Preparation of an antibody capable of detecting multiple subtypes of KPC and application of a kit thereof

Abstract

The present invention discloses the preparation of antibodies capable of detecting multiple subtypes of KPC and the application of its kit, belonging to the field of biomedicine. A kit for detecting common subtypes of KPC and its application provided by the present invention immunizes and prepares murine antibodies using the conserved antigenic epitopes of each subtype of KPC, screens and pairs them in vitro in various ways, and then obtains a pair of antibodies with good specificity, strong affinity and the ability to bind multiple subtypes. The titer of the two antibodies can reach more than 1 million, and the amino acid sequences of their heavy chain variable regions and light chain variable regions are obtained by sequencing. After recombination, rapid and stable expression can be achieved. The product development of the present invention for detecting carbapenem KPC based on the immunochromatography platform has the advantages of high sensitivity, good specificity, good stability, the ability to cover most prevalent KPC subtypes, and rapid and convenient detection, providing important assistance for clinical auxiliary diagnosis and guiding drug use.

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, belonging to the field of biomedicine. Background Art

[0002] Bacterial resistance has become a major challenge in the global public health field. The emergence and prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pan-drug-resistant (PDR) bacteria have posed a major threat to human health. Among the many drug-resistant bacteria faced in clinical practice, the most important are carbapenem-resistant Gram-negative bacteria, especially the rapidly increasing carbapenem-resistant Enterobacterales (CRE) in recent years.

[0003] Infections caused by CRE are significantly associated with higher morbidity and mortality. In bloodstream infections caused by CRE, the mortality rate can be as high as 53.1%, and the disease burden on patients is heavy. The most common CRE strains are Klebsiella pneumoniae and Escherichia coli. In vitro susceptibility test results show that CRE is usually highly sensitive only to tigecycline, polymyxin, and new β-lactamase inhibitor combinations such as ceftazidime / avibactam, highly resistant to most β-lactam antibiotics including carbapenems, highly resistant to quinolones, and has variable resistance to aminoglycosides. For different types of carbapenemases, the in vitro antibacterial activity of ceftazidime / avibactam is different: ceftazidime / avibactam is ineffective against metalloenzymes but sensitive to serine enzymes, AmpC enzymes, and ESBL enzymes. It is reported that for the treatment of CRE bloodstream infections, the initial antibacterial drug treatment has great clinical significance for improving the survival rate of patients. Therefore, rapid detection of carbapenemase-producing Enterobacterales bacteria in bloodstream infections and identification of the carbapenemase type are essential for early optimization of antibacterial drug treatment and improvement of the survival rate.

[0004] The production of Klebsiella pneumoniae carbapenemase (KPC) is the main mechanism of carbapenem resistance. 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 newly discovered KPC gene subtypes has increased rapidly. Currently, a total of 96 KPC-type carbapenemase gene subtypes have been discovered, namely blaKPC-2 type - blaKPC-108 type. The emergence of new KPC gene subtypes has increased the difficulty of clinical anti-infective treatment and laboratory detection. 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. All newly discovered KPC gene subtypes are variants of KPC-2 and KPC-3, and their main mechanisms are deletion, mutation, insertion, and tandem repeat. Taking common gene subtypes as an example, bla KPC-14 subtype is derived from bla KPC-2 by deleting two amino acid variations (glycine at position 242 and threonine at position 243); bla KPC-33 , bla KPC-51 and bla KPC-52 are formed due to the substitution of the amino acid at position 179 (aspartic acid → tyrosine), and are prone to mutation during treatment; bla KPC-53 is formed by the replication of the amino acids in the Ω loop of bla KPC-3 (leucine at position 167 and glutamic acid at position 168); bla KPC-74 is due to the deletion of 6 nucleotides at positions 712 - 717 of the bla KPC-2 gene (deletion of glycine and valine at positions 239 and 240).

[0006] In actual clinical detection, commercially available control kits can only detect common subtypes such as bla KPC-2 , bla KPC-3 , and cannot detect other common KPC-type carbapenemase variants such as bla KPC-14 , bla KPC-31 , bla KPC-33 , bla KPC104 , bla KPC-106 , bla KPC-108 , bla KPC-139 etc., which may lead to adverse consequences such as missed detection of KPC and clinical medication errors. The present invention intends to develop KPC antibody pairs and has developed an immunochromatographic kit that can rapidly detect multiple subtypes of KPC, which can be used to detect multiple subtypes of KPC, aiming to improve the accuracy of auxiliary diagnosis of drug-resistant bacteria and provide important assistance for clinical medication. Summary of the Invention

[0007] In view of the deficiencies of the above-mentioned prior art, the present invention provides a method for preparing antibodies and the application of a kit for detecting multiple subtypes of KPC, aiming to solve the technical problems that existing kits cannot detect common KPC-type carbapenemase variants such as bla KPC-14 , bla KPC-31 , bla KPC-33 , bla KPC51 , bla KPC-52 etc., which may lead to missed detection of KPC and clinical medication errors.

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

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

[0010] (b) SEQ ID NO.7 to 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 some 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 respectively the sequences shown in any one of (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 fungi include yeast or mold, and the bacteria include 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 is to culture 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 product of a biological label or a chemical label, which is an antibody labeled with a labeling agent, and the source of the antibody is any one of the following:

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

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

[0036] In some embodiments, the markers include, but are not limited to, enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, magnetic beads; the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase conjugate, alkaline phosphatase-anti-alkaline phosphatase conjugate, β-galactosidase-anti-β-galactosidase conjugate; 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 Green488, PacificBlue dye, Pacific Orange dye, Texas Red, PerCP dye; the chemiluminescence includes, but is not limited to, isoluminol and its derivatives, acridinium ester and its derivatives, ruthenium terpyridine and its derivatives, etc.; the isotopes include, but are not limited to, iodine labeling; the colloid markers include, but are 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 product of the biological marker or chemical marker described in the sixth technical solution.

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

[0039] In some embodiments, the kit is an immunochromatographic kit, including a test card, and the test card includes: a PVC bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a blotting paper; the sample pad, the conjugate pad, the nitrocellulose membrane and the blotting paper are sequentially overlapped and pasted on the bottom 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 immobilized with a mouse anti-human IgG antibody, and the T line is immobilized 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 application 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 product with a biological or chemical label described in the sixth technical solution in the preparation of a product for detecting KPC-type carbapenemase.

[0041] In certain embodiments, the product includes 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 subtypes of KPC and its application. Mouse-derived antibodies are prepared by immunizing with conserved antigenic epitopes of each KPC subtype, and are screened and paired in vitro by various methods. Then, a pair of antibodies with good specificity, strong affinity and capable of binding multiple subtypes is obtained. The titer of the two antibodies can reach more than 1 million, and the amino acid sequences of the heavy chain variable region and the light chain variable region are obtained by sequencing. After recombination, rapid and stable expression can be achieved. The product development of the present invention for detecting carbapenem KPC based on the immunochromatography platform has the advantages of high sensitivity, good specificity, covering most prevalent KPC subtypes, and rapid and convenient detection, providing important help for clinical assistant diagnosis and guiding drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is the SDS-PAGE diagram of the purified recombinant antibody of the present invention.

[0045] Figure 2 It is the result diagram of detecting multiple subtypes of KPC by the kit of the present invention and the comparative kit. DETAILED DESCRIPTION OF THE EMBODIMENTS

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

[0047] Raw materials used in the examples:

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

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

[0050] 3. LB liquid medium: It is prepared from 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and the pH value needs to be adjusted to 7.3 ± 0.1.

[0051] LB solid medium: Prepared from 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar powder, and 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 immunization process of BALB / c mice was entrusted to Jiangsu Dongkang Biotechnology Co., Ltd.

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

[0055] Step 1: Plasmid construction

[0056] Compare the two common subtypes bla KPC-2 and bla KPC-3 , and comprehensively analyze in combination with the mutation sites of other subtypes. Select the conserved antigenic epitopes of each subtype of KPC, and intercept bla KPC-2 45-165 AA, bla KPC-2 175-240 AA (Uniprot: Q93LQ9), and its amino acid sequences are shown in SEQ ID NO:17, SEQ ID NO:18, 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] DYAVVWPTGRAPIVLAVYTRAPNKDDKHSEAV.

[0063] According to the E. coli codon principle, optimize the above-mentioned dominant antigenic epitope segments into nucleotide sequences, and then perform gene synthesis (completed by GenScript Biotech Corporation).

[0064] The above-mentioned double digestion method was used to ligate HindⅢ and EcoRI to the pcold expression vector to construct an expression plasmid. 100 ng of the expression plasmid was extracted and added to competent Escherichia coli DH5α cells. Then, the competent cells added with the plasmid were subjected to heat shock transformation. The transformed competent cells were added to LB liquid medium and cultured on a shaker at 37°C and 200 rpm for about 30 min, and then taken out. 150 μL of the suspension was aspirated and spread on an LB solid medium plate for overnight culture. After picking monoclonal colonies for expanded culture, the bacterial liquid was collected to extract the plasmid (operated according to the instruction manual of the plasmid extraction kit produced by Sangon Biotech Co., Ltd.), and at the same time sent to General Biosystems for sequencing confirmation to obtain two plasmids pcold-KPC-SUMO.

[0065] Step 2: Preparation of immunogen

[0066] The above two plasmids pcold-KPC-SUMO were transformed into Escherichia coli BL21(DE3) strain. After culturing on an LB plate at 37°C for 12 - 16 h, monoclonal colonies were picked for expanded culture. When the OD600 reached 0.6 - 0.8 during shaking culture, IPTG with a final concentration of 1 mM was added for induction of expression. After 4 h of expression, the cells were collected by centrifugation.

[0067] The cells were resuspended with binding buffer (50 mM Tris pH8.0, 100 mM NaCl, 1 mM PMSF, 0.5 mM EDTA, 1% glycerol) and sonicated for 30 min on ice bath; then the supernatant was collected by centrifugation at high speed of 14,000 rpm for 15 min. The Ni-NTA column was pre-equilibrated with binding buffer, and the expressed antigen was respectively bound to the Ni-NTA column. The Ni-NTA column was washed with imidazole buffer with gradient concentrations to remove the miscellaneous proteins (30, 50, 80, 100 mM imidazole dissolved in 50 mM Tris pH8.0, 100 mM NaCl buffer system respectively). Then, the target protein was eluted with high-concentration imidazole buffer (200 mM imidazole dissolved in 50 mM Tris pH8.0, 100 mM NaCl buffer system), collected in separate tubes, and the size of the target protein was verified by SDS-PAGE electrophoresis. The two expressed protein segments were 45 - 165 AA and 175 - 240 AA, with sizes of 29 kD and 22 kD respectively. The SUMO tag was removed by SUMO protease and dialyzed and concentrated into the binding buffer for standby.

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

[0069] Step 3: Animal immunization

[0070] The above-mentioned target protein immunogen was emulsified with Freund's complete adjuvant. Five SPF-grade BALB / c mice were selected and injected subcutaneously at a dose of 50 μg / mouse. In the second and third weeks, the antigen emulsified with Freund's incomplete adjuvant was injected subcutaneously for immunization at a dose of 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 the change in antibody level in the serum; on the fifth day after the last immunization, blood was collected by removing the mouse's eyeball, and the blood was collected and allowed to stand until the serum was completely separated. The serum was centrifuged at 3000 rpm for 5 minutes, aliquoted, and stored at -70 °C for later use.

[0072] Step 4: Screening and preparation of monoclonal antibodies

[0073] For the immunized BALB / c mice, the eyeballs were removed and blood was collected as the positive control serum; then the mice were sacrificed by cervical dislocation, disinfected with 75% alcohol, the spleens were taken, and spleen cell suspensions (counted under the microscope) were prepared. The spleen cell suspensions were mixed with myeloma cells SP2 / 0 (counted under the microscope) in serum-free DMEM medium. The ratio of the spleen cell suspension to the myeloma cells SP2 / 0 was 5:1. The mixture was centrifuged at 2000 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended. Immediately, pre-warmed 50% PEG4000 was added to fuse the cells. After standing for 1 minute for fusion, serum-free DMEM medium was added to terminate the fusion. The cells were centrifuged after standing at 37 °C for 10 min, the supernatant was removed, and the cells were resuspended in HAT medium and aliquoted into 96-well plates. The cells were cultured in a cell culture incubator for about 10 days until the fused cells covered 20%-50% of the well bottom. The positive clones were screened by the indirect ELISA method.

[0074] The selected pair of hybridoma cells was continuously subcloned using the limiting dilution method until the positive rate of the subcloned cells reached 100%, and then the cells were expanded in culture. The culture conditions were: 37 °C, 220 rpm, 5% CO2, and antibody IgG was prepared. When the cell viability was less than 50%, the culture medium was taken out and collected into a centrifuge bottle, centrifuged at 12000 rpm for 10 min to collect the supernatant, and filtered through a 0.45 μm filter membrane to remove impurities. The Protein G affinity chromatography column was equilibrated with binding buffer, and antibody purification was carried out in this way. After loading the sample, the miscellaneous proteins were washed away with binding buffer, and eluted with 0.1M Gly-HCl pH3.0. Finally, the antibody properties were determined by SDS-PAGE electrophoresis, and after confirmation, it was dialyzed into 50mM Tris pH8.0, 150mM NaCl, 1mM EDTA buffer.

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

[0076] Step Five: Antibody Titer Detection

[0077] The ELISA detection method was used to detect the titer of the antibodies secreted by the positive cell clones: The above-prepared KPC immunogen mixture was diluted with 50mM Na2CO3 pH9.6 buffer to a final concentration of 1 μg / ml, added to a 96-well plate, coated overnight at 4 °C, washed 3 times with 1×PBST, and patted dry; blocked and incubated with PBST containing 2% BSA (purchased from Sigma) for 2 h, washed in the same way, and the antibodies MKN67 and MKN68 secreted by the above positive cell clones were diluted with 1×PBS buffer at gradients of 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, 1:1280000, 1:2560000 to obtain test products with different concentrations. The diluted test products were added to the above enzyme-linked immunosorbent assay (ELISA) plate and incubated at 37 °C for 1 h for the binding reaction; then 1:4000 diluted HRP-goat anti-mouse IgM (purchased from Sigma) was added and incubated at 37 °C for 1 h for the binding reaction; finally, TMB substrate was added and developed color at room temperature in the dark for 10 min, and the reaction was terminated with sulfuric acid. The OD450nm value was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the titers of each antibody are shown in Table 1.

[0078] Table 1 Antibody Titer

[0079]

[0080]

[0081] Step 6: Antibody Pairing and Purification

[0082] The antibodies secreted by the obtained positive clone cells were paired and screened by the ELISA double antibody sandwich method, and the pairing results are shown in Table 2.

[0083] Table 2 Pairing Results

[0084] Serial number Capture antibody Labeled antibody 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 carried out using an immunochromatography platform, and finally a pair of antibodies was determined as shown in Table 3.

[0086] Table 3 Determined Antibodies

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

[0088] Samples were taken and sent to General Biosystems for sequencing.

[0089] The amino acid sequences of the antibody pairs determined by the present invention are as follows:

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

[0091] SEQ ID NO:13:

[0092] QGQMQQSGLELVKPGASVKASCKTCEFTASNNYISWYKQKPGQSLEWIAWIYAGSGG TTYNQDFTGKAQVDVTDSSSTAYMQFSSLTTEDSAIMWNHICARLRWSVPHWYFDVWGA GTTVTVSSAKTTPPSVYPLAP;

[0093] SEQ ID NO:14:

[0094] VVMTQTPLSLPVSFGDQASISCRSDQSLFHSNGNTWLHWFLQKPGQSPKLLIYRVSNR WSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIKRAD.

[0095] Among them, 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 has the amino acid sequence shown in SEQ ID NO:1, HCDR2 has the amino acid sequence shown in SEQ ID NO:2, and HCDR3 has the amino acid sequence shown in SEQ ID NO:3; the three first light-chain CDR regions include: LCDR1 has the amino acid sequence shown in SEQ ID NO:4, LCDR2 has the amino acid sequence shown in SEQ ID NO:5, and LCDR3 has the amino acid sequence shown 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 as shown in SEQ ID NO:15; the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:16;

[0097] SEQ ID NO:15:

[0098] QVQLQQSGPQLGRPGTSVKVSCHASGYTWTSYWMHWVKQRPGQPLEWIGMIDPSDT HTTLNQKMRDKATVMVDKSSSTAYMEVSSPTSEDSAVYYCAREGFFTTIVLPIIYWGQGTV VTV;

[0099] SEQ ID NO:16:

[0100] VVMSQSPSSLAVFWYEKVTMTCRSSQTVVYSGSQKNYLAWYQQKGPCNRKLLIYWA STRESGVPDRFTGSGSGTDMVLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLEL.

[0101] Among them, 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 is the amino acid sequence shown in SEQ ID NO:7, HCDR5 is the amino acid sequence shown in SEQ ID NO:8, and HCDR6 is the amino acid sequence shown in SEQ ID NO:9; the three second light-chain CDR regions include: LCDR4 is the amino acid sequence shown in SEQ ID NO:10, LCDR5 is the amino acid sequence shown in SEQ ID NO:11, and LCDR6 is the amino acid sequence shown 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 IgG antibody was grafted onto the nucleotide sequence of the human IgG heavy-chain constant region framework, and the light-chain variable region VL nucleotide sequence was grafted onto the nucleotide sequence of the human IgG light-chain constant region framework. The obtained heavy-chain variable region and light-chain variable region of the antibody were recombined with the constant region of murine IgG and cloned into the modified mammalian cell expression vector pCMV-C-Flag for expression in Expi 293F cells (the vector was purchased from Beyotime, and the human IgG constant region gene was synthesized by Tsingke Biological Co., Ltd.). The process of grafting to construct the eukaryotic expression plasmid and the reagents or parameters used in each process are not limited, as long as the techniques in the prior art that can achieve the above purposes can be used, and will not be elaborated here. The antibody was purified according to the standard operating procedure for protein purification (as Figure 1 shown), and the anti-KPC recombinant antibody was obtained by expression, still named MKN67 and MKN68.

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

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

[0105] Step 1: While stirring, add 200 - 800 μL of 0.05 - 0.2 M K2CO3 solution drop by drop to 100 mL of colloidal gold solution. After the addition, seal the container and mix gently for 2 - 5 minutes. Then, add anti - KPC carbapenemase monoclonal antibody MKN67 drop by drop. After the addition, seal the container and mix gently for 2 - 5 minutes, and then let it stand and incubate for 30 - 60 minutes. After the incubation, add 20 - 80 mL of blocking solution (1 - 3% BSA) drop by drop, mix for 2 - 5 minutes, let it stand and block for 30 - 60 minutes. After the standing time, seal the container and mix for 2 - 5 minutes again. After blocking, centrifuge with the following parameters: centrifugal force 4000 - 6000 rpm, at 2 - 8 °C, and centrifugation time 20 - 60 min. After centrifugation, remove the supernatant to obtain the gold - labeled precipitate, and store it with the preservation solution to obtain the gold - labeled solution;

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

[0107] Step 3: Add 1 - 10% sucrose solution to anti - KPC carbapenemase monoclonal antibody MKN68, mix well to form the detection line coating solution. Then, add 1 - 10% sucrose solution to goat anti - mouse IgG antibody, mix well to form the quality control line coating solution. Draw the quality control line coating solution and the detection line coating solution on the nitrocellulose membrane at 0.5 - 2 μL / cm, and dry it to obtain the coated plate;

[0108] Step 4: Stick the gold - labeled pad, absorbent paper, and sample pad on the PVC bottom plate in sequence, cut it into test strips with a width of 3 cm and put them into the corresponding cartridges to form the test card.

[0109] Example 3: Comparative application of the multi - subtype KPC detection kit

[0110] This example selects clinically classified and confirmed positive 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-139Bacterial culture sample. Use an inoculation loop to pick up 3 loops of the bacterial sample, and immerse the inoculation loop in the diluent. Shake it back and forth for at least 30 seconds to ensure that the sample is fully eluted into the diluent tube. Cover the diluent tube cap and use a vortex oscillator to mix evenly. Vertically drop 4 drops of the treated sample processing solution into the sample addition hole of the test card in the kit in Example 2, and observe the displayed results within 15 - 20 minutes. Compare with the synchronous detection of the kit (both the comparison kit 1 and the comparison kit 2 used for comparison are purchased from commercially available products). The test results are statistically shown in Table 4 below and Figure 2 as shown.

[0111] Table 4 Test Results of the Kit

[0112]

[0113]

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

[0115] The kit developed by the present invention can detect all common KPC subtypes measured. Among them, the comparison kit 1 will have missed detections of 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 subtypes; the comparison kit 2 will have missed detections of bla KPC-14 , bla KPC-104 , bla KPC-106 , bla KPC-108 , bla KPC-139 subtypes. The kit of the present invention can make up for the missed detection defects, achieve multi-subtype detection, can cover common drug-resistant subtypes in recent years, and provide help for clinical medication diagnosis and treatment, etc.

[0116] Example 4: Performance Evaluation of the Multi-Subtype KPC Detection Kit

[0117] 1. Detection Limit

[0118] Take the calibrated negative sample, prepare a negative matrix, and dilute the KPC-type carbapenemase at multiple concentration gradients, that is, the dilution concentrations are 1000, 800, 600, 500, 400, 300, 200 pg / mL. Repeat the detection 20 times for each concentration gradient using the kit in Example 2 for 3 consecutive days. Observe the color development degree and positive situation of the detection results at each concentration. Take the lowest concentration with a 95% positive detection rate and consistent color development as the detection limit.

[0119] Table 5 Detection Results

[0120]

[0121]

[0122] According to the above result judgment method, it can be seen from Table 5 that through the analysis of the above detection results, when different negative samples are used to dilute the KPC carbapenemase to the detection limit concentration of 600 pg / mL, the positive detection rates are all slightly higher than 95%. Therefore, through verification, the detection limit of the kit of the present invention is finally determined to be 600 pg / mL.

[0123] 2. Stability

[0124] Using positive samples and negative samples with 3 different antibody concentrations as research materials, three batches of the kits described in Example 2 were taken and stored at 2 - 8°C, 20 ± 2°C, and 30 ± 2°C for 27 months respectively. The test times were the 0th month, 4th month, 8th month, 12th month, 16th month, 20th month, 24th month, 26th month, and 27th month to evaluate the stability of the kits. The results are shown in Tables 6 - 8.

[0125] Table 6 Kit Test (Stored at 2 - 8°C)

[0126]

[0127] Table 7 Kit Test (Stored at 20 ± 2°C)

[0128]

[0129] Table 8 Kit Test (Stored at 30 ± 2°C)

[0130]

[0131]

[0132] It can be seen from Tables 6 - 8 that three batches of kits were stored at 2 - 8°C, 20 ± 2°C, and 30 ± 2°C for 27 months respectively, and were tested with strong positive, medium positive, weak positive, and negative samples at the 0th month, 4th month, 8th month, 12th month, 16th month, 20th month, 24th month, 26th month, and 27th month. The test results were good, and the stability of the kits was excellent.

[0133] 3. Analytical Specificity - Cross Substances

[0134] Using 6 KPC-type carbapenemase weakly positive samples and 6 negative samples as research materials, and adding carbapenemase positive samples of other common enzyme types respectively, the kit of Example 2 was used to test and evaluate the cross-reaction of the kit. The results are shown in Table 9.

[0135] Table 9 Specificity test results

[0136]

[0137]

[0138] Judging from Table 9, the carbapenemase positive samples of common enzyme types will not cause cross-reaction to the kit, and the performance is good.

[0139] 4. Cross-interference experiment

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

[0141] Table 10 Interfering substances affecting detection results

[0142]

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

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

Claims

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

12.

2. The antibody according to claim 1, wherein The anti-KPC carbapenemase antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of the heavy chain variable region and the light chain variable region are respectively the sequences shown in any one of (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 according to 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, wherein The host cell includes bacteria, fungi, animal cells or plant cells; the fungi include yeast or mold, and the bacteria include Escherichia coli.

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

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

9. The product according to claim 8, wherein The labeling agent includes one or more of enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, magnetic beads, etc.; the enzymes include one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase conjugate, alkaline phosphatase-anti-alkaline phosphatase conjugate, β-galactosidase-anti-β-galactosidase conjugate; the biotin includes biotin and / or its derivatives; the fluorescein includes one or more of AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, OregonGreen488, Pacific Blue dye, Pacific Orange dye, Texas Red, PerCP dye; the chemiluminescence includes one or more of isoluminol and its derivatives, acridinium ester and its derivatives, ruthenium terpyridine and its derivatives; the isotopes include iodine labeling; the colloid labeling includes colloidal gold, colloidal carbon or colloidal selenium.

10. A kit, characterized in that, The kit contains the antibody described in claim 1 or 2, or the product of the biomarker or chemical label described in 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 kit.

12. The kit according to claim 10 or 11, characterized in that, The kit is an immunochromatographic kit and includes a test strip, which includes: a PVC bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and a blotting paper; the sample pad, conjugate pad, nitrocellulose membrane, and blotting paper are sequentially lapped and pasted on the bottom 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 immobilized with a mouse anti-human IgG antibody, and the T line is immobilized 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.

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

14. The application according to claim 13, wherein The product includes reagents, kits, detection chips, or biosensors.

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