Preparation of antibody capable of detecting multi-subtype KPC and use of kit thereof

Abstract

The present invention belongs to the field of biomedicine. Disclosed in the present invention are preparation of an antibody capable of detecting multi-subtype KPC and the use of a kit thereof. Provided in the present invention are a kit for detecting common subtypes of KPC and the use thereof. Murine antibodies are prepared by means of the conservative antigen epitope immunization of each subtype of KPC, and in-vitro screening and pairing are performed in various ways so as to obtain an antibody pair with good specificity and strong affinity and which is capable of binding to multiple subtypes. The two antibodies can achieve a titer of 1,000,000 or more, and the amino acid sequences of a heavy chain variable region and a light chain variable region thereof are obtained by means of sequencing, thus rapid and stable expression can be achieved after recombination. In the present invention, the product development for carbapenem KPC detection based on an immunochromatography platform has the advantages of high sensitivity, good specificity, good stability, being able to cover multiple prevalent KPC subtypes, and rapid and convenient detection, and provides notable assistance in terms of clinical auxiliary diagnosis and medication guidance.

Description

Preparation of an antibody for detecting multiple KPC subtypes and its application in a kit Technical Field

[0001] This invention relates to the preparation of an antibody capable of detecting multiple subtypes of KPC and the application of its reagent kit, belonging to the field of biomedicine. Background Technology

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

[0003] CRE (carbapenemitis) infections are significantly associated with high morbidity and mortality rates, with a mortality rate as high as 53.1% in bloodstream infections caused by CRE, resulting in a heavy disease burden for patients. The most common CRE strains are *Klebsiella pneumoniae* and *Escherichia coli*. In vitro drug susceptibility testing shows that CRE is typically only highly sensitive to tigecycline, polymyxins, and novel β-lactamase inhibitor combination preparations such as ceftazidime / avibactam. It is highly resistant to most β-lactam antibiotics, including carbapenems, and also highly resistant to quinolones, with varying resistance to aminoglycosides. Furthermore, the in vitro antibacterial activity of ceftazidime / avibactam varies against different types of carbapenemases: ceftazidime / avibactam is ineffective against metalloenzymes but sensitive to serine enzymes, AmpC enzymes, and ESBL enzymes. It has been reported that initial antimicrobial therapy is of significant clinical importance in improving patient survival rates for the treatment of CRE bloodstream infections. Therefore, rapid detection of carbapenemase-producing Enterobacteriaceae in bloodstream infections and identification of carbapenemase types are essential for early optimization of antimicrobial therapy and improvement of survival rates.

[0004] The production of carbapenemase (KPC) in Klebsiella pneumoniae is a major mechanism of carbapenem resistance. Ceftazidime / avibactam (CZA) is considered a promising combination of β-lactamase inhibitors with activity against serine β-lactamases (including KPC).

[0005] Since 2020, the number of newly discovered KPC genotypes has increased rapidly. Currently, 96 KPC-type carbapenemase genotypes have been identified, ranging from blaKPC-2 to blaKPC-108. The emergence of new KPC genotypes has increased the difficulty of clinical anti-infective treatment and laboratory testing. A study published in *Front Cell Infect Microbiol* in 2020 showed that blaKPC-2 (51.6%) was the most common carbapenemase subtype among CRE strains isolated from adult and pediatric patients. Currently discovered new KPC genotypes are all variants of KPC-2 and KPC-3, and their main mechanisms are deletion, mutation, insertion, and tandem duplication. Taking common gene subtypes as examples, the blaKPC-14 subtype is derived from the mutation of blaKPC-2 by the deletion of two amino acids (242 glycine and 243 threonine); the formation of blaKPC-33, blaKPC-51, and blaKPC-52 is due to the substitution of amino acid at position 179 (aspartic acid → tyrosine), and they are prone to mutation during treatment; blaKPC-53 is formed by the replication of the Ω-ring amino acid of blaKPC-3 (167 leucine and 168 glutamic acid); and blaKPC-74 is the deletion of 6 nucleotides at positions 712-717 of the blaKPC-2 gene (deletion of glycine and valine at positions 239 and 240). Technical issues

[0006] In actual clinical testing, existing commercially available control kits can only detect common subtypes such as blaKPC-2 and blaKPC-3, and cannot detect other common KPC carbapenemase variants such as blaKPC-14, blaKPC-31, blaKPC-33, blaKPC104, blaKPC-106, blaKPC-108, and blaKPC-139. This can lead to undetected KPCs and errors in clinical medication. This invention aims to develop KPC antibody pairs and a rapid immunochromatographic kit for detecting multiple KPC subtypes, thereby improving the accuracy of auxiliary diagnosis of drug-resistant bacteria and providing significant assistance for clinical medication. Technical solutions

[0007] To address the shortcomings of the existing technologies, this invention provides an antibody preparation and kit for detecting multiple KPC subtypes. The purpose is to solve the technical problem that existing kits cannot detect common KPC carbapenemase variants such as blaKPC-14, blaKPC-31, blaKPC-33, blaKPC51, and blaKPC-52, which can lead to missed detection of KPC and incorrect clinical medication.

[0008] The first technical solution provided by this 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, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in any one of (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 some embodiments, the anti-KPC carbapenemase antibody includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are as 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 polynucleotides 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 some embodiments, the host cell includes, but is not limited to, bacteria, fungi, animal cells, or plant cells.

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

[0031] In some 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 carbapenemase antibody, wherein the method involves culturing the host cells 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 this invention is a biomarker or chemically marked product, wherein the product is an antibody marked by a marker, and the antibody originates from any of the following sources:

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

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

[0036] In some embodiments, the labeling agents include, but are not limited to, enzymes, biotinylate, luciferase, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridges, alkaline phosphatase-antialkaline phosphatase bridges, and β-galactosidase-antiβ-galactosidase bridges; the biotinylate class includes, but is not limited to, biotin and its derivatives; the luciferase class includes, but is not limited to, AF350, AF488, AF532, AF546, AF555, and AF56. 8. AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, PerCP dye; the chemiluminescent dyes include, but are not limited to, isoluminol and its derivatives, acridine esters and their derivatives, ruthenium terpyridine and its derivatives, etc.; the isotope dyes include, but are not limited to, iodine labeling; the colloidal labelings 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 reagent kit containing the antibody described in the first technical solution or the biomarker or chemically labeled product described in the sixth technical solution.

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

[0039] In some embodiments, the kit is an immunochromatographic kit, comprising a test card, the 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 adhered to the base plate; the conjugate pad is sprayed with antibody-labeled tracer markers, C-lines and T-lines, wherein the C-line is fixed with mouse anti-human IgG antibody, and the T-line is fixed with antibodies, the heavy chain variable region amino acid sequence shown in SEQ ID NO:13 and the light chain variable region amino acid sequence shown in SEQ ID NO:14.

[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 biomarker or chemically labeled product described in the sixth technical solution in the preparation of a product for detecting KPC type carbapenemase.

[0041] In some embodiments, the product includes reagents, kits, detection chips, or biosensors. Beneficial effects

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

[0043] This invention provides a kit for detecting common KPC subtypes and its applications. Mouse antibodies are prepared by immunization using conserved epitopes of various KPC subtypes. These antibodies are then screened and paired in vitro using various methods to obtain a pair of antibodies with high specificity, strong affinity, and the ability to bind to multiple subtypes. The two antibodies can achieve titers exceeding 1 million. The amino acid sequences of their heavy and light chain variable regions are obtained through sequencing, and rapid and stable expression can be achieved after recombination. This invention, based on an immunochromatographic platform for the detection of carbapenem KPC, offers advantages such as high sensitivity, high specificity, coverage of most prevalent KPC subtypes, and rapid and convenient detection, providing significant assistance for clinical auxiliary diagnosis and medication guidance. Attached Figure Description

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

[0045] Figure 2 shows the results of detecting multiple subtypes of KPC using the kit of the present invention and the comparative kit. Embodiments of the present invention

[0046] The preferred embodiments of the present invention are described below. 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] Raw materials used in the examples:

[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 from 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar powder, with the pH adjusted to 7.3 ± 0.1.

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

[0053] 4. The immunization process for BALB / c mice was commissioned to Jiangsu Dongkang Biotechnology Co., Ltd.

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

[0055] Step 1: Plasmid Construction

[0056] By comparing the two common subtypes, blaKPC-2 and blaKPC-3, and combining a comprehensive analysis of mutation sites in other subtypes, conserved antigenic epitopes of each KPC subtype were selected, and blaKPC-3 was extracted. KPC-2 45-165AA, bla KPC-2 175-240AA (Uniprot: Q93LQ9), its amino acid sequence is shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19:

[0057] SEQ ID NO:17:

[0058] SEQ ID NO:18:

[0059] SEQ ID NO:19:

[0060] Based on the E. coli codon principle, the above-mentioned dominant antigenic epitope segments were optimized into nucleotide sequences, and then the gene was synthesized (completed by Genscript Biotech).

[0061] The above-mentioned method uses a double enzyme digestion method to ligate HindIII and EcoRI into the pcold expression vector to construct the expression plasmid. 100 ng of the expression plasmid was extracted and added to *E. coli* DH5α competent cells. The competent cells after plasmid addition were then heat-shock transformed. The transformed competent cells were added to LB liquid medium and cultured on a shaker at 37°C and 200 rpm for approximately 30 min. Then, 150 μL of the suspension was taken out and spread onto LB solid medium plates for overnight culture. Single colonies were picked and expanded, and the bacterial culture was collected to extract plasmids (according to the instructions of the plasmid extraction kit produced by Sangon Biotech). Simultaneously, the samples were sent to General Biotech for sequencing confirmation, yielding two plasmids: pcold-KPC-SUMO.

[0062] Step 2: Immunogen Preparation

[0063] The two plasmids pcold-KPC-SUMO were transformed into Escherichia coli BL21(DE3) strain. After culturing on LB plates at 37°C for 12–16 h, single colonies were picked and expanded. When the OD600 reached 0.6–0.8, IPTG at a final concentration of 1 mM was added to induce expression. After 4 h of expression, the bacterial cells were collected by centrifugation.

[0064] Bacterial cells were resuspended in binding buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 1 mM MMSF, 0.5 mM EDTA, 1% glycerol) and sonicated on ice for 30 min. The supernatant was then collected by centrifugation at 14,000 rpm for 15 min. Ni-NTA columns were pre-equilibrated with binding buffer, and the expressed antigen was bound to the Ni-NTA column. Contaminating proteins were washed with gradient concentrations of imidazole buffer (30, 50, 80, and 100 mM imidazole dissolved in 50 mM Tris, pH 8.0, and 100 mM NaCl buffer). The target protein was then eluted with high-concentration imidazole buffer (200 mM imidazole dissolved in 50 mM Tris, pH 8.0, and 100 mM NaCl buffer), collected in separate tubes, and its size was verified by SDS-PAGE electrophoresis. The two expressed protein fragments were 45-165 AA and 175-240 AA, with sizes of 29 kDa and 22 kDa, respectively. The SUMO tag was removed using SUMO protease and then concentrated by dialyzing into binding buffer for later use.

[0065] Due to the small molecular weight of the target protein, 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 solution and incubated at room temperature for 1 hour. Excess SPDP was washed away using a HiTrap desalting column. Finally, the target peptide was added to the conjugated BSA-SPDP system and incubated overnight at room temperature. The immunogen was obtained by mixing BSA-KPC (45-165) and BSA-KPC (175-240) in equal proportions.

[0066] Step 3: Animal Immunization

[0067] The target protein immunogen was emulsified with Freund's complete adjuvant. Five SPF-grade BALB / c mice were selected and injected subcutaneously with 50 μg / mouse. In the second and third weeks, the antigen emulsified with Freund's incomplete adjuvant was injected subcutaneously with 50 μg / mouse. In the fourth week, the antigen protein solution was injected to boost the immunization.

[0068] 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 eyeballs of mice. 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.

[0069] Step 4: Monoclonal Antibody Screening and Preparation

[0070] BALB / c mice that had been immunized had their eyes removed and blood collected as positive control serum. The mice were then euthanized by cervical dislocation, disinfected with 75% alcohol, and the spleen was harvested. A spleen cell suspension (microscopically counted) was prepared and mixed with myeloma SP2 / 0 cells (microscopically counted) in serum-free DMEM medium at a ratio of 5:1. The mixture was centrifuged at 2000 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended. Preheated 50% PEG4000 fusion cells were immediately added, and after 1 minute of incubation, serum-free DMEM medium was added to terminate the fusion. The cells were incubated 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 fusion cells covered 20%-50% of the bottom of the wells. Positive clones were screened using an indirect ELISA method.

[0071] A pair of hybridoma cells selected using the limiting dilution method was serially subcloned until the positive rate of subcloned cells reached 100%, and then the cells were expanded for culture. The culture conditions were: 37℃, 220 rpm, 5% CO2, for the preparation of antibody IgG. When the cell viability was less than 50%, the culture medium was collected in a centrifuge flask, centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.45 μm filter to remove impurities. The Protein G affinity chromatography column was equilibrated with binding buffer, and antibody purification was performed using this method. After loading, contaminating proteins were washed away with binding buffer, followed by elution with 0.1 M mgly-HCl pH 3.0. Finally, the antibody properties were determined by SDS-PAGE electrophoresis. After confirmation, the antibody was dialyzed into a 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA buffer.

[0072] 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. Serum collected from immunized mice after enucleation served as the positive control, and the supernatant from cultured SP2 / 0 myeloma cells served as the negative control. A total of 18 positive cell clones secreted antibodies. They were named as follows: MKN02, MKN26, MKN35, MKN38, MKN50, MKN52, MKN67, MKN68, MKN99, MKN102, MKN105, MKN134, MKN135, MKN156, MKN160, MKN169, MKN181, and MKN186.

[0073] Step 5: Antibody titer testing

[0074] The titer of antibodies secreted by positive cell clones was detected by ELISA: The prepared KPC immunogen mixture was diluted with 50 mM Na2CO3 pH 9.6 buffer to a final concentration of 1 μg / ml, added to a 96-well plate, coated overnight at 4°C, washed three 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 manner, and the above-mentioned positive cell clone secreted antibodies MKN67 and MKN68 were serially diluted with 1×PBS buffer at concentrations of 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, 1:1280000, and 1:2560000 to obtain test samples of different concentrations. The diluted test sample was added to the above-mentioned ELISA plate and incubated at 37°C for 1 hour. Then, HRP-goat anti-mouse IgM diluted 1:4000 (purchased from Sigma) was added and incubated at 37°C for 1 hour. Finally, TMB substrate was added and the reaction was carried out at room temperature in the dark for 10 minutes, and the reaction was terminated with sulfuric acid. The OD450nm value was measured by an ELISA reader, and the titers of each antibody are shown in Table 1.

[0075] Table 1 Antibody titer

[0076] Step Six: Antibody Pairing and Purification

[0077] The obtained positive clone cells were screened for paired antibodies using the ELISA double-antibody sandwich method. The pairing results are shown in Table 2.

[0078] Table 2 Matching Results

[0079] Further screening was conducted using an immunochromatographic platform, and a pair of antibodies was finally identified as shown in Table 3.

[0080] Table 3 Identifying Antibodies

[0081] The samples were sent to General Biotech for sequencing.

[0082] The amino acid sequence of the antibody pair determined in this invention is as follows:

[0083] 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;

[0084] SEQ ID NO:13:

[0085] SEQ ID NO:14:

[0086] The antibody MKN67 comprises three first heavy chain CDR regions and three first light chain CDR regions. The three first heavy chain CDR regions are: HCDR1 (amino acid sequence shown in SEQ ID NO:1), HCDR2 (amino acid sequence shown in SEQ ID NO:2), and HCDR3 (amino acid sequence shown in SEQ ID NO:3). The three first light chain CDR regions are: LCDR1 (amino acid sequence shown in SEQ ID NO:4), LCDR2 (amino acid sequence shown in SEQ ID NO:5), and LCDR3 (amino acid sequence shown in SEQ ID NO:6). 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.

[0087] 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;

[0088] SEQ ID NO:15:

[0089] SEQ ID NO:16:

[0090] The antibody MKN68 comprises three second heavy chain CDR regions and three second light chain CDR regions. The three second heavy chain CDR regions are: HCDR4 (amino acid sequence shown in SEQ ID NO:7), HCDR5 (amino acid sequence shown in SEQ ID NO:8), and HCDR6 (amino acid sequence shown in SEQ ID NO:9); the three second light chain CDR regions are: LCDR4 (amino acid sequence shown in SEQ ID NO:10), LCDR5 (amino acid sequence shown in SEQ ID NO:11), and LCDR6 (amino acid sequence shown in SEQ ID NO:12). 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.

[0091] Using molecular construction methods, the heavy chain variable region of the above-mentioned IgG antibody was grafted onto the nucleoside sequence of the human IgG heavy chain constant region backbone, and the VL nucleotide sequence of the light chain variable region was grafted onto the nucleotide sequence of the human IgG light chain constant region backbone. 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 (vector purchased from Beyotime, human IgG constant region gene synthesized from Qingke Biotechnology Co., Ltd.). The process of grafting and constructing eukaryotic expression plasmids and the reagents or parameters used in each process are not limited. Any existing technology that can achieve the above objectives is acceptable and will not be elaborated here. Antibody purification was performed according to the standard operating procedure for protein purification (as shown in Figure 1), and the anti-KPC recombinant antibody was obtained by expression, still named MKN67 and MKN68.

[0092] Example 2: Preparation of a multi-subtype KPC detection kit

[0093] This invention utilizes the aforementioned antibody pairs to develop a kit for detecting multiple KPC subtypes based on an immunochromatographic platform. The preparation method of the kit is as follows:

[0094] Step 1: While stirring, add 200-800 μL of 0.05-0.2 MK2CO3 solution dropwise to 100 mL of colloidal gold solution. After addition, seal the container and mix slowly for 2-5 minutes. Then, add the anti-KPC carbapenemase monoclonal antibody MKN67 dropwise. After addition, seal the container and mix slowly for 2-5 minutes. Incubate for 30-60 minutes. After incubation, add 20-80 mL of blocking buffer (1-3% BSA) dropwise, mix for 2-5 minutes, and incubate for 30-60 minutes. After incubation, seal the container and mix for another 2-5 minutes. After blocking, centrifuge at 4000-6000 rpm, 2-8℃, and for 20-60 minutes. After centrifugation, discard the supernatant to obtain the gold-labeled precipitate. Preserve the precipitate in preservation solution to obtain the gold-labeled solution.

[0095] Step 2: Spray the gold labeling solution onto the glass cellulose membrane and dry it to make a gold labeling pad;

[0096] Step 3: Add 1-10% sucrose solution to the anti-KPC carbapenemase monoclonal antibody MKN68, mix well to form the detection line coating solution, then add 1-10% sucrose solution to the goat anti-mouse IgG antibody, mix well to form the control line coating solution, and streak the control line coating solution and the detection line coating solution at 0.5-2 μL / cm onto the nitrocellulose membrane, dry to obtain the coated plate;

[0097] Step 4: Attach the gold label pad, absorbent paper, and sample pad to the PVC base plate in sequence, cut them into 3cm wide test strips, and insert them into the corresponding card holders to form a test card.

[0098] Example 3: Comparative Application of Multi-Subtype KPC Detection Kits

[0099] This embodiment uses 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-139For bacterial culture samples, pick up 3 loops of bacterial sample using an inoculation loop, immerse the inoculation loop in the diluent, and shake back and forth to mix for at least 30 seconds to ensure the sample is fully eluted into the diluent tube. Cap the diluent tube and vortex until homogeneous. Vertically add 4 drops of the treated sample solution to the sample well of the test card in Example 2. Observe the results within 15-20 minutes. Simultaneous detection with the control kit (Control Kit 1 and Control Kit 2 were both purchased commercially available products). The detection results are shown in Table 4 and Figure 2 below.

[0100] Table 4. Test results of the reagent kit

[0101] Note: √ indicates detectable, × indicates undetectable.

[0102] The kit developed in this invention can detect all common KPC subtypes, while kit 1 will show 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 false negatives; in contrast, kit 2 may have bla KPC-14 bla KPC-104 bla KPC-106 bla KPC-108 bla KPC-139 The kit of this invention can compensate for the deficiency of missed detection of subtypes, realize the detection of multiple subtypes, and cover common drug-resistant subtypes in recent years, providing assistance for clinical drug diagnosis and treatment.

[0103] Example 4: Performance evaluation of the multi-subtype KPC detection kit

[0104] 1. Detection limit

[0105] Take the labeled negative samples and prepare a negative matrix. Perform multiple concentration gradient dilutions of KPC carbapenemase, namely, dilution concentrations of 1000, 800, 600, 500, 400, 300, and 200 pg / mL. Repeat the test 20 times for each concentration gradient using the kit in Example 2. Detect for 3 consecutive days and observe the color development and positive status of the test results for each concentration. The lowest concentration with a positive detection rate of 95% and consistent color development is taken as the limit of detection.

[0106] Table 5 Test Results

[0107] Based on the above result judgment method, as can be seen from Table 5, through the analysis of the above detection results, when different negative samples were used to dilute KPC carbapenemase to the detection limit concentration of 600 pg / mL, the positive detection rate was slightly higher than 95%. Therefore, through verification, the detection limit of the kit of the present invention was finally determined to be 600 pg / mL.

[0108] 2. Stability

[0109] Using positive and negative samples with three different antibody concentrations as research materials, three batches of the kit described in Example 2 were stored at 2–8℃, 20±2℃, and 30±2℃ for 27 months, respectively. Testing was conducted 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.

[0110] Table 6. Reagent kit testing (store at 2–8°C)

[0111] Table 7. Reagent kit testing (store at 20±2℃)

[0112] Table 8. Reagent kit testing (store at 30±2℃)

[0113] As can be seen from Tables 6-8, the three batches of reagent kits were stored at 2-8℃, 20±2℃ and 30±2℃ for 27 months, respectively. At 0 months, 4 months, 8 months, 12 months, 16 months, 20 months, 24 months, 26 months and 27 months, strong positive, medium positive, weak positive and negative samples were tested, respectively. The test results were good and the reagent kits had excellent stability.

[0114] 3. Analysis of specificity – cross-substances

[0115] Six weakly positive KPC carbapenemase samples and six negative samples were used as research materials. Other common carbapenemase positive samples were added to these samples, and the cross-reactivity of the kit was evaluated using the kit described in Example 2. The results are shown in Table 9.

[0116] Table 9 Specificity Test Results

[0117] Based on Table 9, it can be determined that carbapenemase-positive samples of common enzyme types do not exhibit cross-reactivity with the kit, indicating good performance.

[0118] 4. Cross-interference experiment

[0119] Negative matrix and KPC carbapenemase borderline positive samples were selected as base samples and a certain concentration of interfering substance was added to confirm whether a certain concentration of interfering substance would affect the test results.

[0120] Table 10 Results of Detection of Interfering Substances

[0121] The results in Table 10 demonstrate that the kit can effectively resist interference from both endogenous and exogenous substances.

[0122] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined 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, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in any one of (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 carbapenemase antibody includes a heavy chain variable region and a light chain variable region, the amino acid sequences of which are as 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 of claim 1 or 2.

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

5. A host cell expressing the antibody of claim 1 or 2, or containing the polynucleotide of claim 3, or transformed with the expression vector of 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 carbapenemase antibody, characterized in that, The method is to culture the host cells as described in claim 5 or 6 to obtain a culture containing the antibody as described in claim 1 or 2.

8. A product marked with a biomarker or a chemical marker, characterized in that, The product is an antibody labeled with a marker, and the antibody originates from any of the following sources: (1) The antibody according to claim 1 or 2; (2) The culture of the host cell as described in claim 5 or 6.

9. The product according to claim 8, characterized in that, The labeling agents include, but are not limited to, enzymes, biotinylate, luciferase, 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-antialkaline phosphatase bridges, and β-galactosidase-antiβ-galactosidase bridges; the biotinylate class includes, but is not limited to, biotin and its derivatives; the luciferase class includes, but is not limited to, AF350, AF488, AF532, AF546, AF555, and AF568. AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, PerCP dye; the chemiluminescent dyes include, but are not limited to, isoluminol and its derivatives, acridine esters and their derivatives, ruthenium terpyridine and its derivatives; the isotope dyes include, but are not limited to, iodine labeling; the colloidal labeling includes, but is not limited to, colloidal gold, colloidal carbon, and colloidal selenium.

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

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

12. The kit according to claim 10 or 11, characterized in that, The kit is an immunochromatographic kit, comprising a test card, which includes: 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 adhered to the base plate; the conjugate pad is sprayed with antibody-labeled tracer markers, C-lines and T-lines, wherein the C-line is fixed with mouse anti-human IgG antibody, and the T-line is fixed with antibodies, wherein the heavy chain variable region amino acid sequence is fixed with SEQ ID NO:13, and the light chain variable region amino acid sequence is fixed with SEQ ID NO:

16.

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

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

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