prepared by mixing Gram positive or negative bacteria with a concentrated platelet reagent.
  • FIG. 3 shows the results of detection for Staphylococcus epidermidis as a representative Gram positive bacterium and Pseudomonas aeruginosa as a Gram negative bacterium, respectively.
  • the contaminations by the tested microorganisms were also detected at a level of 10 5 cfu/mL or less. Therefore it was found that the detection kit prepared in Example 4 could detect PGN in the samples such as concentrated platelet reagents, as well as a purified PGN.
  • the PCR-reaction solution was prepared by mixing 2.5 ul of cDNA, 5 pmol of primer, polymerase (Ex Tag, Takara RRo1AM), 1.5 mM of MgCl 2 , 1 mM of dNTP mixture, and a PCR buffer.
  • the PCR conditions were as follows: initial denaturation of 94 °C for 5 minutes; 35 cycles of 94 °C for 1 minute, 50 °C for 1 minute, and 72 °C for 2 minutes; and followed by a final extension of 72 °C for 5 minutes.
  • the amplified PCR products were subjected to electrophoresis on 1 % agarose gel containing SYBR (Invitrogen S-33102). As a result, two amplified DNA products were identified at positions corresponding about 400 bp of each of the heavy and light chains, based on a 100 bp standard DNA (1 Kb plus ladder, Invitrogen 10787).
  • the gene fragments were recovered and purified using AxyPrep DNA gel extraction kit (Axygen AP-GX-250).
  • the purified gene fragments were subcloned into pCR2.1-TOPO vector (Invitrogen) and then the resulting vectors were introduced into E. coli DH5-T1 (Invitrogen) to obtain transformants.
  • the obtained transformants were cultured overnight in LB medium supplemented with 50 ug/ml of ampicillin. Plasmids were extracted from the transformants using Wizard plus SV minipreps DNA purification system (Promega) and then treated with restriction enzyme EcoR I (NEB) to obtain clones containing the same size of gene fragments.
  • CDR complementarity determining region
  • the CDRs of heavy chain and light chain variable regions of the anti-PGRP antibody obtained in Example 1 were shown in Table 6.
  • the amino acid sequence of the heavy chain variable region thereof; and the cDNA sequence encoding the same were SEQ ID NOs: 4 and 5, respectively.
  • the amino acid sequence of the light chain variable region; and the cDNA sequence encoding the same were SEQ ID NOs: 9 and 10, respectively.
  • the CDRs of heavy chain and light chain variable regions of anti-PGN-PGRP-complex antibody obtained in Example 1 were shown in Table 7.
  • the amino acid sequence of the heavy chain variable region thereof; and the cDNA sequence encoding the same were SEQ ID NOs: 14 and 15, respectively.
  • the amino acid sequence of the light chain variable region; and the cDNA sequence encoding the same were SEQ ID NOs: 19 and 20, respectively.
  • Tables 6 and 7 show the amino acid sequences, the nucleotide sequences and CDRs in heavy chain and light chain variable regions of the anti-PGRP antibody and the anti-PGN-PGRP-complex antibody obtained in Example 1.
  • nucleotide sequences analyzed in the above i.e. cDNA sequences of SEQ ID NOs: 5, 10, 15 and 20 were identified as a novel gene.
  • Isotypes of the anti-PGRP antibody and the anti-PGN-PGRP-complex antibody were identified with enzyme-linked immuno-sorbent assay (ELISA).
  • ELISA enzyme-linked immuno-sorbent assay
  • 1 ug/mL of each antibody solution 100 uL was added to each well coated with PGN-PGRP-complex of an ELISA plate, which was then incubated at 37 °C for 2 hours.
  • a solution of mouse MonoAb ID kit (Zymed 90-6550) (100 uL) was added to each well, which was then incubated at 37 °C for 2 hours.
  • 100 uL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each well and allowed color-generation at a room temperature for 3 minutes.
  • Each optical density (OD) was measured using ELISA reader at 405 nm.
  • OD optical density
  • the heavy chains of both of the anti-PGRP antibody and the anti-PGN-PGRP-complex antibody belong to IgG1-type and the light chains thereof belong to kappa-type.
  • Isoelectric point (pI) of the anti-PGRP antibody was in the range of 6.5 to 7.0 and pI of the anti-PGN-PGRP-complex antibody was in the range of 5.9 to 6.2.

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Abstract

La présente invention concerne un nécessaire de détection d'une infection bactérienne comprenant un anticorps monoclonal se liant de façon spécifique à une protéine de reconnaissance du peptidoglycane, ainsi qu'un anticorps monoclonal se liant de façon spécifique à un complexe de peptidoglycane et de protéine de reconnaissance du peptidoglycane. On peut détecter, au moyen de ce nécessaire, une infection bactérienne et ce, avec une grande spécificité et une grande affinité de liaison. Ces anticorps peuvent même, par exemple, détecter 20 pg de peptidoglycane de façon sélective. La présente invention concerne également des anticorps monoclonaux, c'est-à-dire un anticorps monoclonal se liant de façon spécifique à une protéine de reconnaissance du peptidoglycane et un anticorps monoclonal se liant, de façon spécifique, à un complexe de peptidoglycane et de protéine de reconnaissance du peptidoglycane, lesdits anticorps monoclonaux pouvant être utilisés dans un nécessaire de détection d'une infection bactérienne.
PCT/KR2010/007710 2009-11-05 2010-11-03 Nécessaire de détection d'une infection bactérienne comprenant des anticorps monoclonaux inédits Ceased WO2011055968A2 (fr)

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US8981061B2 (en) 2001-03-20 2015-03-17 Novo Nordisk A/S Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof
US9000127B2 (en) 2012-02-15 2015-04-07 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
WO2016070001A1 (fr) * 2014-10-31 2016-05-06 Jounce Therapeutics, Inc. Méthodes de traitement d'états pathologiques avec des anticorps qui se lient à b7-h4
US9663568B2 (en) 2012-02-15 2017-05-30 Novo Nordisk A/S Antibodies that bind peptidoglycan recognition protein 1
US10179814B2 (en) 2014-07-17 2019-01-15 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
US10189904B2 (en) 2012-02-15 2019-01-29 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
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US12239503B2 (en) 2022-08-17 2025-03-04 Union Hospital Magnetic-field-adjustable micromagnetic orthodontic accelerator
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CN120737201B (zh) * 2025-09-01 2025-11-18 北京大学口腔医学院 一种靶向pglyrp1的单克隆抗体及其制备方法和应用

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KR101016689B1 (ko) * 2007-12-28 2011-02-25 주식회사유한양행 펩티도글리칸 인식 신호 전달에 관여하는 단백질, 이를코딩하는 유전자, 및 이를 포함하는 박테리아 감염 검출용키트

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US10906965B2 (en) 2012-02-15 2021-02-02 Novo Nordisk A/S Methods of treating autoimmune disease or chronic inflammation wtih antibodies that bind peptidoglycan recognition protein 1
US9000127B2 (en) 2012-02-15 2015-04-07 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US9663568B2 (en) 2012-02-15 2017-05-30 Novo Nordisk A/S Antibodies that bind peptidoglycan recognition protein 1
US10150809B2 (en) 2012-02-15 2018-12-11 Bristol-Myers Squibb Company Antibodies that bind peptidoglycan recognition protein 1
US10906975B2 (en) 2012-02-15 2021-02-02 Novo Nordisk A/S Methods of treating autoimmune disease or chronic inflammation with antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US10189904B2 (en) 2012-02-15 2019-01-29 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US11072654B2 (en) 2014-07-17 2021-07-27 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
US10179814B2 (en) 2014-07-17 2019-01-15 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
US12116408B2 (en) 2014-07-17 2024-10-15 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
US10626176B2 (en) 2014-10-31 2020-04-21 Jounce Therapeutics, Inc. Methods of treating conditions with antibodies that bind B7-H4
WO2016070001A1 (fr) * 2014-10-31 2016-05-06 Jounce Therapeutics, Inc. Méthodes de traitement d'états pathologiques avec des anticorps qui se lient à b7-h4
US11155618B2 (en) 2018-04-02 2021-10-26 Bristol-Myers Squibb Company Anti-TREM-1 antibodies and uses thereof
US11919954B2 (en) 2018-04-02 2024-03-05 Bristol-Myers Squibb Company Anti-TREM-1 antibodies and uses thereof
US11952420B2 (en) 2018-04-02 2024-04-09 Bristol-Myers Squibb Company Nucleic acids encoding anti-TREM-1 antibodies
US12239503B2 (en) 2022-08-17 2025-03-04 Union Hospital Magnetic-field-adjustable micromagnetic orthodontic accelerator
CN115825415A (zh) * 2022-09-28 2023-03-21 珠海重链生物科技有限公司 阻断剂和体外免疫诊断产品、应用
CN120718152A (zh) * 2025-09-01 2025-09-30 北京大学口腔医学院 一种特异性结合pglyrp1的单克隆抗体及其制备方法和应用
CN120737201B (zh) * 2025-09-01 2025-11-18 北京大学口腔医学院 一种靶向pglyrp1的单克隆抗体及其制备方法和应用
CN120718152B (zh) * 2025-09-01 2025-11-18 北京大学口腔医学院 一种特异性结合pglyrp1的单克隆抗体及其制备方法和应用

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