KR101251023B1 - Kit for detecting bacterial infection comprising novel monoclonal antibodies - Google Patents

Abstract

The present invention provides a bacterial infection detection kit comprising a monoclonal antibody that specifically binds to a peptidoglycan recognition protein and a monoclonal antibody that specifically binds to a complex of a peptidoglycan and a peptidoglycan recognition protein. To provide. The kit can detect bacteria with significantly high specificity and sensitivity, and specifically detects peptidoglycans present at levels of about 20 pg / mL. The present invention also relates to a monoclonal antibody, ie, a monoclonal antibody that specifically binds to a peptidoglycan recognition protein and a complex of peptidoglycan and peptidoglycan recognition protein usefully used in the kit for detecting bacterial infection. Provided are monoclonal antibodies that specifically bind.

Bacteria, peptidoglycan, peptidoglycan recognition protein, monoclonal antibody

Description

Kit for detecting bacterial infection comprising novel monoclonal antibodies

The present invention relates to a kit for detecting bacterial infection, and more particularly, to a kit for detecting bacterial infection comprising a novel monoclonal antibody useful for detecting peptidoglycan of bacteria in a sample.

Recent genetic engineering studies show that Drosophila PGRP-SA and Drosophila PGRP-SD, proteins that recognize the peptidoglycan (PGN) of Drosophila melanogaster , activate the Toll pathway (Michel, T., Reichhart). , JM, Hoffmann, JA & Royet, J. (2001) Nature 414, 756-759 and Bischoff, V., Vignal, C., Boneca, IG, Michel, T., Hoffmann, JA & Royet, J. (2004 ) Nat Immunol 5, 1175-1180), Drosophila PGRP-LC and Drosophila It has been reported that PGRP-LE is a receptor for the Imd pathway (Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, JA, Ferrandon, D. & Royet , J. (2002) Nature 416, 640-644; Choe, KM, Werner, T., Stoven, S., Hultmark, D. & Anderson, KV (2002) Science 296, 359-362; and Takehana, A. , Katsuyama, T., Yano, T., Oshima, Y., Takada, H., Aigaki, T. & Kurata, S. (2002) Proc Natl Acad Sci USA 99, 13705-13710). Meanwhile, Drosophila Gram-negative bacteria binding protein 1 (Drosophila GNBP1) is an immune phenotype of loss-of-function mutant Drosophila Indistinguishable from PGRP-SA, this indicates that two proteins are required to activate the Toll pathway in response to infection of Gram-positive bacteria (Gobert, V., Gottar, M., Matskevich, AA, Rutschmann, S Royet, J., Belvin, M., Hoffmann, JA & Ferrandon, D. (2003) Science 302, 2126-2130; Pili-Floury, S., Leulier, F., Takahashi, K., Saigo, K ., Samain, E., Ueda, R. & Lemaitre, B. (2004) J Biol Chem 279, 12848-12853; and Wang, L., Weber, AN, Atilano, ML, Filipe, SR, Gay, NJ & Ligoxygakis, P. (2006) EMBO J 25, 5005-5014). However, the mechanism at the molecular level of the upstream portion of the Toll pathway in Gram-negative bacterial recognition is not yet clear.

Pro-phenoloxisase (pro-PO) activating cascades that induce melaninization of invading pathogens are another important inherent immune defense mechanism in invertebrates, which are PGN and β-1,3- Caused by glucan (Cerenius, L. & Soderhall, K. (2004) Immunol Rev 198, 116-126 and Kanost, MR, Jiang, H. & Yu, XQ (2004) Immunol Rev 198, 97-105). Similar to the complement system of vertebrates, the pro-PO cascade is a plasma proteolytic cascade. Therefore, the pro-PO system is a good research model for biochemical studies of PGN and β-1,3-glucan recognition and subsequent signaling in cell-free conditions.

The inventors of the Drosophila Tenebrio molitor peptidoglycan recognition protein (PGRP) has been identified that shows high sequence homology with PGRP-SA. The PGRP, named PGRP-SA, has been shown to activate the Lys-PGN-dependent pro-PO system in Tenebrio insects (WO2007 / 081067). In addition, the inventors have isolated new proteins involved in the pro-PO system through the biochemical approach using the Drosophila Toll pathway in vivo, the in vitro pro-PO system, and recombinant PGRP-SA. It has been shown that it is possible to detect bacterial infection in samples such as blood, and also to prepare a soluble linearized Lys-type peptidoglycan (SLPG) that can be used as a standard for Lys-PGN. A method has been developed (WO2008 / 096994).

The present inventors have previously reported a water-soluble linear lysine-type peptidoglycan (SLPG) and peptidoglycan recognition protein (PGRP) to detect accurately and quickly bacterial infections in a sample. Various studies were conducted to fabricate the kit. As a result, monoclonal antibodies capable of detecting PGRP and PGN-PGRP complexes with remarkably high specificity and sensitivity were isolated, and the monoclonal antibodies were immunochromatographic or enzyme-linked immnuo-. When applied to sorbent assay (ELISA), it was found that bacterial infection in samples can be detected quickly and accurately. In addition, the present inventors analyzed the amino acid sequence and the nucleotide sequence of the antigen-binding site of the monoclonal antibody, that is, heavy and light chain variable region, and analyzed the complementarity determining site from them.

Accordingly, the present invention provides a bacterial infection comprising a monoclonal antibody (anti-PGRP antibody) that specifically binds to the PGRP and a monoclonal antibody (anti-PGN-PGRP complex antibody) that specifically binds to the PGN-PGRP complex. It is an object to provide a kit for detection.

In addition, the present invention provides a monoclonal antibody useful in a kit for detecting bacterial infection, that is, an anti-PGRP antibody and an anti-PGN-PGRP complex antibody, amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody and base sequences encoding the same. It aims to do it.

Another object of the present invention is to provide a recombinant vector into which the cDNA gene encoding the variable region of the heavy or light chain of the monoclonal antibody is inserted, and a transformant transformed with the recombinant vector.

According to one aspect of the invention, the peptidoglycan recognition protein (PGRP) of SEQ ID NO: 21; Monoclonal antibodies (anti-PGRP antibodies) that specifically bind to peptidoglycan recognition proteins; And a monoclonal antibody (anti-PGN-PGRP complex antibody) that specifically binds to a complex of a peptidoglycan and a peptidoglycan recognition protein.

Preferably, the kit for detecting bacterial infection of the present invention comprises a peptidoglycan recognition protein (PGRP) of SEQ ID NO: 21; An anti-PGRP antibody wherein the complementarity determining regions of the heavy chain variable regions are the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 6, 7, and 8; And an anti-PGN-PGRP complex antibody wherein the complementarity determining regions of the heavy chain variable regions are the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 16, 17, and 18; It may include.

In one embodiment, the heavy chain variable region of the anti-PGRP antibody is the amino acid sequence of SEQ ID NO: 4, and the light chain variable region of the anti-PGRP antibody is the amino acid sequence of SEQ ID NO: 9. In another embodiment, the heavy chain variable region of the anti-PGN-PGRP complex antibody is an amino acid sequence of SEQ ID NO: 14, and the light chain variable region of the anti-PGN-PGRP complex antibody is an amino acid sequence of SEQ ID NO: 19.

In the kit for detecting bacterial infection according to the present invention, the peptidoglycan in the sample may be detected by immunochromatographic assay or enzyme-linked immuno-sorbent assay (ELISA). In one embodiment, the peptidoglycan in the sample is detected by immunochromatography, and the kit may be in the form of a strip.

According to another aspect of the invention, the complementarity determining regions of the heavy chain variable region are the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 6, 7, and 8, Monoclonal antibodies (anti-PGRP antibodies) that specifically bind to peptidoglycan recognition proteins are provided. In the anti-PGRP antibody, the heavy chain variable region may be an amino acid sequence of SEQ ID NO: 4, and the light chain variable region may be an amino acid sequence of SEQ ID NO: 9. In addition, the anti-PGRP antibody may be an amino acid sequence of the heavy chain variable region is encoded by the cDNA of SEQ ID NO: 5, the light chain variable region may be an amino acid sequence encoded by the cDNA of SEQ ID NO: 10.

According to another aspect of the invention, the complementarity determining regions of the heavy chain variable regions are the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 16, 17, and 18 There is provided a monoclonal antibody (anti-PGN-PGRP complex antibody) that specifically binds to a complex of peptidoglycan and peptidoglycan recognition protein. In the anti-PGN-PGRP complex antibody, the heavy chain variable region may be an amino acid sequence of SEQ ID NO: 14, and the light chain variable region may be an amino acid sequence of SEQ ID NO: 19. The anti-PGN-PGRP complex antibody may be an amino acid sequence in which the heavy chain variable region is encoded by cDNA of SEQ ID NO: 15, and an amino acid sequence in which the light chain variable region is encoded by cDNA of SEQ ID NO: 20.

According to another aspect of the invention, as a recombinant vector, a recombinant vector pC1120-VH into which a cDNA gene having a nucleotide sequence of SEQ ID NO: 5 is inserted; A recombinant vector pC1120-VL having a cDNA gene having a nucleotide sequence of SEQ ID NO: 10 inserted therein; A recombinant vector pC2172-VH into which a cDNA gene having the nucleotide sequence of SEQ ID NO: 15 is inserted; And a recombinant vector pC2172-VL having a cDNA gene having a nucleotide sequence of SEQ ID NO: 20 inserted therein.

According to another aspect of the present invention, a transformant transformed with the recombinant vector includes: transformant E. coli DH5α / pC1120-VH (accession number: KCTC 11572BP) transformed with recombinant vector pC1120-VH; Transformant E. coli DH5α / pC1120-VL (Accession No .: KCTC 11571BP) transformed with recombinant vector pC1120-VL; Transformant E. coli DH5α / pC2172-VH transformed with recombinant vector pC2172-VH (Accession Number: KCTC 11574BP); And transformant E. coli DH5α / pC2172-VL (Accession No .: KCTC 11573BP) transformed with recombinant vector pC2172-VL.

Monoclonal antibodies according to the present invention, ie anti-PGRP antibody and anti-PGN-PGRP complex antibody, can detect PGRP and PGN-PGRP complexes with remarkably high specificity and sensitivity, specifically about 20 pg / mL The PGN existing as can also be selectively detected. Therefore, when the monoclonal antibodies are applied to an immunochromatography assay or an enzyme-linked immnuo-sorbent assay, ELISA, blood samples, such as blood for transfusion, in which PGN is present at an extremely low concentration. In addition, bacterial infection can be detected quickly and accurately.

The present invention provides monoclonal antibodies that can be used to detect bacterial infections by selectively recognizing bacterial cell wall components, PGN.

After the inventors have prepared a number of hybridized cellines using SLPG (WO2008 / 096994) and PGRP (WO2007 / 081067) developed by the present inventors, Many monoclonal antibodies were isolated from them. The present inventors selected about 10 monoclonal antibody secreting cell lines with high reactivity to the PGRP and PGN-PGRP complexes among the isolated monoclonal antibodies, among which the sensitivity and specificity of the PGRP or PGN-PGRP complexes are particularly excellent. Hybridoma cell lines producing monoclonal antibodies were obtained. The present inventors synthesized cDNA of heavy and light chain genes by extracting RNA from the hybridoma cell line, and then subjected to a polymerase chain reaction (PCR) using the template as a template to perform heavy chain and light chain cDNA of variable regions. The genes were isolated and analyzed for amino acid and base sequences of complementarity determining regions (CDRs) that recognize antigens, ie, PGN or PGN-PGRP complexes.

Thus, in one aspect of the invention, the invention provides variable regions of the heavy and light chains of monoclonal antibodies that specifically bind to PGRP, genes encoding them, and monoclonal antibodies comprising them.

That is, the present invention provides a heavy chain variable region of a monoclonal antibody that specifically binds to PGRP having the amino acid sequences of SEQ ID NOs: 1, 2, and 3 as complementarity determining regions. Preferably, the heavy chain variable region may have an amino acid sequence of SEQ ID NO. The present invention also provides a cDNA gene encoding the heavy chain variable region, preferably a cDNA gene having a nucleotide sequence of SEQ ID NO: 5.

The present invention also provides a light chain variable region of a monoclonal antibody that specifically binds to PGRP having the amino acid sequences of SEQ ID NOs: 6, 7, and 8 as complementarity determining regions. Preferably, the light chain variable region may have an amino acid sequence of SEQ ID NO. The present invention also provides a cDNA gene encoding the light chain variable region, preferably a cDNA gene having a nucleotide sequence of SEQ ID NO: 10.

The present invention also provides a monoclonal antibody comprising the heavy and light chain variable region. Specifically, in the present invention, the complementarity determining region of the heavy chain variable region is an amino acid sequence of SEQ ID NOs: 1, 2, and 3, and the complementarity determining region of the light chain variable region is specific to PGRP, which is an amino acid sequence of SEQ ID NOs: 6, 7, and 8 A monoclonal antibody (anti-PGRP antibody) that binds to an antibody, preferably, the heavy chain variable region of the anti-PGRP antibody is an amino acid sequence of SEQ ID NO: 4, the light chain variable region of the anti-PGRP antibody is SEQ ID NO: Amino acid sequence of 9.

Further, in one aspect of the present invention, the present invention provides variable regions of heavy and light chains of monoclonal antibodies that specifically bind to PGN-PGRP complexes, genes encoding them, and monoclonal antibodies comprising them.

That is, the present invention provides a heavy chain variable region of a monoclonal antibody that specifically binds to the PGN-PGRP complex having amino acid sequences of SEQ ID NOs: 11, 12, and 13 as complementarity determining regions. Preferably, the heavy chain variable region may have an amino acid sequence of SEQ ID NO: 14. The present invention also provides a cDNA gene encoding the heavy chain variable region, preferably a cDNA gene having a nucleotide sequence of SEQ ID NO: 15.

The present invention also provides a light chain variable region of a monoclonal antibody that specifically binds to the PGN-PGRP complex having the amino acid sequences of SEQ ID NOs: 16, 17, and 18 as complementarity determining regions. Preferably, the light chain variable region may have an amino acid sequence of SEQ ID NO: 19. The present invention also provides a cDNA gene encoding the light chain variable region, preferably a cDNA gene having a nucleotide sequence of SEQ ID NO: 20.

The present invention also provides a monoclonal antibody comprising the heavy and light chain variable region. Specifically, PGN-PGRP wherein the complementarity determining region of the heavy chain variable region is the amino acid sequence of SEQ ID NOs: 11, 12, and 13, and the complementarity determining region of the light chain variable region is the amino acid sequence of SEQ ID NO: 16, 17, and 18 A monoclonal antibody (anti-PGN-PGRP complex antibody) that specifically binds to the complex, preferably, the heavy chain variable region of the anti-PGN-PGRP complex antibody is the amino acid sequence of SEQ ID NO: 14, wherein the anti- The light chain variable region of the PGRP antibody is the amino acid sequence of SEQ ID NO: 19.

In one aspect of the invention, there is provided a recombinant vector inserted with a gene encoding the variable region of the light and / or heavy chain of a monoclonal antibody that specifically recognizes the PGRP and PGN-PGRP complexes. The recombinant vector can be prepared by inserting the gene into a known plasmid vector, for example, a plasmid vector such as pCR2.1-TOPO (Invitrogen, USA). Specifically, the production vector is a recombinant vector obtained by inserting the genes into plasmid vectors of pCR2.1-TOPO (Invitrogen, USA), respectively, and a recombinant DNA cDNA gene having a nucleotide sequence of SEQ ID NO: 5 is inserted. Vector pC1120-VH; A recombinant vector pC1120-VL having a cDNA gene having a nucleotide sequence of SEQ ID NO: 10 inserted therein; A recombinant vector pC2172-VH into which a cDNA gene having the nucleotide sequence of SEQ ID NO: 15 is inserted; And a recombinant vector pC2172-VL into which a cDNA gene having a nucleotide sequence of SEQ ID NO: 20 is inserted.

The recombinant vectors can be prepared by transforming Escherichia coli, such as, for example, DH5α T1 (Invitrogen, USA). Accordingly, the present invention provides an E. coli transformant transformed with the recombinant vectors, specifically, transformant E. coli DH5α / pC1120-VH (accession number: KCTC) transformed with the recombinant vector pC1120-VH. 11572BP); Transformant E. coli DH5α / pC1120-VL (Accession No .: KCTC 11571BP) transformed with recombinant vector pC1120-VL; Transformant E. coli DH5α / pC2172-VH transformed with recombinant vector pC2172-VH (Accession Number: KCTC 11574BP); Transformant E. coli DH5α / pC2172-VL (Accession No .: KCTC 11573BP) transformed with recombinant vector pC2172-VL is provided.

Recovery of the recombinant vector from the transformant was recovered using the Wizard plus SV minipreps DNA purification system (Promega). However, the recombinant vector can be recovered using other known methods.

The complementarity determining regions of the monoclonal antibodies according to the present invention, ie, the complementarity determining regions of the heavy and light chains of monoclonal antibodies specifically recognizing PGRP; And complementarity determining regions of the heavy and light chains of monoclonal antibodies that specifically recognize the PGN-PGRP complex (eg, SEQ ID NOs: 1, 2, 3, 6, 7 and 8; or SEQ ID NOs: 11, 12, 13, 16, 17 and 18) can be used in fusion to human antibodies (CDR grafting). In addition, in the monoclonal antibody according to the present invention, a framework region other than the complementarity determining region may be modified or substituted as appropriate.

A monoclonal antibody comprising a variable region according to the present invention, ie, a monoclonal antibody comprising a variable region of a heavy chain and a light chain of a monoclonal antibody that specifically recognizes PGRP; And monoclonal antibodies comprising variable regions of heavy and light chains of monoclonal antibodies that specifically recognize the PGN-PGRP complex, the gene encoding the variable regions (eg, SEQ ID NOs: 5 and 10; or SEQ ID NO: Each monoclonal antibody can be obtained by fusing 15 and 20) into a human antibody gene or by substituting with a variable region of a human antibody. In addition, it can also be utilized in the form of an antibody fragment containing the variable region, if necessary.

The monoclonal antibody that specifically recognizes the PGRP and / or the monoclonal antibody that specifically recognizes the PGN-PGRP complex may be immunochromatographic or enzyme-linked immnuo-sorbent assay, ELISA. Can be used as a kit for detecting bacterial infection. That is, the present invention provides a kit for detecting bacterial infection, wherein the kit for detecting is a peptidoglycan recognition protein of SEQ ID NO: 21; Monoclonal antibodies (anti-PGRP antibodies) that specifically bind to peptidoglycan recognition proteins; And monoclonal antibodies (anti-PGN-PGRP complex antibodies) that specifically bind to a complex of peptidoglycan and peptidoglycan recognition protein. Preferably, the kit for detecting bacterial infection comprises a peptidoglycan recognition protein (PGRP) of SEQ ID NO: 21; An anti-PGRP antibody wherein the complementarity determining regions of the heavy chain variable regions are the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 6, 7, and 8; And an anti-PGN-PGRP complex antibody wherein the complementarity determining regions of the heavy chain variable regions are the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 16, 17, and 18; It may include. In one embodiment, the heavy chain variable region of the anti-PGRP antibody is the amino acid sequence of SEQ ID NO: 4, and the light chain variable region of the anti-PGRP antibody is the amino acid sequence of SEQ ID NO: 9. In another embodiment, the heavy chain variable region of the anti-PGN-PGRP complex antibody is an amino acid sequence of SEQ ID NO: 14, and the light chain variable region of the anti-PGN-PGRP complex antibody is an amino acid sequence of SEQ ID NO: 19. If necessary, a reagent for pretreatment of the sample may be included to improve sensitivity and specificity. For example, the detection kit of the present invention may include a reagent that facilitates exposure and / or binding of an effective binding site of peptidoglycan present in the sample by pretreating the sample with trichloroacetic acid, NaOH, or the like. have.

For example, the detection kit of the present invention may be composed of a detection kit by immunochromatography. In this case, the peptidoglycan recognition protein of SEQ ID NO: 21 of (i) may be a solution or powder in a separate container. Or it may be in the form of a sensitization pad (sensitization pad) and the like, (i) and (ii) may be produced as a detection kit in the form of a strip containing a colored particle pad and an antibody droplet membrane, respectively. Schematic diagram of the detection kit of the strip form is as shown in Figure 1, with reference to Figure 1, the detection kit of the present invention will be described as follows: a sample pad; A fine particle pad having the sample pad fixed thereto, wherein a part of the sample pad is overlapped and fixed to one end of the fine particle pad; And a support plate on which the particle pad, two kinds of antibody drop membranes, and an absorption pad are fixed, wherein an opposite surface of the particle pad in a portion where the sample pad is not overlapped is fixed to one end of the support plate, and the absorption pad is Overlaid and fixed on the other end of the support plate, the two antibody droplet membranes consist of a support plate located in the middle on the support plate. The sample pad is a portion in which a sample obtained by adding PGRP to a sample is instilled. As a material of the sample pad, glass fiber (Millipore, USA) may be used. The particulate pad is formed by binding an anti-PGRP antibody (i.e., the antibody of (i)) with the colored fine particles to one end of the support plate. The colored fine particles can be used without limitation, the colored fine particles that can visually identify the reaction results without binding the antibody to inhibit the reactivity of the antibody, for example, can be used colloidal gold. The support plate may be made of a polyethylene material including an adhesive capable of bonding the membrane and the pad, and the absorbent pad is fixed on the end of the support plate opposite to the particulate pad fixing portion. The absorption pad induces the absorption of the sample in the capillary phenomenon, it can be configured using a cellulose material (Pall, USA). Anti-PGN-PGRP complex antibody (i.e., the antibody of (ii)) is dropped and fixed in the sample pad side membrane of the two antibody droplet membranes formed on the support plate, and used to confirm whether PGN is detected. The remaining one antibody droplet membrane is configured, for example, by dropping an anti-mouse immunoglobulin antibody to indicate the end of the test or a control result line. When PGN is present in the sample, when the sample is treated with PGRP, a PGN-PGRP complex is formed, and when the sample on which the complex is formed is loaded on a sample pad, the PGN-PGRP complex is moved by a capillary phenomenon. The colored microparticles immobilized on it react with the bound monoclonal antibody. The conjugate continues to move through the stationary phase (ie, the membrane) and binds to the anti-PGN-PGRP complex antibody immobilized on the membrane in the form of a sandwich. If no PGN is present in the sample, the PGN-PGRP complex is not formed even if the sample is treated with PGRP. Therefore, when the sample is loaded into the sample pad, the pigment is not bound to the monoclonal antibody to which the particulate matter is bound and detected. Passing through the line, the colored particulate line is observed only at the end line on which the anti-mouse immunoglobulin antibody has been deposited.

In addition, the detection kit of the present invention may be configured as a detection kit by an enzyme-linked immnuo-sorbent assay (ELISA). In this case, the peptidoglycan recognition protein of SEQ ID NO: 21 of (i) may be present in a separate container in the form of a solution, powder, etc., and the anti-PGN-PGRP complex antibody [ie, the antibody of (ii) ] Can be manufactured to be fixed to the analytical plate. Schematic diagram of the detection kit of the above type is shown in Figure 2, with reference to Figure 2, describes the detection kit of the present invention as follows: Anti-PGN-PGRP complex monoclonal antibody is fixed to the assay plate The assay plate may be a conventional ELISA detection plate. When PGN is present in the sample, when the sample is treated with PGRP, a PGN-PGRP complex is formed, and when the sample with the complex is added to the plate, it binds to the monoclonal antibody immobilized on the plate. The peptidoglycan in the sample can be detected by color development using an antibody having a binding site different from the immobilized antibody (eg, an anti-PGRP antibody having a binding substrate bound thereto). However, when no PGN is present in the sample, the binding reaction of the antibody does not occur, and thus color development does not occur. The color substrate may be a color substrate commonly used for ELISA detection, for example, a color substrate such as horseradish peroxidase (HRP). In addition, the ELISA detection method can also quantitatively analyze PGN in a sample through a PGN standard calibration curve.

In the kit for detection of the present invention, the sample is blood for transfusion, blood of mammals including humans, food such as vegetables, meat, fruits, cooked or uncooked food, tap water, ground water, rain water, sterile water Products, etc., and all samples for the detection of other microorganisms. Preferably, the detection kit of the present invention may be usefully used for the detection of bacterial infection in blood for transfusion or in the blood of mammals including humans.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the invention, and the invention is not limited by the following examples.

Example 1 Preparation of Monoclonal Antibodies

(1) Immunity and Cell Fusion

100 ug of SLPG (WO2008 / 096994) as PGN; And 100 ug of PGRP (WO2007 / 081067) of SEQ ID NO: 21 to 150 uL of phosphate buffered saline (PBS), and the same amount of complete Freund's adjuvant (Sigma F5881) The mixed solution emulsified by mixing was first injected into the abdominal cavity of 7-week old Balb / c mice. Two weeks later, 150 μL of PBS mixed with 100 ug of PGN and PGRP were mixed with the same amount of incomplete Freund's adjuvant (Sigma F5506), and the mixture was emulsified intraperitoneally.

After 3 days, the blood was collected from the mouse and antibody formation was confirmed by ELISA as follows: 96-well plate for ELISA analysis (Nunc 469949) 100 μL of 1 ug / mL PGRP was added to each pore, followed by 15 at 4 ° C. It was fixed by reacting for over time. Thereafter, 350 μL of 0.5% casein-PBS buffer was added to each of the pores, followed by reaction at 37 ° C. for 1 hour and 30 minutes. The blood collected from the mouse was allowed to stand at 37 ° C. for 20 minutes, and then centrifuged to collect a supernatant, or serum, to prepare a sample diluted 1,000-fold or 10,000-fold, and added to each pore of the prepared ELISA plate for 37 hours. After reacting at ℃, 100 μL of horseradish peroxidase (HRP) labeled goat anti-mouse IgG-HRP (Bio-rad 170-6516) was diluted 1,000-fold. Each reaction was added at 37 ° C. for 1 hour. Then, 100 μL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each pore, followed by color development at room temperature for 3 minutes, followed by optical density at a wavelength of 405 nm using an ELISA meter. density, OD) was measured. Mice confirmed reactivity 10 days after the last immunization, 200 μL of PBS mixed with 100 ug each of PGN and PGRP was administered to the tail vein. Three days later, the spleen cells of the mouse were extracted and fused with Sp2 / O (ATCC CRL-1581) cells using 50% polyethylene glycol 1500 (Roche 783641).

(2) Screening of antibody-producing cell lines and purification of monoclonal antibodies

HAT (0.1 mM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine, Invitrogen 31062-037) was added to select the fused cells using a selection medium capable of selecting only cells fused with spleen cells of the mouse. Cells secreting antibodies to the PGRP and PGN-PGRP complexes were selected and isolated into monoclonal cells.

Reactivity to PGN-PGRP complexes from fusion cell groups prepared from 2.5 x 10 ⁸ splenocytes obtained through immunization were measured as follows: 96-well plate for ELISA analysis (Nunc 469949) 1 μg / mL concentration in each pore 100 μL of PGN was added and reacted at 4 ° C. for at least 15 hours to fix. Thereafter, 350 μL of 0.5% casein-PBS buffer was added to each of the pores, followed by reaction at 37 ° C. for 1 hour and 30 minutes. 100 μL of PGRP at a concentration of 1 ug / mL was added to the PGN-fixed plate, and then reacted at 37 ° C. for 1 hour to prepare a plate to which the PGN-PGRP complex was fixed. 100 μL of the culture medium of each pore identified as a colony growing in a selective medium was added to each pore of the prepared ELISA plate and reacted at 37 ° C. for 2 hours, followed by HRP-labeled goat anti-rat Immunoglobulin paper (goat anti-mouse IgG-HRP, Bio-rad 170-6516) was diluted 1,000-fold and added to 100 μL and reacted for 1 hour at 37 ℃. Then, 100 μL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each pore, followed by color development at room temperature for 3 minutes, followed by optical density at a wavelength of 405 nm using an ELISA meter. density, OD) was measured.

As a result of the above reactivity measurement, 10 kinds of monoclonal antibody-secreting cell lines showing high activity were selected. Among them, two kinds of cell lines showing remarkably high activity were separated from other cell lines, and these were anti-PGRP and anti- PGN-PGRP complex activity was shown.

Cultures of the separated antibody-secreting cell lines were centrifuged at 6,000 x g to remove cells and precipitates, and the supernatants were recovered. Antibodies were isolated and purified from the culture supernatant using a column filled with Protein A sepharose 4 (Amersham 17-0974-02) resin.

Example 2 Response Specificity Analysis of Monoclonal Antibodies

Binding specificity and reactivity to the antibody obtained in Example 1 were analyzed by ELISA.

(i) PGN fixation plate: 96-well plate (Nunc 469949) for ELISA analysis After inserting 100 μL of PGN at 1 μg / mL concentration in each pore, the reaction was fixed at 4 ° C. for at least 15 hours. Thereafter, 300 μL of 0.5% casein-PBS buffer was added to each of the pores, followed by reaction at 37 ° C. for 1 hour and 30 minutes to prepare a plate fixed with PGN.

(ii) PGRP Fixing Plate: In the same manner as in (i) above, 100 uL of PGRP diluted to a concentration of 0.001 to 1 ug / mL was added to a 96-well plate, and then reacted and fixed at 4 ° C. for 15 hours or more. Thereafter, 275 μL of 0.5% casein-PBS buffer was added to each of the pores, followed by reaction at 37 ° C. for 1 hour and 30 minutes to prepare a plate having PGRP fixed thereto.

(iii) binding specificity measurement

PGRP was added at a concentration of 0.001 to 1 ug / mL in the PGN fixation plate treated with casein-PBS buffer in (i), followed by reaction at 37 ° C. for 1 hour. Thereafter, 10 antibodies selected and purified in Example 1 were added to each pore of the prepared plate at a concentration of 1 μg / mL, respectively, and reacted at 37 ° C. for 2 hours, followed by HRP-labeled goat anti- Rat immunoglobulin (goat anti-mouse IgG-HRP, Bio-rad 170-6516) was diluted 1,000-fold and added to each 100 μL and reacted for 1 hour at 37 ℃. Then, 100 μL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each pore, followed by color development at room temperature for 3 minutes, followed by optical density at a wavelength of 405 nm using an ELISA meter. , OD) was selected to select anti-PGN-PGRP complex monoclonal antibody having excellent binding to the PGN-PGRP complex.

In (ii), the 10 kinds of antibodies selected and purified in Example 1 above were treated to casein-PBS buffer-treated PGRP fixation plates at 100 μL each at a concentration of 1 ug / mL, respectively, for 2 hours. The goat anti-mouse immunoglobulin (goat anti-mouse IgG-HRP, Bio-rad 170-6516) labeled with HRP was diluted 1,000-fold and 100 μL was added for 1 hour at 37 ° C. Reacted. Then, 100 μL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each pore, followed by color development at room temperature for 3 minutes, followed by optical density at a wavelength of 405 nm using an ELISA meter. , OD) was selected to select anti-PGRP monoclonal antibody with excellent binding to PGRP.

The anti-PGN-PGRP complex antibody binding to the PGN-PGRP complex was named C2-172, and the anti-PGRP antibody binding to PGRP was named C1-120, and the results are shown in Table 1 below.

Sensitivity of Anti-PGN-PGRP Complex Antibody (C2-172) and Anti-PGRP Antibody (C1-120) PGRP
(ug / mL) Anti-PGN-PGRP Complex Antibody
(C2-172) Anti-PGRP antibody
(C1-120) PGN Pretreatment
(PGN fixing plate) PGN Untreated
(PGRP fixed
plate) PGN Pretreatment
(PGN lock
plate) PGN Untreated
(PGRP fixed
plate) 1,000 0.931 0.010 0.794 0.286 0.500 0.871 0.010 0.918 0.218 0.250 0.732 0.007 0.788 0.152 0.125 0.591 0.014 0.573 0.105 0.063 0.439 0.004 0.476 0.060 0.031 0.259 0.000 0.290 0.043 0.016 0.145 0.000 0.153 0.013 0.008 0.077 0.000 0.093 0.015 0.004 0.034 0.000 0.035 0.000 0.002 0.011 0.000 0.012 0.000 0.001 0.000 0.000

From the results of Table 1, the anti-PGN-PGRP complex (C2-172) antibody obtained in accordance with the present invention did not show a reactivity in the plate containing only PGRP, reactive only in the plate PGN pre-treated and PGRP added thereto Indicated. Through this, it can be seen that the anti-PGN-PGRP complex antibody (C2-172) according to the present invention specifically binds to the PGN-PGRP complex.

Anti-PGN-PGRP Complex Antibody (C2-172) and Anti-PGRP Antibody (C1-120) did not bind to PGN, and Anti-PGRP Antibody was specific to binding to both PGRP in its form or in complex Indicated.

(iv) Confirmation of PGN binding reactivity of PGRP

PGN binding of the PGRP of the present invention and binding characteristics of gram positive / negative PGN were confirmed by ELISA using the anti-PGRP monoclonal antibody (C1-120). According to the preparation method of the PGN sample disclosed in WO2008 / 096994, Staphylococcus aureus was used as a PGN sample derived from Gram-positive bacteria, and Escherichia as Gram-negative bacteria-derived PGN sample. PGN (Invivogen, USA) extracted from Escherichia coli was used.

PGN at a concentration of 1 μg / mL derived from Gram-positive bacteria and Gram-negative bacteria, respectively, was placed in 100 μL of pores for each 96-well plate (Nunc 469949) for ELISA analysis, and then reacted and fixed at 4 ° C. for 15 hours or more. Thereafter, 275 μL of 0.5% casein-PBS buffer was added to each of the pores, followed by reaction at 37 ° C. for 1 hour and 30 minutes to prepare a plate having PGN fixed thereto. Thereafter, 100 μL of PGRP at a concentration of 1 ug / mL was added to each pore, followed by reaction at 37 ° C. for 1 hour. The anti-PGRP monoclonal antibody (C1-120) purified in Example 1 was added to each of the pores of the prepared plate at a concentration of 1 μg / mL by reaction at 37 ° C. for 2 hours, and then HRP was labeled. Goat anti-mouse immunoglobulin (goat anti-mouse IgG-HRP, Bio-rad 170-6516) was diluted 1,000-fold and added to each 100 μL and reacted for 1 hour at 37 ℃. Then, 100 μL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each pore, followed by color development at room temperature for 3 minutes, followed by optical density at a wavelength of 405 nm using an ELISA meter. , OD) was measured to determine the reactivity, the results are shown in Table 2 below.

PGN Reactivity of PGRP PGN Reactivity origin division Staphylococcus aurius
( Staphylococcus aureus ) Gram positive has exist Escherichia coli
( Escherichia coli ) Gram voice has exist

From the results in Table 2, it was confirmed that the PGRP obtained according to the present invention binds to both Gram-positive and Gram-negative PGN.

Example 3: PGN Detection Using ELISA

For PGN detection using ELISA, 0.5 ug of the anti-PGN-PGRP complex antibody (C2-172) obtained in Example 1 was reacted at each pore of the assay plate (Nunc 469949) at 4 ° C. for at least 15 hours to fix the assay plate. Then, 300 μL of 0.5% casein-PBS buffer was added and reacted at 37 ° C. for 1 hour 30 minutes. 50 uL of PBS diluted with 400 ng of PGRP and 50 uL of PBS mixed with 10-320 pg of SLPG (a water-soluble polymerizable lysine-type peptidoglycan obtained according to WO2008 / 096994) After mixing and reacting for 30 minutes at room temperature, it was added to each pore of the plate prepared above and reacted at 37 ° C. for 2 hours. After the reaction, the pores were washed three times with PBS containing 0.05% Triton X-100 to remove unreacted substances, and 1 ug of HRP-labeled anti-PGN-PGRP complex antibody or anti-PGRP antibody was added, respectively. The reaction was carried out at 37 ° C. for 1 hour. HRP labeling of each antibody was performed using a Peroxidase labeling Kit (Dojindo, Japan), and prepared according to the manufacturer's manual. After the reaction, 100 μL of HRP substrate solution (horseradish peroxidase substrate kit, Bio-rad 172-1064) was added to each pore, followed by color development at room temperature for 3 minutes, followed by optical density at a wavelength of 405 nm using an ELISA meter. , OD) was measured. In addition, instead of the reaction of PGRP and PGN, Gram-positive, Gram-negative bacteria such as Staphylococcus aureus and Escherichia coli were incubated for 12 to 24 hours in a conventional culture medium. Next, the PGN component of the bacterial cell wall was extracted. 50 μL of sterile distilled water was added to the bacterial precipitates recovered by centrifugation at 10,000 rpm for 15 minutes, and 50 μL of 40% trichloroacetic acid was added thereto, followed by heat treatment at 90 ° C. for 15 minutes. After the heat treatment, the supernatant was removed by centrifugation under the same conditions. 0.1 mL of 0.1 N NaOH was added to the recovered precipitate, followed by heat treatment at 90 ° C. for 15 minutes. After heat treatment, the pH of the sample was neutralized using 10 uL of 0.1 M sodium phosphate solution. The microbial extract obtained in this manner was treated for each concentration, and the optical density was measured by the same method as above. The results are shown in Tables 3 and 4, and the reactivity in the following table is a relative measurement calculated based on the results of the PGRP single treatment group.

Purified PGN Reactivity by PGN Detection Using ELISA Refined PGN (pg) Reactivity C1-120-HRP C2-172-HRP 320 1.86 1.87 80 0.48 0.64 40 0.19 0.23 20 0.10 0.15 10 0.00 0.04 PGRP only processing 0.00 0.00

Microbial Extract Reactivity of PGN Detection Using ELISA Microbial Extract (CFU) Reactivity C1-120-HRP C2-172-HRP 1 x 10 ⁶ 7.47 5.05 1 x 10 ⁵ 0.86 0.22 1 x 10 ⁴ 0.40 0.00 1 x 10 ³ 0.00 0.00 PGRP only treatment group 0.00 0.00

From the results of Table 3 and Table 4, the reactivity was proportional to the concentration in the applied PGN concentration range, it can be seen that the detection at 20 pg PGN level.

Example 4 Preparation of Detection Device Using Immunochromatography

(1) Preparation of monoclonal antibody-binding colored fine particle fixing pad

About 40 nm of colloidal gold was used to prepare monoclonal antibody-binding colored fine particles. The anti-PGRP antibody obtained in Example 1 was added to 6 ug per 1 mL of colloidal gold while stirring for 100 minutes while stirring 100 mL of colloidal gold having an maximum absorbance optical density (O.D.) of about 1. After the reaction, the casein solution was added to a final concentration of 0.2% and reacted for 1 hour. The reaction was centrifuged at 9,000 rpm for 15 minutes, the supernatant was removed and the precipitate was taken up and stabilized (1% casein, 3% sucrose, 50 mM Tris buffer (pH 9), 50 mM borate buffer (pH 9), 0.5 % Triton X-100, 0.5% Tween-20). The suspension was adjusted to a concentration of 5 based on the maximum absorbance at 400 nm to 800 nm, and the solution was lyophilized after sufficiently wetted with a pad of fine particles.

(2) Preparation of Antibody Immobilized Membrane

In order to make the result line which can visually observe the reaction result, the anti-PGN-PGRP complex antibody obtained in Example 1 was made to a concentration of 2 mg / mL, and 0.8 uL / cm was added to the membrane formed on the fixed support plate. It was fixed by dropping as much as possible. As the fixed support plate, a support plate made of PVC / PE was used. PBS containing 0.5% sucrose was used for the drop antibody solution. An end line indicating the end of the reaction was dripped at the top of the result line. 2 mg / mL goat anti-mouse immunoglobulin antibody was used for the end point drop, and the drop condition was applied in the same manner as the antibody drop in the result line.

(3) Manufacture of detection device

The detection apparatus of the present invention included a sample pad to which a sample is applied and an absorption pad for inducing and maintaining absorption of the sample. Glass pads (Millipore, USA) were used as sample pads, and cellulose materials (Pall, USA) were used as absorbent pads. The antibody-binding colored fine particle pad prepared in (1) was bonded to the supporting plate to which the antibody drop membrane prepared in (2) was fixed so as to overlap, and the sample pad was overlapped with the colored fine particle pad in the longitudinal direction. The absorbent pad was bonded to overlap the membrane on top of the membrane to make a detection device.

Example 5: PGN Detection Using Immunochromatography

Purified PGN was detected using the PGN detection device prepared in Example 4. A reaction sample was prepared by mixing 100 ng of PGRP 10 uL with 90 uL of PBS containing an amount of purified PGN and applied to a sample pad of the detection device. As a result of the test, a sample line containing about 40 ng of purified PGN was confirmed by visual observation in the evaluated apparatus.

In order to evaluate the reactivity of the detection device against the PGN derived from microorganisms in addition to the purified PGN, dilutions of Gram-positive bacteria and Gram-negative bacteria were prepared as test bacteria, and then, 1 mL was taken and centrifuged (13,400 rpm, 10 minutes), and the supernatant. Was removed. 100 uL of trichloroacetic acid was added to the recovered precipitate, and the resultant was heat-treated at 90 ° C for 15 minutes. After the heat treatment, the supernatant was removed by centrifugation under the same conditions. 0.1 mL of 0.1 N NaOH was added to the recovered precipitate, followed by heat treatment at 90 ° C. for 15 minutes. After the heat treatment, the pH of the sample was neutralized using 10 uL of 0.1 M sodium phosphate solution, and then loaded into the detection device prepared in Example 4. The detection device prepared in Example 4 was confirmed to be responsive to PGN derived from microorganisms even at the level of 10 ⁵ cfu / mL or less in both Gram positive and Gram negative bacteria.

Example 6: PGN Detection of Microbial Contaminated Concentrated Platelet Samples

In order to confirm the detection of Gram-positive or Gram-negative bacteria-derived PGN in samples contaminated with microorganisms, the reactivity of the detection apparatus was confirmed by artificially mixing Gram-positive or Gram-negative bacteria with concentrated platelet preparations.

(1) pretreatment of samples

Samples were prepared by mixing 0.1 mL of a test cell dilution containing Gram-positive and Gram-negative bacteria to 0.5 mL of concentrated platelet preparation, and prepared to have a strain concentration of 10 ⁴ , 10 ⁵ , and 10 ⁶ cfu / mL. The test bacteria used are shown in Table 5.

Test bacteria Test bacteria KCTC Number Gram stain Bacillus subtilis 3135 positivity Staphylococcus aureus 3881 Staphylococcus epidermidis 3958 Streptococcus pyogenes 3984 Enterobacter cloacae 2361 voice Escherichia coli 2441 Klebsiella pneumoniae 2208 Klebsiella oxytoca 1686 Pseudomonas aeruginosa 1750 Serratia marcescens 12457

0.4 mL of platelet lysate (0.1 N NaOH, 4% Triton X-100) and 0.1 mL of methanol were added to the prepared sample, followed by stirring, followed by centrifugation (13,400 rpm, 10 minutes) to remove the supernatant. 50 uL of sterile distilled water was added to the recovered precipitate and suspended, and 50 uL of 40% trichloroacetic acid was added thereto, followed by heat treatment at 90 ° C. for 15 minutes. After the heat treatment, the supernatant was removed by centrifugation under the same conditions. 0.1 mL of 0.1 N NaOH was added to the recovered precipitate, followed by heat treatment at 90 ° C. for 15 minutes. After heat treatment, the pH of the sample was neutralized using 10 uL of 0.1 M sodium phosphate solution.

(2) PGN detection

100 ng of PGRP was mixed with 110 uL of the pretreated sample in (1), loaded on a sample pad of a detection apparatus, and the sample was developed to determine the presence or absence of a detection line. The development of the sample was judged by the presence of the end line, and the presence of the detection line was confirmed 30 and 60 minutes after the application of the sample. As a result, contamination of the test bacteria was detected at the level of 10 ⁵ cfu / mL or less within 60 minutes, showing the same result in both Gram-positive and Gram-negative bacteria. Figure 4 shows the detection results of the representative Gram-positive strain Staphylococcus epidermidis and Gram-negative strain Pseudomonas aeruginosa . All other strains also detected contamination of the test bacteria at levels below 10 ⁵ cfu / mL. Therefore, it can be seen that the detection device prepared in Example 4 can be analyzed not only in purified PGN but also in samples such as concentrated platelet preparations.

Example 7: Analysis of Antibody Genes

Each hybridoma clone identified as producing the antibody in Example 1 was cultured to recover 1 × 10 ⁷ cells and used a Trizol plus RNA purification kit (Invitrogen 12183-555). Total RNA was isolated from total nucleic acid. Total cDNA synthesis was performed using a cloned AMV first-strand cDNA synthesis kit (Invitrogen 12328-040). Polymerase chain reaction (PCR) was performed using the synthesized cDNA as a template. Ig-Primer sets (mouse Ig-primer set, Novagen 69831-3) were used as primers for the polymerase reaction. The polymerase reaction solution was prepared by mixing 5 pmol of primer, polymerase (Ex Tag, Takara RR01AM), 1.5 mM MgCl ₂ , 1 mM dNTP mixture, and polymerase buffer in 2.5 μl of cDNA. The polymerase reaction was first performed at 94 ° C. for 5 minutes, secondly at 94 ° C. for 1 minute, at 50 ° C. for 1 minute, and at 72 ° C. for 2 minutes. The reaction was carried out at 72 ° C. for 5 minutes.

The amplified PCR product was subjected to 1% agarose gel electrophoresis containing SYBR (SYBR, Invitrogen S-33102), and 400bp each of heavy and light chains based on 100bp standard DNA (1Kb plus ladder, Invitrogen 10787). The presence of the amplified gene fragment at the position was confirmed.

The agarose gel site including the heavy and light chain gene fragments obtained in the above procedure was cut to recover the gene fragments using an AxyPrep DNA gel extraction kit (Axygen AP-GX-250). The purified gene fragment was subcloned into the pCR2.1-TOPO vector (Invitrogen), and then transformed into E. coli DH5α-T1 (Invitrogen) to obtain a transformant. The transformants obtained were incubated overnight in LB medium containing 50 μg / ml of ampicillin, and then plasmids were recovered using a Wizard plus SV minipreps DNA purification system (Promega). The recovered plasmid was digested with restriction enzyme EcoR I (NEB) to obtain clones into which gene fragments of the same size were inserted.

After pure purification of the plasmid of each of the clones, the base sequence of each clone was analyzed by requesting from Genotech (Korea), a sequencing company. Complementary determining regions (CDRs) of variable region amino acids were analyzed according to H. Linda and B. Jurgen, Profiles for the analysis of immunoglobulin sequences: comparion of V gene subgroups, Protein Science 4: 306-310, 1995. Was performed.

Complementarity determining sites of the heavy and light chain variable regions of the anti-PGRP antibody obtained in Example 1 are shown in Table 6, and the amino acid sequence of the heavy chain variable region and the base sequence of the cDNA gene encoding the same are shown in SEQ ID NOs: 4 and 5, respectively. The amino acid sequence of the light chain variable region and the nucleotide sequence of the cDNA gene encoding the same are as shown in SEQ ID NOs: 9 and 10, respectively. In addition, the complementarity determining sites of the heavy and light chain variable regions of the anti-PGN-PGRP complex antibody obtained in Example 1 are shown in Table 7 below, and the amino acid sequence of the heavy chain variable region and the base sequence of the cDNA gene encoding the same are respectively sequenced. Nos. 14 and 15, and the amino acid sequence of the light chain variable region and the nucleotide sequence of the cDNA gene encoding the same are the same as SEQ ID NO: 19 and 20, respectively.

The amino acid sequence, base sequence, and complementarity determining sites of the heavy and light chain variable regions of the anti-PGRP antibody and the anti-PGN-PGRP complex antibody obtained in Example 1 are shown in Tables 6 and 7.

Complementarity Determining Site Sequences of Anti-PGRP Antibodies SEQ ID NO: order Heavy chain CDR1 One DYYMD CDR2 2 YINPNDGGTIYNQKFKG CDR3 3 ERADGYSYSFDY Light chain CDR1 6 LASQTIGTWLA CDR2 7 AATSLAD CDR3 8 RQFYSTPW

Complementarity Determining Site Sequences of Anti-PGN-PGRP Complex Antibodies SEQ ID NO: order Heavy chain CDR1 11 ECYMH CDR2 12 WIDPASGNTIFDPKFQG CDR3 13 YYDDYDEGFTY Light chain CDR1 16 SASSSVSYMY CDR2 17 STSNLAS CDR3 18 QQRSTYPY

The nucleotide sequence analyzed above, that is, the nucleotide sequences of SEQ ID NOs: 5, 10, 15, and 20, was searched through NCBI's Blast (Basic Local Alignment Search Tool), and found to be a novel gene.

Example 8: Isotyping of Monoclonal Antibodies

The isotype for the anti-PGRP antibody and the anti-PGN-PGRP complex antibody was confirmed by enzyme immunoassay. 100 μl of 1 μg / ml antibody solution was added to each well of the assay plate coated with PGN-PGRP complex and reacted at 37 ° C. for 2 hours. Then, 100 μl of a mouse MonoAb ID kit (Zymed 90-6550) solution was added to each well, followed by reaction at 37 ° C. for 2 hours, and 100 μl of HRP substrate solution, followed by color development at room temperature for 3 minutes. Optical density was measured at 405 nm using ELISA-reader. As a result, both the anti-PGRP antibody and the anti-PGN-PGRP complex antibody showed that the heavy chain was IgG1 type and the light chain was kappatype. On the other hand, the pI value of the anti-PGRP antibody (isoelectirc point) was found to be 6.5 to 7.0, and the pI value of the anti-PGN-PGRP complex antibody was found to be 5.9 to 6.2.

1 is a schematic diagram of an example and a detection mechanism of a detection kit by immunochromatography. In FIG. 1, antibody G represents an anti-PGRP antibody with chromophore binding, a red antibody represents an anti-PGN-PGRP complex antibody, and a blue antibody represents a goat anti-mouse antibody antibody.

2 is a schematic diagram of an example of a detection kit and a detection mechanism by enzyme immunoassay. In FIG. 2, red antibodies represent anti-PGN-PGRP complex antibodies, and green antibodies represent anti-PGRP antibodies with chromophores bound.

3 is a gram-positive strain Staphylococcus epidermidis (A) and a gram-negative strain Pseudomonas aeruginosa as a detection kit by immunochromatography prepared according to the present invention. The result of detecting a strain infection in the sample containing (B) is shown.

<110> YUHAN CORPORATION <120> Kit for detecting bacterial infection comprises novel monoclonal          antibodies <130> PN0281 <160> 21 <170> Kopatentin 1.71 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 1 Asp Tyr Tyr Met Asp   1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 2 Tyr Ile Asn Pro Asn Asp Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys   1 5 10 15 Gly     <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 3 Glu Arg Ala Asp Gly Tyr Ser Tyr Ser Phe Asp Tyr   1 5 10 <210> 4 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> variable region of heavy chain <400> 4 Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile          35 40 45 Gly Tyr Ile Asn Pro Asn Asp Gly Gly Thr Ile Tyr Asn Gln Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Glu Lys Ser Ser Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys                  85 90 95 Ala Arg Glu Arg Ala Asp Gly Tyr Ser Tyr Ser Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser         115 120 <210> 5 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> variable region of heavy chain <400> 5 gaggtcctgc tgcaacagtc tggacctgag gtggtgaagc ctggggcttc agtgaagata 60 ccctgcaagg cttctggata cacattcact gactactaca tggactgggt gaaacagagc 120 catggaaaga gccttgagtg gattggatat attaatccta acgatggtgg aactatctac 180 aaccagaagt tcaagggcaa ggccacattg actgtagaga agtcctccag cacagcctac 240 atggagctcc gcagcctgac atctgaggac actgcagtct atttctgtgc aagagagagg 300 gctgatggtt actcttactc ctttgactac tggggccaag gcaccactct cacagtctcc 360 tca 363 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 6 Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala   1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 7 Ala Ala Thr Ser Leu Ala Asp   1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 8 Arg Gln Phe Tyr Ser Thr Pro Trp   1 5 <210> 9 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> variable region of light chain <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly   1 5 10 15 Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile          35 40 45 Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala  65 70 75 80 Glu Asp Phe Val Ser Tyr Tyr Cys Arg Gln Phe Tyr Ser Thr Pro Trp                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg             100 105 <210> 10 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> variable region of light chain <400> 10 gacattcaga tgacccagtc tcctgcctcc cagtctgcat ctctgggaga aagtgtcacc 60 atcacatgcc tggcaagtca gaccattggt acatggttag cgtggtatca gcagaaacca 120 gggaaatctc ctcagctcct gatttatgct gcaaccagct tggcagatgg ggtcccatca 180 aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct 240 gaagattttg taagttatta ctgtcgacaa ttttatagta ctccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa acgg 324 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 11 Glu Cys Tyr Met His   1 5 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 12 Trp Ile Asp Pro Ala Ser Gly Asn Thr Ile Phe Asp Pro Lys Phe Gln   1 5 10 15 Gly     <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 13 Tyr Tyr Asp Asp Tyr Asp Glu Gly Phe Thr Tyr   1 5 10 <210> 14 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> variable region of heavy chain <400> 14 Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly   1 5 10 15 Ala Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Glu              20 25 30 Cys Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp          35 40 45 Ile Gly Trp Ile Asp Pro Ala Ser Gly Asn Thr Ile Phe Asp Pro Lys      50 55 60 Phe Gln Gly Lys Ala Ser Leu Thr Ser Asp Thr Ser Ser Asn Thr Ala  65 70 75 80 Tyr Leu Gln Leu Tyr Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr                  85 90 95 Cys Ala Arg Tyr Tyr Asp Tyr Asp Glu Gly Phe Thr Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ala         115 120 <210> 15 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> variable region of heavy chain <400> 15 tcagaggttc agctgcagca gtctggggct gagcttgtga ggccaggggc cttagtcaag 60 ttgtcctgca aagcttctgg cttcaacatt aaagagtgtt atatgcactg ggtgaagcag 120 aggcctggac aaggcctgga gtggattgga tggattgatc ctgcgagtgg taatactata 180 tttgacccga agttccaggg caaggccagt ctaacatcag acacatcctc caacacagcc 240 tacctgcagc tctacagcct gacatctgag gacactgccg tctattactg tgctagatac 300 tatgattacg acgagggctt tacttactgg ggccaaggga ctctggtcac tgtctctgca 360                                                                          360 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 16 Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr   1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 17 Ser Thr Ser Asn Leu Ala Ser   1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> complementary determining region <400> 18 Gln Gln Arg Ser Thr Tyr Pro Tyr   1 5 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> variable region of light chain <400> 19 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly   1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met              20 25 30 Tyr Trp Phe His Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr          35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Ala Arg Phe Ser Gly Ser      50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu  65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Tyr Thr                  85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg             100 105 <210> 20 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> variable region of light chain <400> 20 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 ataacctgca gtgccagctc aagtgtaagt tacatgtact ggttccacca gaagccaggc 120 acttctccca agctctggat ttatagtaca tccaacctgg cttctggagt ccctgctcgc 180 ttcagtggca gtggatctgg gacctcttac tctctgacaa tcagccgaat ggaggctgaa 240 gatgctgcca cttattactg ccagcaaagg agtacttacc cgtacacgtt cggagggggg 300 accaagctgg aaataaaacg g 321 <210> 21 <211> 193 <212> PRT <213> Tenebrio molitor <400> 21 Met Leu Leu Ala Thr Ile Ala Arg Gly Val Tyr Gln Ile Ser Ala Leu   1 5 10 15 Ser Gly Ser Thr Ile Pro Arg Ile Cys Pro Glu Ile Ile Ser Arg Thr              20 25 30 Arg Trp Gly Ala Arg Thr Pro Leu Glu Val Asp Tyr Ser Leu Ile Pro          35 40 45 Ile Glu Asn Val Val Val His His Thr Val Thr His Thr Cys Asp Ser      50 55 60 Glu Ser Glu Cys Ala Thr Leu Leu Arg Asn Val Gln Asn Phe His Met  65 70 75 80 Glu Asn Leu Glu Phe His Asp Ile Gly Tyr Asn Phe Leu Val Ala Gly                  85 90 95 Asp Gly Gln Ile Tyr Glu Gly Ala Gly Trp His Lys Val Gly Ala His             100 105 110 Thr Arg Gly Tyr Asn Thr Arg Ser Leu Gly Leu Ala Phe Ile Gly Asn         115 120 125 Phe Thr Ser Gln Leu Pro Val Gln Lys Gln Leu Lys Val Ala Lys Asp     130 135 140 Phe Leu Gln Cys Gly Val Glu Leu Gly Glu Leu Ser Lys Asn Tyr Lys 145 150 155 160 Leu Phe Gly Ala Arg Gln Val Ser Ser Thr Ser Ser Pro Gly Leu Lys                 165 170 175 Leu Tyr Arg Glu Leu Gln Asp Trp Pro His Phe Thr Arg Ser Pro Pro             180 185 190 Lys      

Claims (12)

Peptidoglycan recognition protein (PGRP) of SEQ ID NO: 21; Monoclonal antibodies (anti-PGRP antibodies) that specifically bind to peptidoglycan recognition proteins; And a monoclonal antibody (anti-PGN-PGRP complex antibody) that specifically binds to a complex of a peptidoglycan and a peptidoglycan recognition protein. The method of claim 1, wherein the peptidoglycan recognition protein (PGRP) of SEQ ID NO: 21; An anti-PGRP antibody wherein the complementarity determining regions of the heavy chain variable regions are the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 6, 7, and 8; And an anti-PGN-PGRP complex antibody wherein the complementarity determining regions of the heavy chain variable regions are the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and the complementarity determining regions of the light chain variable regions are the amino acid sequences of SEQ ID NOs: 16, 17, and 18; Kit for detecting bacterial infection comprising. The bacterium of claim 1 or 2, wherein the heavy chain variable region of the anti-PGRP antibody is an amino acid sequence of SEQ ID NO: 4, and the light chain variable region of the anti-PGRP antibody is an amino acid sequence of SEQ ID NO: 9 Infection detection kit. The heavy chain variable region of claim 1 or 2, wherein the heavy chain variable region of the anti-PGN-PGRP complex antibody is an amino acid sequence of SEQ ID NO: 14, and the light chain variable region of the anti-PGN-PGRP complex antibody is an amino acid sequence of SEQ ID NO: 19 Kit for detecting bacterial infection, characterized in that. The bacterial infection according to claim 1 or 2, wherein the peptidoglycan in the sample is detected by an immunochromatographic assay or an enzyme-linked immuno-sorbent assay (ELISA). Detection kit. The kit for detecting bacterial infection according to claim 5, wherein the peptidoglycan in the sample is detected by immunochromatography and the form of the kit is in the form of a strip. delete delete delete The complementarity determining regions of the heavy chain variable region are the amino acid sequences of SEQ ID NOs: 11, 12, and 13, and the complementarity determining regions of the light chain variable region are the amino acid sequences of SEQ ID NOs: 16, 17, and 18, and peptidoglycan. Monoclonal antibody that specifically binds to a complex with a recognition protein. The monoclonal antibody according to claim 10, wherein the heavy chain variable region is an amino acid sequence of SEQ ID NO: 14, and the light chain variable region is an amino acid sequence of SEQ ID NO: 19. The monoclonal antibody according to claim 11, wherein the heavy chain variable region is an amino acid sequence encoded by cDNA of SEQ ID NO: 15, and the light chain variable region is an amino acid sequence encoded by a cDNA of SEQ ID NO: 20.

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