Preparation of antibody capable of detecting multi-subtype KPC and application of kit of antibody

Abstract

The invention discloses preparation of an antibody capable of detecting multi-subtype KPC and application of a kit of the antibody, and belongs to the field of biological medicine. According to the kit for detecting the common subtypes of KPC and the application of the kit, the mouse antibodies are prepared through immunization of conserved antigen epitopes of all the subtypes of KPC, in-vitro screening and pairing are carried out in multiple modes, then an antibody pair which is good in specificity and high in affinity and can be combined with multiple subtypes is obtained, the titer of the two antibodies can reach one million or above, and the kit is high in specificity, good in affinity and capable of being used for detecting the common subtypes of KPC. Amino acid sequences of a heavy chain variable region and a light chain variable region are obtained through sequencing, and rapid and stable expression can be realized after recombination. The product for detecting the carbapenem KPC is developed on the basis of an immunochromatography platform, and the kit has the advantages of high sensitivity, good specificity, good stability, capability of covering most epidemic KPC subtypes and rapidness and convenience in detection, and provides important help for clinical auxiliary diagnosis and medication guidance.

Description

Technical Field

[0001] The present invention relates to the preparation of an antibody capable of detecting multiple subtypes of KPC and the application of a kit thereof, and belongs to the field of biomedicine. Background Art

[0002] Bacterial resistance has become a major challenge to global public health. The emergence and prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pandrug-resistant (PDR) bacteria pose a significant threat to human health. Among the many drug-resistant bacteria encountered clinically, the most important are carbapenem-resistant Gram-negative bacteria, particularly carbapenem-resistant Enterobacteriales (CRE), which have rapidly increased in recent years.

[0003] Infections caused by CRE are significantly associated with high morbidity and mortality. The mortality rate for CRE-induced bloodstream infections can be as high as 53.1%, resulting in a significant burden of disease for patients. Klebsiella pneumoniae and Escherichia coli are the most common CRE strains. In vitro antimicrobial susceptibility tests show that CRE are generally sensitive only to tigecycline, polymyxins, and new β-lactamase inhibitor combinations such as ceftazidime / avibactam. They are highly resistant to most β-lactam antibiotics, including carbapenems, as well as to quinolones, with varying resistance to aminoglycosides. Furthermore, ceftazidime / avibactam exhibits varying in vitro antimicrobial activity against different carbapenemases: ceftazidime / avibactam is ineffective against metalloenzymes but sensitive to serine enzymes, AmpC enzymes, and ESBL enzymes. Initial antimicrobial therapy has been reported to be clinically significant in improving patient survival in the treatment of CRE bloodstream infections. Therefore, rapid detection of carbapenemase-producing Enterobacteriales in bloodstream infections and identification of carbapenemase types are essential for early optimization of antimicrobial therapy and improvement of survival rates.

[0004] Carbapenemase (KPC) production is the main mechanism of carbapenem resistance in Klebsiella pneumoniae. Ceftazidime / avibactam (CZA) is considered a promising β-lactam-β-lactamase inhibitor combination with activity against serine β-lactamases, including KPC.

[0005] Since 2020, the number of new KPC gene subtypes discovered has increased rapidly. Currently, a total of 96 KPC carbapenemase gene subtypes have been found, namely blaKPC-2 type to blaKPC-108 type. The emergence of new KPC gene subtypes has increased the difficulty of clinical anti-infection treatment and laboratory testing. A study published in Front Cell Infect Microbiol in 2020 showed that among CRE strains isolated from adult and pediatric patients, blaKPC-2 (51.6%) is the most common carbapenemase subtype. The new KPC gene subtypes discovered so far are all variants of KPC-2 and KPC-3, and their main mechanisms are deletion, mutation, insertion and tandem duplication. Taking the common gene subtypes as an example, bla KPC-14 Subtype is bla KPC-2 The mutation results from the deletion of two amino acids (glycine 242 and threonine 243); bla KPC-33 、bla KPC-51 and bla KPC-52 The formation of bla is due to the substitution of amino acid 179 (aspartic acid → tyrosine), and it is easy to mutate during treatment; KPC-53 It's bla KPC-3 The Ω ring amino acids are replicated (167 leucine and 168 glutamic acid); bla KPC-74 is bla KPC-2 Six nucleotides were deleted at positions 712-717 of the gene (glycine and valine at positions 239 and 240 were deleted).

[0006] In actual clinical testing, the control kits available on the market can only detect bla KPC-2 、bla KPC-3 Common subtypes are tested, but bla KPC-14 、bla KPC-31 、bla KPC-33 、bla KPC104 、bla KPC-106 、bla KPC-108 、bla KPC-139 Other common KPC-type carbapenemase variants can lead to missed KPC detection, clinical medication errors, and other adverse consequences. This invention proposes the development of KPC antibody pairs and an immunochromatographic kit for rapid detection of multiple KPC subtypes. This can be used to detect multiple KPC subtypes, thereby improving the accuracy of auxiliary diagnosis of drug-resistant bacteria and providing important assistance for clinical medication. Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides an antibody preparation capable of detecting multiple subtypes of KPC and the application of the kit thereof, the purpose of which is to solve the problem that the existing kit cannot detect subtypes of KPC such as bla KPC-14 、bla KPC-31 、bla KPC-33 、bla KPC51 、bla KPC-52 Common KPC-type carbapenemase variants such as β-lactamase and β-lactamase may lead to technical problems such as missed KPC detection and clinical medication errors.

[0008] The first technical solution provided by the present invention is an anti-KPC carbapenemase antibody, wherein the anti-KPC carbapenemase antibody comprises a heavy chain and a light chain; the heavy chain comprises HCDR1, HCDR2, and HCDR3, and the light chain comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively as shown in any one of the groups (a) to (b):

[0009] (a) SEQ ID NO.1~SEQ ID NO.6;

[0010] (b) SEQ ID NO.7~SEQ ID NO.12;

[0011] SEQ ID NO: 1: SNNYISWY;

[0012] SEQ ID NO:2:WIYAGSGGTTYNQDFTG;

[0013] SEQ ID NO:3: CARLRWSVPHWYFDV;

[0014] SEQ ID NO:4: RSDQSLFHSNGNTWLH;

[0015] SEQ ID NO:5: RVSNRWS;

[0016] SEQ ID NO:6: SQSTHVPFTF;

[0017] SEQ ID NO:7:TSYWMHWV;

[0018] SEQ ID NO:8: MIDPSDTHTTLNQKMRD;

[0019] SEQ ID NO:9:EGFFTTIVLPIIY;

[0020] SEQ ID NO:10: RSSQTVVYSGSQKNYLA;

[0021] SEQ ID NO: 11: WASTRESG;

[0022] SEQ ID NO: 12: QQYYNYPLT.

[0023] In certain embodiments, the anti-KPC carbapenemase antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are as shown in any one of the groups (c) to (d):

[0024] (c) SEQ ID NO: 13 and SEQ ID NO: 14;

[0025] (d) SEQ ID NO:15 and SEQ ID NO:16.

[0026] The second technical solution provided by the present invention is a polynucleotide encoding the antibody described in the first technical solution.

[0027] The third technical solution provided by the present invention is an expression vector carrying the polynucleotide described in the second technical solution.

[0028] The fourth technical solution provided by the present invention is a host cell expressing the antibody described in the first technical solution, or containing the polynucleotide described in the second technical solution, or transformed with the expression vector described in the third technical solution.

[0029] In certain embodiments, the host cell includes, but is not limited to, bacteria, fungi, animal cells, or plant cells.

[0030] In certain embodiments, the fungus comprises yeast or mold, and the bacteria comprises Escherichia coli.

[0031] In certain embodiments, the animal cells include but are not limited to 293 cells.

[0032] The fifth technical solution provided by the present invention is a method for preparing an anti-KPC type carbapenemase antibody, which comprises culturing the host cell described in the fourth technical solution to obtain a culture containing the antibody described in the first technical solution.

[0033] The sixth technical solution provided by the present invention is a biologically or chemically labeled product, wherein the product is an antibody labeled with a marker, and the antibody is derived from any one of the following:

[0034] (1) The antibody described in the first technical solution;

[0035] (2) The culture of the host cells described in the fourth technical solution.

[0036] In certain embodiments, the label includes but is not limited to enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, β-galactosidase-anti-β-galactosidase bridge; the biotin includes but is not limited to biotin and its derivatives; the fluorescein includes but is not limited to AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, PerCP dyes; the chemiluminescent type includes but is not limited to isoluminol and its derivatives, acridinium esters and their derivatives, terpyridine ruthenium and its derivatives, etc.; the isotope type includes but is not limited to iodine labeling; the colloidal labeling includes but is not limited to colloidal gold, colloidal carbon, colloidal selenium, etc.

[0037] The seventh technical solution provided by the present invention is a kit, which contains the antibody described in the first technical solution or the biomarker or chemically labeled product described in the sixth technical solution.

[0038] In certain embodiments, the kit includes an enzyme-linked immunosorbent assay kit and an immunofluorescence assay kit.

[0039] In certain embodiments, the kit is an immunochromatography kit, comprising a test card, comprising: a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially overlapped and pasted on the base plate; the conjugate pad is sprayed with a tracer marker labeled with an antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 13 and a light chain variable region amino acid sequence as shown in SEQ ID NO: 14, a C line, and a T line, wherein the C line is fixed with a mouse anti-human IgG antibody, and the T line is fixed with an antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 15 and a light chain variable region amino acid sequence as shown in SEQ ID NO: 16.

[0040] The eighth technical solution provided by the present invention is the use of the antibody described in the first technical solution, or the polynucleotide described in the second technical solution, or the expression vector described in the third technical solution, or the host cell described in the fourth technical solution, or the method described in the fifth technical solution, or the biomarker or chemically labeled product described in the sixth technical solution in the preparation of a product for detecting KPC-type carbapenemases.

[0041] In certain embodiments, the product comprises a reagent, a kit, a detection chip, or a biosensor.

[0042] The technical effects of the present invention are as follows:

[0043] The present invention provides a kit for detecting common KPC subtypes and its application. Mouse antibodies are prepared by immunization using conserved antigenic epitopes from various KPC subtypes. These antibodies are then screened and paired in vitro using various methods to obtain a pair of antibodies with good specificity, strong affinity, and the ability to bind to multiple subtypes. The two antibodies can achieve a titer of over 1 million. The amino acid sequences of their heavy and light chain variable regions are sequenced, and after recombination, rapid and stable expression can be achieved. The present invention develops a product for carbapenem KPC detection based on an immunochromatographic platform. The product has the advantages of high sensitivity, good specificity, coverage of most prevalent KPC subtypes, and rapid and convenient detection, providing important assistance for clinical auxiliary diagnosis and medication guidance. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is an SDS-PAGE diagram of the recombinant antibody of the present invention after purification.

[0045] Figure 2 Graph showing the results of detecting multiple subtypes of KPC using the kit of the present invention and a comparative kit. DETAILED DESCRIPTION

[0046] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0047] The raw materials used in the embodiment are:

[0048] 1. The pcold expression vector was purchased from Takara.

[0049] 2. Escherichia coli DH5α and Escherichia coli BL21 (DE3) were purchased from Takara.

[0050] 3. LB liquid medium: Prepared with 10 g / L peptone, 5 g / L yeast extract, and 5 g / L sodium chloride. The pH value needs to be adjusted to 7.3 ± 0.1.

[0051] LB solid medium: Prepared with 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar powder. The pH value needs to be adjusted to 7.3 ± 0.1.

[0052] Serum-free DMEM medium was purchased from Gibco;

[0053] 4. The implementation of the BALB / c mouse immunization process was entrusted to Jiangsu Dongkang Biological Co., Ltd.

[0054] Example 1 Preparation of anti-KPC monoclonal antibodies MKN67 and MKN68

[0055] Step 1: Plasmid construction

[0056] Comparison of two common subtypes of bla KPC-2 and bla KPC-3 , and combined with comprehensive analysis of mutation sites of other subtypes, we selected conserved antigen epitopes of each KPC subtype and intercepted bla KPC-2 45-165AA, bla KPC-2 175-240AA (Uniprot: Q93LQ9), the amino acid sequence of which is shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19:

[0057] SEQ ID NO: 17:

[0058] TNLVAEPFAKLEQDFGGSIGVYAMDTGSGATVSYRAEERFPLCSSFKGFLAAAVLARSQ QQAGLLDTPIRYGKNALVPWSPISEKYLTTGMTVAELSAAAVQYSDNAAANLLLKELGGPA GLTAFMRSIGDTTFRLDRWE;

[0059] SEQ ID NO: 18:

[0060] QRQQFSSPRAVTESLQKLTLGSALAAPQRQQFVDWLKGNTTGNHRIRAAVPADWAVG DKTGTCGVY;

[0061] SEQ ID NO: 19:

[0062] DYAVVWPTGAPIVLAVYTRAPNKDDKHSEAV.

[0063] The above dominant antigen epitope segments were optimized into nucleotide sequences according to the E. coli codon principle, and then gene synthesis was performed (completed by GenScript Biotech).

[0064] The double enzyme digestion method was used to connect HindⅢ and EcoRI to the pcold expression vector to construct an expression plasmid. 100 ng of the expression plasmid was extracted and added to the Escherichia coli DH5α competent cells. The competent cells after the addition of the plasmid were then heat-shocked and transformed. The transformed competent cells were added to LB liquid culture medium and cultured in a shaker at 37°C and 200 rpm for about 30 minutes. After removal, 150 uL of the suspension was aspirated and spread on an LB solid culture medium plate for overnight culture. After picking a single clone colony for expansion and culture, the bacterial solution was collected to extract the plasmid (according to the instructions of the plasmid extraction kit produced by Sangon Biotechnology Co., Ltd.), and sent to Universal Biotechnology for sequencing confirmation to obtain two plasmids, pcold-KPC-SUMO.

[0065] Step 2: Immunogen preparation

[0066] The two plasmids pcold-KPC-SUMO were transformed into Escherichia coli BL21 (DE3) strain and cultured on LB plates at 37°C for 12-16 h. Single clones were picked and expanded. The culture was shaken until the OD600 reached 0.6-0.8, and IPTG at a final concentration of 1 mM was added to induce expression. After 4 h of expression, the bacteria were collected by centrifugation.

[0067] Resuspend the cells in binding buffer (50mM Tris pH 8.0, 100mM NaCl, 1mM PMSF, 0.5mM EDTA, 1% glycerol) and sonicate on ice for 30 minutes. Centrifuge at 14,000 rpm for 15 minutes and collect the supernatant. Pre-equilibrate a Ni-NTA column with binding buffer and bind the expressed antigen to the column. Wash away contaminants with a gradient of imidazole buffer (30, 50, 80, and 100mM imidazole in 50mM Tris pH 8.0, 100mM NaCl). Elute the target protein with a high-concentration imidazole buffer (200mM imidazole in 50mM Tris pH 8.0, 100mM NaCl) and collect the resulting protein. Verify the size of the target protein by SDS-PAGE. The two expressed protein segments, 45-165 AA and 175-240 AA, were sized at 29 kD and 22 kD, respectively. The SUMO tag was removed using SUMO protease and the protein was dialyzed and concentrated into binding buffer for later use.

[0068] Because the target protein has a small molecular weight, BSA was chosen as the carrier protein for the immunogen. The synthesized peptide was conjugated to BSA using the SPDP conjugation method: a DMSO solution with a final concentration of 20 mM SPDP was used. BSA was dissolved in PBS-EDTA and allowed to stand at room temperature for 1 hour. The solution was then passed through a HiTrap desalting column to remove excess SPDP. Finally, the target peptide was added to the conjugated BSA-SPDP system and incubated overnight at room temperature. The immunogen was then obtained by mixing BSA-KPC (45-165) and BSA-KPC (175-240) in equal proportions.

[0069] Step 3: Animal immunization

[0070] The above target protein immunogen was emulsified with Freund's complete adjuvant, and 5 SPF-grade BALB / c mice were selected for subcutaneous injection at 50 μg / mouse. In the second and third weeks, the antigen emulsified with Freund's incomplete adjuvant was subcutaneously injected at 50 μg / mouse. In the fourth week, the antigen protein solution was injected for booster immunization.

[0071] Before each immunization, blood was collected from the tail vein to detect changes in serum antibody levels; on the fifth day after the last immunization, blood was collected from the mouse eyeballs, the blood was collected and allowed to stand until the serum was completely separated, and the serum was centrifuged at 3000 rpm for 5 minutes and packaged into aliquots and frozen at -70°C for later use.

[0072] Step 4: Monoclonal antibody screening and preparation

[0073] After immunization, BALB / c mice were eyeballed and blood was collected to serve as a positive control serum. The mice were then killed by cervical dislocation and disinfected with 75% alcohol. The spleens were removed and a splenocyte suspension (counted under a microscope) was prepared. The suspension was mixed with myeloma SP2 / 0 cells (counted under a microscope) in serum-free DMEM medium at a ratio of 5:1. The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was removed, and the suspension was resuspended. Preheated 50% PEG4000 was immediately added to fuse the cells. After standing for 1 minute, serum-free DMEM medium was added to terminate the fusion. The cells were allowed to stand at 37°C for 10 minutes, centrifuged, the supernatant was removed, and the cells were resuspended in HAT medium. The cells were aliquoted into 96-well plates and cultured in a cell culture incubator for approximately 10 days until the fused cells covered 20%-50% of the well bottom. Positive clones were screened by indirect ELISA.

[0074] The selected hybridoma cell pair was serially subcloned using the limiting dilution method until the subclonal cell positive rate reached 100%, after which the cells were expanded. Antibody IgG was produced at 37°C, 220 rpm, and 5% CO2. When cell viability was less than 50%, the culture medium was removed and collected into a centrifuge bottle. The supernatant was centrifuged at 12,000 rpm for 10 minutes and filtered through a 0.45 μm filter to remove impurities. Antibody purification was performed using a Protein G affinity chromatography column equilibrated with binding buffer. After loading, the column was washed with binding buffer to remove impurities and eluted with 0.1 M Gly-HCl, pH 3.0. Finally, the antibody was characterized by SDS-PAGE electrophoresis and, after confirmation, dialyzed into 50 mM Tris, pH 8.0, 150 mM NaCl, and 1 mM EDTA buffer.

[0075] The antigen mixture collected in step (3) was coated onto a 96-well plate, and HRP-labeled rabbit anti-mouse IgG was used as a secondary antibody. Serum collected from enucleated immunized mice served as a positive control, and supernatant from cultured myeloma SP2 / 0 cells served as a negative control. A total of 18 positive cell clones secreting antibodies were obtained. They were named: MKN02, MKN26, MKN35, MKN38, MKN50, MKN52, MKN67, MKN68, MKN99, MKN102, MKN105, MKN134, MKN135, MKN156, MKN160, MKN169, MKN181, and MKN186.

[0076] Step 5: Antibody titer detection

[0077] The titer of antibodies secreted by positive cell clones was detected by ELISA: the KPC immunogen mixture prepared above was diluted with 50mM Na2CO3 pH9.6 buffer to a final concentration of 1μg / ml, added to a 96-well plate, coated overnight at 4°C, washed three times with 1×PBST, and patted dry; blocked with PBST containing 2% BSA (purchased from Sigma) and incubated for 2h. After washing in the same manner, the above-mentioned positive cell clones secreted antibodies MKN67 and MKN68 were diluted with 1×PBS buffer at 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, 1:1280000, and 1:2560000 to obtain different concentrations of test products. The diluted test sample was added to the above-mentioned ELISA plate and incubated at 37°C for 1 hour. A 1:4000 dilution of HRP-goat anti-mouse IgM (purchased from Sigma) was then added and incubated at 37°C for 1 hour. Finally, TMB substrate was added and color was developed at room temperature in the dark for 10 minutes. The reaction was terminated with sulfuric acid. OD 450 nm values ​​were measured using a microplate reader. The titers of the antibodies are shown in Table 1.

[0078] Table 1 Antibody titer

[0079]

[0080]

[0081] Step 6: Antibody Pairing and Purification

[0082] The ELISA double antibody sandwich method was used to screen the paired antibodies secreted by the positive clone cells. The paired results are shown in Table 2.

[0083] Table 2 Pairing results

[0084] Serial number Capture antibody Labeled antibodies 1 MKN26 MKN50 2 MKN50 MKN99 3 MKN67 MKN68 4 MKN102 MKN38 5 MKN134 MKN156 6 MKN169 MKN99 7 MKN181 MKN134

[0085] Further screening was performed using an immunochromatographic platform, and a pair of antibodies were finally identified as shown in Table 3.

[0086] Table 3 Identified Antibodies

[0087] Serial number Capture antibody Labeled antibodies 1 MKN67 MKN68

[0088] The samples were sent to General Biotech for sequencing.

[0089] The amino acid sequences of the antibodies identified in the present invention are as follows:

[0090] Antibody MKN67: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14;

[0091] SEQ ID NO: 13:

[0092] QGQMQQSGLELVKPGASVKASCKTCEFTASNNYISWYKQKPGQSLEWIAWIYAGSGG TTYNQDFTGKAQVDVTDSSSTAYMQFSSLTTEDSAIMWNHICARLRWSVPHWYFDVWGA GTTVTVSSAKTTPPSVYPLAP;

[0093] SEQ ID NO: 14:

[0094] VVMTQTPLSLPVSFGDQASISCRSDQSLFHSNGNTWLHWFLQKPGQSPKLLIYRVSNR WSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIKRAD.

[0095] Antibody MKN67 includes three first heavy chain CDR regions and three first light chain CDR regions. The three first heavy chain CDR regions include: HCDR1 with the amino acid sequence set forth in SEQ ID NO:1, HCDR2 with the amino acid sequence set forth in SEQ ID NO:2, and HCDR3 with the amino acid sequence set forth in SEQ ID NO:3. The three first light chain CDR regions include: LCDR1 with the amino acid sequence set forth in SEQ ID NO:4, LCDR2 with the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 with the amino acid sequence set forth in SEQ ID NO:6. The specific sequence information is as follows: SEQ ID NO:1: SNNYISWY. SEQ ID NO:2: WIYAGSGGTTYNQDFTG. SEQ ID NO:3: CARLRWSVPHWYFDV. SEQ ID NO:4: RSDQSLFHSNGNTWLH. SEQ ID NO:5: RVSNRWS. SEQ ID NO:6: SQSTHVPFTF.

[0096] Antibody MKN68: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16;

[0097] SEQ ID NO: 15:

[0098] QVQLQQSGPQLGRPGTSVKVSCHASGYTWTSYWMHWVKQRPGQPLEWIGMIDPSDT HTTLNQKMRDKATVMVDKSSSTAYMEVSSPTSEDSAVYYCAREGFFTTIVLPIIYWGQGTV VTV;

[0099] SEQ ID NO: 16:

[0100] VVMSQSPSSLAVFWYEKVTMTCRSSQTVVYSGSQKNYLAWYQQKGPCNRKLLIYWA STRESGVPDRFTGSGSGTDMVLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLEL.

[0101] Antibody MKN68 includes three second heavy chain CDR regions and three second light chain CDR regions. The three second heavy chain CDR regions include: HCDR4 with the amino acid sequence set forth in SEQ ID NO:7, HCDR5 with the amino acid sequence set forth in SEQ ID NO:8, and HCDR6 with the amino acid sequence set forth in SEQ ID NO:9. The three second light chain CDR regions include: LCDR4 with the amino acid sequence set forth in SEQ ID NO:10, LCDR5 with the amino acid sequence set forth in SEQ ID NO:11, and LCDR6 with the amino acid sequence set forth in SEQ ID NO:12. The specific sequence information is as follows: SEQ ID NO:7: TSYWMHWV. SEQ ID NO:8: MIDPSDTHTTLNQKMRD. SEQ ID NO:9: EGFFTTIVLPIIY. SEQ ID NO:10: RSSQTVVYSGSQKNYLA. SEQ ID NO:11: WASTRESG. SEQ ID NO:12: QQYYNYPLT.

[0102] By molecular construction methods, the heavy chain variable region of the above-mentioned IgG antibody was grafted onto the nucleotide sequence of the human IgG heavy chain constant region skeleton, and the light chain variable region VL nucleotide sequence was grafted onto the nucleotide sequence of the human IgG light chain constant region skeleton. The antibody heavy chain variable region and light chain variable region obtained by sequencing were recombined with the constant region of mouse IgG and cloned into the modified mammalian cell expression vector pCMV-C-Flag for expression and recombination in Expi 293F cells (the vector was purchased from Beyotime, and the human IgG constant region gene was synthesized by Qingke Biotechnology Co., Ltd.). The process of grafting to construct a eukaryotic expression plasmid and the reagents or parameters used in each process are not limited. As long as the technology in the existing technology can achieve the above purpose, it will not be repeated here, and the antibody purification is carried out according to the standard operating procedures for protein purification (such as Figure 1 As shown), the anti-KPC recombinant antibodies were expressed and were still named MKN67 and MKN68.

[0103] Example 2: Preparation of a multi-subtype KPC detection kit

[0104] The present invention uses the above-mentioned antibody pair to develop a kit for detecting multiple subtypes of KPC based on an immunochromatographic platform. The preparation method of the kit is as follows:

[0105] Step 1: Add 200-800 μL of 0.05-0.2M K2CO3 solution dropwise to 100mL of colloidal gold solution while stirring. After the addition is complete, seal the container and mix slowly for 2-5 minutes. After mixing, add the anti-KPC carbapenemase monoclonal antibody MKN67 dropwise. After the addition is complete, seal the container and mix slowly for 2-5 minutes. Incubate for 30-60 minutes. After the incubation is completed, add 20-80mL of blocking solution (1-3% BSA) dropwise, mix for 2-5 minutes, and let it stand for 30-60 minutes. After the standing time is over, continue to seal and mix for 2-5 minutes. After the blocking is completed, centrifuge and set the parameters: centrifugal force 4000-6000rpm, 2-8℃, centrifugation time 20-60min. After the centrifugation is completed, remove the supernatant to obtain the gold-labeled precipitate, and preserve it with preservation solution to obtain the gold-labeled solution;

[0106] Step 2: Spray the gold-labeled solution onto the glass cellulose membrane and dry it to make a gold-labeled pad;

[0107] Step 3: Add 1-10% sucrose solution to the anti-KPC carbapenemase monoclonal antibody MKN68 and mix well to form a test line coating solution. Then, add 1-10% sucrose solution to the goat anti-mouse IgG antibody and mix well to form a control line coating solution. Streak 0.5-2 μL / cm of the control line coating solution and the test line coating solution onto a nitrocellulose membrane and dry it to obtain a coating plate.

[0108] Step 4: Stick the gold label pad, absorbent paper, and sample pad on the PVC base plate in sequence, cut them into 3cm wide test strips and put them into the corresponding card shell to form a test card.

[0109] Example 3: Comparative Application of Multi-Subtype KPC Detection Kits

[0110] In this example, bla KPC-1 、bla KPC-2 、bla KPC-3 、bla KPC-14 、bla KPC-25 、bla KPC-31 、bla KPC-33 、bla KPC-51 、bla KPC-52 、bla KPC-71 、bla KPC-77 、bla KPC-104 、bla KPC-106 、bla KPC-108 、bla KPC-139For bacterial culture samples, pick 3 loops of bacterial samples with an inoculating loop and immerse the inoculating loop in the diluent, shake it back and forth, and mix it for at least 30 seconds to ensure that the sample is fully eluted in the diluent tube. Cover the diluent tube with a cap and oscillate it evenly using a vortex oscillator. Vertically add 4 drops of treated sample treatment solution to the sample well of the test card in the kit in Example 2, observe the results within 15-20 minutes, and perform simultaneous detection with the comparison kit (the comparison kits 1 and 2 used were purchased from commercially available products). The test results are shown in Tables 4 and 4 below. Figure 2 shown.

[0111] Table 4 Kit test results

[0112]

[0113]

[0114] Note: √ means detectable, × means undetectable.

[0115] The kit developed by the present invention can detect all common KPC subtypes tested, among which the comparison kit 1 will have bla KPC-14 、bla KPC-31 、bla KPC-33 、bla KPC-52 、bla KPC-71 、bla KPC-77 、bla KPC-104 、bla KPC-106 、bla KPC-108 、bla KPC-139 Subtype missed detection; compared to kit 2, there will be bla KPC-14 、bla KPC-104 、bla KPC-106 、bla KPC-108 、bla KPC-139 The kit of the present invention can make up for the missed detection of subtypes, realize multi-subtype detection, cover the common drug-resistant subtypes in recent years, and provide assistance for clinical drug diagnosis and treatment.

[0116] Example 4: Performance evaluation of a multi-subtype KPC detection kit

[0117] 1. Detection limit

[0118] A calibrated negative sample was taken to prepare a negative matrix, and the KPC-type carbapenemase was diluted in multiple concentration gradients, i.e., the dilution concentrations were 1000, 800, 600, 500, 400, 300, and 200 pg / mL. Each concentration gradient was repeatedly tested 20 times using the kit of Example 2. The detection was continued for 3 days, and the color development and positivity of the test results at each concentration were observed. The lowest concentration with a positive detection rate of 95% and consistent color development was used as the detection limit.

[0119] Table 5 Test results

[0120]

[0121]

[0122] According to the above result judgment method, it can be seen from Table 5 that through analysis of the above test results, when KPC-type carbapenemases were diluted to a detection limit concentration of 600 pg / mL using different negative samples, the positive detection rate was slightly higher than 95%. Therefore, through verification, it was finally determined that the detection limit of the kit of the present invention was 600 pg / mL.

[0123] 2. Stability

[0124] Positive and negative samples at three different antibody concentrations were used as research materials. Three batches of the kit described in Example 2 were stored at 2-8°C, 20±2°C, and 30±2°C for 27 months. Testing was performed at 0, 4, 8, 12, 16, 20, 24, 26, and 27 months to evaluate the stability of the kit. The results are shown in Tables 6-8.

[0125] Table 6 Test kit (storage 2-8°C)

[0126]

[0127] Table 7 Test kit (storage at 20±2°C)

[0128]

[0129] Table 8 Test kit (stored at 30±2°C)

[0130]

[0131]

[0132] As can be seen from Tables 6 to 8, the three batches of test kits were stored at 2-8°C, 20±2°C and 30±2°C for 27 months, respectively. Strongly positive, moderately positive, weakly positive and negative samples were used for testing at 0 months, 4 months, 8 months, 12 months, 16 months, 20 months, 24 months, 26 months and 27 months, respectively. The test results were good, and the test kits were extremely stable.

[0133] 3. Analytical Specificity – Cross-Substances

[0134] Six KPC-type carbapenemase-positive samples and six KPC-type carbapenemase-negative samples were used as research materials. Carbapenemase-positive samples of other common carbapenemase types were added to evaluate the cross-reactivity of the kit using the kit described in Example 2. The results are shown in Table 9.

[0135] Table 9 Specificity test results

[0136]

[0137]

[0138] According to Table 9, the common carbapenemase-positive samples did not cross-react with the kit, indicating good performance.

[0139] 4. Cross-interference experiment

[0140] Negative matrix and KPC-type carbapenemase critical positive samples were selected as basic samples and a certain concentration of interfering substances were added to confirm whether a certain concentration of interfering substances would affect the test results.

[0141] Table 10 Interfering substances affect test results

[0142]

[0143] The results in Table 10 show that the kit can effectively resist the interference of endogenous and exogenous substances.

[0144] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Anti-KPC type carbapenemase antibody, characterized in that: The anti-KPC type carbapenemase antibody comprises a heavy chain and a light chain; the heavy chain comprises HCDR1, HCDR2, and HCDR3, and the light chain comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively the sequences shown in any one of the groups (a) to (b): (a) SEQ ID NO.1~SEQ ID NO.6; (b) SEQ ID NO.7 to SEQ ID NO.

12.

2. The antibody according to claim 1, characterized in that The anti-KPC type carbapenemase antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively as shown in any one of the groups (c) to (d): (c) SEQ ID NO: 13 and SEQ ID NO: 14; (d) SEQ ID NO:15 and SEQ ID NO:

16.

3. A polynucleotide encoding the antibody according to claim 1 or 2.

4. An expression vector carrying the polynucleotide of claim 3.

5. A host cell expressing the antibody according to claim 1 or 2, or containing the polynucleotide according to claim 3, or transformed with the expression vector according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell includes bacteria, fungi, animal cells or plant cells; optionally, the fungi include yeast or mold, and the bacteria include Escherichia coli.

7. A method for preparing an anti-KPC type carbapenemase antibody, characterized in that: The method comprises culturing the host cell according to claim 5 or 6 to obtain a culture containing the antibody according to claim 1 or 2.

8. A biologically or chemically labeled product, characterized in that: The product is an antibody labeled with a marker, and the source of the antibody is any of the following: (1) The antibody according to claim 1 or 2; (2) A culture of the host cell according to claim 5 or 6.

9. The product according to claim 8, characterized in that The markers include but are not limited to enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; optionally, the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridges, alkaline phosphatase-anti-alkaline phosphatase bridges, and β-galactosidase-anti-β-galactosidase bridges; the biotins include but are not limited to biotin and its derivatives; the fluoresceins include but are not limited to AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green488, Pacific Blue dye, Pacific Orange dye, Texas Green488, and fluorophores. Red, PerCP dyes; the chemiluminescent type includes but is not limited to isoluminol and its derivatives, acridinium esters and their derivatives, terpyridine ruthenium and its derivatives; the isotope type includes but is not limited to iodine labeling; the colloidal labeling includes but is not limited to colloidal gold, colloidal carbon, and colloidal selenium.

10. A kit, characterized in that: The kit contains the antibody of claim 1 or 2 or the biomarker or chemically labeled product of claim 8 or 9.

11. The kit according to claim 10, characterized in that The kit includes an enzyme-linked immunosorbent assay kit and an immunofluorescence assay kit.

12. The kit according to claim 10 or 11, characterized in that The kit is an immunochromatography kit, comprising a detection card, wherein the detection card comprises: a PVC base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a blotting paper; the sample pad, the conjugation pad, the nitrocellulose membrane and the blotting paper are overlapped and pasted on the base plate in sequence; the conjugation pad is sprayed with a tracer marker labeled with an antibody whose amino acid sequence of a heavy chain variable region is shown in SEQ ID NO: 13 and whose amino acid sequence of a light chain variable region is shown in SEQ ID NO: 14, a C line and a T line, wherein the C line is fixed with a mouse anti-human IgG antibody, and the T line is fixed with an antibody whose amino acid sequence of a heavy chain variable region is shown in SEQ ID NO: 15 and whose amino acid sequence of a light chain variable region is shown in SEQ ID NO:

16.

13. Use of the antibody according to claim 1 or 2, or the polynucleotide according to claim 3, or the expression vector according to claim 4, or the host cell according to claim 5 or 6, or the method according to claim 7, or the biomarker or chemically labeled product according to claim 8 or 9 in the preparation of a product for detecting KPC-type carbapenemase.

14. The use according to claim 13, characterized in that The products include reagents, test kits, detection chips or biosensors.

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