Preparation of anti-MCR-1 and anti-MCR-2 monoclonal antibody and development and application of kit of anti-MCR-1 and anti-MCR-2 monoclonal antibody

Abstract

The invention discloses preparation of an anti-MCR-1 and anti-MCR-2 monoclonal antibody and development and application of a kit of the anti-MCR-1 and anti-MCR-2 monoclonal antibody, and belongs to the technical field of biological detection. The antibody disclosed by the invention can be used for detecting MCR-1 and MCR-2 proteins, and the detection efficiency of polymyxin drug resistance can be properly improved. The screened antibody can be efficiently paired, shows an excellent synergistic effect in a double-antibody sandwich method, and is strong in signal and low in background. Through surface plasmon resonance detection, the affinity constants of the antibody to MCR-1 and MCR-2 proteins reach a nanomole level, high sensitivity of detection is ensured, the detection limit of recombinant proteins can reach 0.01 ng / mL, and the detection limit of bacteria can reach 104 CFU / mL. The whole detection process only needs 10-15 minutes, the operation is simple, professional equipment and training are not needed, and the method is very suitable for on-site rapid screening. The kit is simple in component, low in production cost and easy to produce and popularize on a large scale.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation of a monoclonal antibody against MCR-1 and MCR-2 and the development and application of its kit, belonging to the field of biological detection technology. BACKGROUND

[0002] Bacterial drug resistance has become a major challenge in the field of global public health. The emergence and prevalence of multi-drug resistant (MDR), extensively drug resistant (XDR), and even pandrug-resistant (PDR) bacteria pose a significant threat to human health, especially the rapidly increasing carbapenem-resistant Enterobacteriaceae (CRE) in recent years.

[0003] Polymyxins have become the last line of defense for treating carbapenem-resistant Gram-negative bacterial infections. However, with the widespread use of polymyxins in clinical and animal husbandry, the problem of drug resistance has become increasingly prominent. Polymyxins are a group of cyclic peptide antibiotics produced by Paenibacillus polymyxa, consisting of components A, B, C, D, E, etc. Its antibacterial mechanism is divided into two stages: first, polymyxins interact with lipopolysaccharide (LPS) present in the outer membrane (OM), causing the outer membrane to swell; then, through the "self-promoted uptake" mechanism, it penetrates the outer membrane, causing the physical integrity of the cell membrane phospholipid bilayer to be destroyed, resulting in osmotic imbalance and killing bacteria. The outer membrane of Gram-negative bacteria plays a key role as a permeability barrier, controlling the entry and exit of most antibiotics and harmful substances into cells. Polymyxins can bind to the free phosphate of the lipoprotein in the sensitive bacterial plasma membrane, reducing the surface tension of the plasma membrane and increasing its permeability, causing the plasma membrane to lose its barrier function and causing the outflow of purines, pyrimidines, and nucleotides in the cell, leading to bacterial cell death.

[0004] In 2015, Chinese scientists first reported a mobile plasmid-mediated polymyxin resistance gene MCR-1 / 2, which mainly exists in the Enterobacteriaceae family. In 2016, research results showed that MCR-1 is carried by a plasmid independent of the bacterial chromosome and can be horizontally transferred between enteric flora, thereby conferring polymyxin resistance to the recipient bacteria, causing global public health concern. MCR-1 is a phosphoethanolamine transferase. Since the discovery of the MCR-1 gene, multiple MCR families (MCR-1 to MCR-8) have been identified, of which MCR-1 and MCR-2 have approximately 81% protein sequence homology, and lower homology with other MCR families. MCR-1 protein is anchored to the periplasm of the plasma membrane, and bacterial LPS is synthesized in the cytoplasm and converted to periplasm by ABC transporter MSBA. Lipid A in LPS is covalently modified by phosphoethanolamine in the periplasm. The modified LPS has reduced affinity for polymyxins, conferring resistance to polymyxins.

[0005] Currently, the main methods for detecting MCR-1 drug-resistant bacteria include: microbiological drug susceptibility test, which is complicated and time-consuming (usually 18-24 hours). Molecular biology methods (such as PCR, real-time fluorescent PCR), which have high sensitivity, but require professional experimental equipment and personnel, and are high in cost, and are difficult to popularize in primary laboratories or on-site detection. In addition, there is genome sequencing, but its cost is high, and the data analysis is complex, which is not suitable for large-scale screening. Recently, there have been studies to develop an immunodetection method for MCR-1, which uses a sandwich ELISA method. This method still requires laboratory equipment and multiple steps, which is difficult to meet the needs of on-site rapid detection.

[0006] Therefore, there is an urgent need in the art for a method that can quickly, simply and highly specifically detect MCR-1 and MCR-2 proteins or polymyxin-resistant bacteria for clinical diagnosis, food safety monitoring and environmental health monitoring. SUMMARY

[0007] In view of the above problems of the prior art, the present application provides a preparation of a monoclonal antibody of MCR-1 / 2 and development and application of a kit thereof, aiming to solve the technical problem that there is currently a lack of a method that can quickly, simply and highly specifically detect MCR-1 and MCR-2 proteins or polymyxin-resistant bacteria.

[0008] The first technical solution provided by the present application is an anti-MCR-1 and / or MCR-2 protein antibody, which comprises a light chain variable region and a heavy chain variable region, the light chain variable region has a light chain CDR composed of CDR L1, CDR L2, CDR L3, and the heavy chain variable region has a heavy chain CDR composed of CDR H1, CDR H2, CDR H3, the amino acid sequences of the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR H3 are shown in any one of (A)~(B): (A) SEQ ID NO: 4~6, SEQ ID NO: 8~10; (B) SEQ ID NO: 12~14, SEQ ID NO: 16~18.

[0009] In some embodiments, the amino acid sequences of the light chain variable region and the heavy chain variable region are as shown in any one of (C)~(D): (C) SEQ ID NO: 3, SEQ ID NO: 7; (D) SEQ ID NO: 11, SEQ ID NO: 15.

[0010] The second technical solution provided by the present application is a gene encoding the anti-MCR-1 and / or MCR-2 protein antibody of the first technical solution.

[0011] The third technical solution provided by the present application is a recombinant vector carrying the gene of the second technical solution.

[0012] In some embodiments, the recombinant vector uses plasmid pcDNA3.1 as an expression vector.

[0013] The fourth technical solution provided by the present application is a recombinant cell expressing the anti-MCR-1 and / or MCR-2 protein antibody of the first technical solution, or containing the gene of the second technical solution, or transformed with the recombinant vector of the third technical solution.

[0014] In some embodiments, the recombinant cell uses fungi, bacteria, animal cells or plant cells as hosts.

[0015] In some embodiments, the fungi include yeast or mold, and the bacteria include Escherichia coli.

[0016] In some embodiments, the animal cells include but are not limited to 293 cells.

[0017] The fifth technical solution provided by the present application is a method for preparing an anti-MCR-1 and / or MCR-2 protein antibody, which comprises culturing the recombinant cell of the fourth technical solution to obtain a culture containing the anti-MCR-1 and / or MCR-2 protein antibody of the first technical solution.

[0018] The sixth technical solution provided by the present application is a product of a biological marker or a chemical marker, which is an antibody labeled by a marker, and the antibody is derived from any one of the following: (1) the anti-MCR-1 and / or MCR-2 protein antibody of the first technical solution; (2) the culture of the recombinant cell of the fourth technical solution.

[0019] 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.

[0020] The seventh technical solution provided by the present invention is a kit containing the anti-MCR-1 and / or MCR-2 protein antibodies described in the first technical solution or the biomarker or chemically labeled products described in the sixth technical solution.

[0021] In some embodiments, the kit includes an enzyme-linked immunosorbent assay (ELISA) kit and an immunochromatographic assay kit.

[0022] In some embodiments, the kit includes 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 adhered to the base plate; the conjugate pad is coated with a tracer marker of the detection antibody, a C-line, and a T-line, wherein the C-line is fixed with mouse anti-human IgG antibody, and the T-line is fixed with capture antibody; the detection antibody is an MCR-1 / MCR-2 protein antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO:3 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:7; the capture antibody is an MCR-1 / MCR-2 protein antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO:11 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:15.

[0023] The eighth technical solution provided by the present invention is the application of the anti-MCR-1 and / or MCR-2 protein antibody described in the first technical solution, or the gene described in the second technical solution, or the expression recombinant vector described in the third technical solution, or the recombinant 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 products for detecting MCR-1 and / or MCR-2 proteins.

[0024] In some embodiments, the product includes reagents, kits, detection chips, or biosensors.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: Broad spectrum: The monoclonal antibody provided by this invention can detect both MCR-1 and MCR-2 proteins, which can appropriately improve the detection efficiency of polymyxin resistance. The screened antibodies can pair efficiently and exhibit excellent synergistic effects in the double antibody sandwich method, with strong signal and low background.

[0026] High sensitivity: Surface plasmon resonance (SPR) detection shows that the antibody's affinity constants for both MCR-1 and MCR-2 proteins reach the nanomolar (nM) level, ensuring high detection sensitivity. The detection limit for recombinant proteins is as low as 0.01 ng / mL, and the detection limit for bacteria is as low as 10 ng / mL. 4 CFU / mL.

[0027] Fast and convenient: The entire testing process takes only 10-15 minutes, is simple to operate, requires no professional equipment or training, and is very suitable for rapid on-site screening.

[0028] Low cost: The reagent kit has simple components, low production cost, and is easy to mass-produce and promote. Attached Figure Description

[0029] Figure 1 This is a comparison of the MCR-1 (210-541AA) and MCR-2 (217-538AA) sequences in specific embodiment 1 of the present invention.

[0030] Figure 2 The purification results of antibodies MK-3C3 and MK-5D6 in Specific Example 1 of this invention are shown.

[0031] Figure 3 This is a graph showing the sensitivity detection results of the reagent kit in specific embodiment 2 of the present invention.

[0032] Figure 4 This is a graph showing the specific detection results of the reagent kit in specific embodiment 2 of the present invention. Detailed Implementation

[0033] 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.

[0034] Raw materials used in the examples: 1. pET28a vector and pcDNA3.1 vector were purchased from Takara.

[0035] 2. Escherichia coli DH5α and Escherichia coli BL21 (DE3) were purchased from Takara.

[0036] 3. LB liquid medium: prepared with 10 g / L peptone, 5 g / L yeast extract, and 5 g / L sodium chloride, with the pH adjusted to 7.3±0.1; 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, with the pH adjusted to 7.3±0.1; Serum-free DMEM medium was purchased from Gibco; fetal bovine serum was purchased from Sijiqing; and trypsin was purchased from Gibco.

[0037] 4. RPMI-1640 culture medium was purchased from Nanjing Beyotime Biotechnology Co., Ltd.

[0038] 5. HAT culture medium was purchased from Guangzhou Aolong Biotechnology Co., Ltd.

[0039] 6. The immunization process for BALB / c mice was commissioned to Jiangsu Dongkang Biotechnology Co., Ltd.

[0040] Example 1: Preparation of Monoclonal Antibodies 1. Immunity and Fusion Recombinant MCR-1 protein: MCR-1 protein 210-541AA was obtained by expression and purification using an E. coli expression system (amino acid sequence referenced from GenBank: ALU62862.1, as shown in SEQ ID NO.1).

[0041] SEQ ID NO.1: YKKASAPKDTIYHAKDAVQATKPDMRKPRLVVFVVGETARADHVSFNGYERDTFPQLAKIDGVTNFSNVTSCGTSTAYSVPCMFSYLGADEYDVDTAKYQENVLDTLDRLGVSILWRDNNSDSKGVMDKLPKAQFADYKSATNNAICNTNPYNECRDVGMLVGLDD FVAANNGKDMLIMLHQMGNHGPAYFKRYDEKFAKFTPVCEGNELAKCEHQSLINAYDNALLATDDFIAQSIQWLQTHSNAYDVSMLYVSDHGESLGENGVYLHGMPNAFAPKEQRSVPAFFWTDKQTGITPMATDTVLTHDAITPTLLKLFDVTADKVKDRTAFIR.

[0042] Recombinant MCR-2 protein: MCR-2 protein 217-538AA was obtained by expression and purification using an E. coli expression system (amino acid sequence reference Uniprot: A0A1C3NEV1, as shown in SEQ ID NO.2).

[0043] SEQ ID NO.2: YKKATAPTDTIYHAKDAVQTTKPSERKPRLVVFVVGETARADHVQFNGYGRETFPQLAKVDGLANFSQVTSCGTSTAYSVPCMFSYLGQDDYDVDTAKYQENVLDTLDRLGVGILWRDNNSDSKGVMDKLPATQYFDYKSATNNTICNTNPYNECRDVGMLVGLDD YVSANNGKDMLIMLHQMGNHGPAYFKRYDEQFAKFTPVCEGNELAKCEHQSLINAYDNALLATDDFIAKSIDWLKTHEANYDVAMLYVSDHGESLGENGVYLHGMPNAFAPKEQRAVPAFFWSNNTTFKPTASDTVLTHDAITPTLLKLFDVTAGKVKDRAAFIQ.

[0044] The target genes (SEQ ID NO: 19, 20) were ligated into the pET28a expression vector using BamHI and EcoRI via double enzyme digestion to construct recombinant plasmids pET28a-MCR-1 and pET28a-MCR-2, respectively. The recombinant plasmids pET28a-MCR-1 and pET28a-MCR-2 were transformed into *Escherichia coli* BL21(DE3) strain and cultured on LB agar plates at 37°C for 12–16 h. Single colonies were picked and expanded, and the culture was incubated with shaking until the OD600 reached 0.6–0.8. IPTG was then added at a final concentration of 0.5 mM to induce expression. After 4 h of expression, the bacterial cells were collected by centrifugation. The cells were lysed by sonication under ice bath, and the supernatant was collected by high-speed centrifugation. The target proteins MCR-1 and MCR-2 were purified using a Ni-NTA column. The purity of the purified proteins was verified by SDS-PAGE >95%, and the concentration was tested by the BCA method. The purified proteins were aliquoted and stored at -80°C.

[0045] Sequence and structural analysis revealed high homology and structural similarity between SEQ ID NO.1 and SEQ ID NO.2. Therefore, they were synthesized as immunogens to prepare polymyxin-resistant bacteria capable of detecting both genotypes. Six- to eight-week-old female BALB / c mice, SPF grade, were selected as experimental animals and housed in a standard environment. For the initial immunization, 60 µg of recombinant MCR-1 protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites (4-6 points on the back), 50 µL per site. Two booster immunizations were administered two weeks later, for a total of two booster immunizations. Each booster consisted of 50-100 µg of protein emulsified with an equal volume of Freund's incomplete adjuvant and injected using the same method as the initial immunization. Seven to ten days after the second booster immunization, blood was collected via the tail vein, and serum was separated. Antibody titers were determined using an indirect ELISA at a 1:10 ratio. 4 A dilution with an OD450nm value >1.0 and significantly higher than the negative control (pre-immunization serum) was considered acceptable. Finally, the mice with the highest titer were selected, and 50 µg of purified protein without adjuvant (dissolved in sterile PBS) was injected intraperitoneally 3 days before cell fusion as a final booster.

[0046] Spleens were aseptically harvested from immunized mice and placed in pre-chilled serum-free RPMI-1640 medium. The cells were homogenized using a syringe core and passed through a 200-mesh cell sieve. The cell suspension was collected, centrifuged at 1000 rpm for 5 minutes, and treated with erythrocyte lysis buffer to remove red blood cells. The cells were washed, resuspended, and counted for later use. SP2 / 0 cells were prepared by routine passage in RPMI-1640 medium containing 10% fetal bovine serum 3 days before fusion, ensuring the cells were in the logarithmic growth phase and viability >95%. Spleen cells were mixed with SP2 / 0 cells at a ratio of 5:1 to 10:1, centrifuged, and the supernatant was discarded. The cell pellet was placed in a 37°C water bath, and 1 mL of 50% PEG-1500 (w / v) was slowly added over 1 minute with gentle stirring. Then, 20 mL of pre-warmed serum-free medium was slowly added over 2 minutes to terminate the fusion. After centrifugation, the cells were gently resuspended in RPMI-1640 complete medium containing 20% ​​fetal bovine serum, 1×HAT supplement, 1% sodium pyruvate, 1% non-essential amino acids, and 2 mM L-glutamine. The cell suspension was seeded into 96-well cell culture plates at approximately 100 µL per well (containing approximately 1×10⁻⁶ g of glutamine). 5 (Spleen cells). Place in an incubator at 37℃ and 5% CO2 saturated humidity for static culture.

[0047] 2. Screening, subcloning, and stability assessment Starting 4-5 days after fusion, half the HAT medium was replaced with fresh medium every 2-3 days. After 10-14 days of culture, clear clones were visible under a microscope. Initial screening was performed using an indirect ELISA method. Recombinant MCR-1 and MCR-2 proteins were diluted to 1 µg / mL with carbonate coating buffer (pH 9.6), and 100 µL was added to each well of a 96-well microplate, incubated overnight at 4°C. After coating, the plates were washed with 1×PBST, and then 200 µL of PBST containing 5% skim milk (0.05% Tween-20) was added to each well, and the plates were blocked at 37°C for 1 hour. After washing with 1×PBST, 100 µL of the culture supernatant was added to the corresponding well, along with a positive control (immunized mouse serum) and a negative control (SP2 / 0 culture supernatant and non-immunized mouse serum), and incubated at 37°C for 1 hour. After washing with 1×PBST, HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) was added, and the plates were incubated at 37°C for 1 hour. After washing with 1×PBST, TMB substrate solution was added for color development, and the reaction was terminated with 2M H2SO4. The absorbance was read at 450 nm. Wells with an OD value ≥ 2.1 times the mean of the negative control were considered positive.

[0048] For strongly positive wells (typically with the highest OD values), subcloning should be performed immediately using limiting dilution. Cells from positive wells should be gently blown off, counted, and serially diluted to a final concentration of approximately 3-5 cells / mL. Seeds should be placed into new 96-well plates at 100 µL per well. Irradiated-inactivated BALB / c mouse spleen cells (approximately 1 × 10⁻⁶ cells) should be pre-added to the culture plate as a feeder layer. 4 (Cells / well). After culturing for 7-10 days, mark the wells where a single clone has grown. When the clone has grown to more than 1 / 3 of the bottom area of ​​the well, collect the supernatant again for indirect ELISA screening. Repeat the above process at least 3 times until the positivity rate of all positive subclones in the wells is 100% to ensure that hybridoma cells of monoclonal origin are obtained.

[0049] The resulting monoclonal hybridoma cell lines were continuously passaged (20-30 generations), with each generation cryopreserved as a backup. Antibody titers were periodically measured using the supernatant from the logarithmic growth phase. Stable clones with titer fluctuations within ±15% were selected. Cell cryopreservation and the establishment of a tertiary cell bank were prepared. 10-20 vials of the selected monoclonal cells were expanded and cryopreserved to form the primary cell bank (PCB). One vial was thawed from the PCB, expanded, and cryopreserved to form the master cell bank (MCB). Before establishment, sterility, mycoplasma testing, and antibody secretion function verification were required. One vial was thawed from the MCB, expanded on a small scale, and cryopreserved to form the working cell bank (MCB), which was directly used for subsequent production.

[0050] 3. Antibody production and purification Healthy 8-10 week old BALB / c mice were sensitized for 7-10 days by intraperitoneal injection of 0.5 mL of liquid paraffin. Hybridoma cells in the logarithmic growth phase were resuspended in PBS and counted. Each mouse was injected intraperitoneally with 5 × 10⁵ mL of liquid paraffin. 5 ~1×10 6Cells (0.5-1 mL volume). 7-14 days post-inoculation, after significant abdominal distension in mice, aspirate ascites using a sterile syringe needle. Multiple collections can be performed at 2-3 day intervals. Immediately place the collected ascites on ice, centrifuge at 3000 rpm for 10 minutes, collect the supernatant, and freeze at -20°C or -80°C for later use. After thawing, filter the ascites through a 0.45 µm filter membrane. Dilute with an equal volume of Binding Buffer (20 mM sodium phosphate, 0.15 M NaCl, pH 7.0). Perform Protein G affinity chromatography, equilibrating the column with 5-10 column volumes of Binding Buffer. Load the pretreated ascites at a flow rate of 1-2 mL / min, then wash with Binding Buffer until the A280 baseline stabilizes to remove non-specific binders. Finally, elute with 0.1 M glycine-HCl buffer (pH 2.7), and immediately neutralize the eluent in the collection tube with 1 / 10 volume of 1 M Tris-HCl (pH 9.0). Combine the elution peaks, place them in a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyze overnight with PBS (pH 7.4) at 4°C, changing the buffer 2-3 times during the process. Concentrate to a high concentration (5-10 mg / mL) using an ultrafiltration centrifuge tube.

[0051] Antibodies MK-5D6 and MK-3C3 were obtained by filtration through a 0.22 µm sterile filter membrane. Concentrations were determined, and the antibodies were aliquoted as needed, labeled, and stored at -80°C. Purified antibodies underwent SDS-PAGE (reduced and non-reduced) band analysis to ensure purity >95%, and concentrations were determined. After aliquoting, the antibodies were stored at -80°C.

[0052] Example 2: Antibody Characterization and Pairing Screening Antibody subtype identification and sequencing: The monoclonal antibody subtype identification kit from Detai Biotechnology was used for detection. The results are shown in Table 1. It was determined that the antibodies MK-5D6 and MK-3C3 obtained in Example 1 are both IgG1 subtypes with the κ light chain. After purification, the antibodies were sent to Qingke Biotechnology for sequencing, and the obtained sequences are shown in Table 2 below.

[0053] Table 1: Antibody Subtype Identification Results

[0054] Table 2: Summary of Antibody Variable Region Sequences

[0055] Affinity assay: A Cytiva series SPR instrument and Series S CMS chip were used. Recombinant MCR-1 protein was diluted to 10 µg / mL with acetate buffer at pH 5.0 and immobilized onto the chip surface via amine coupling. Surface plasmon resonance (SPR) analysis showed that the dissociation constant (KD) between antibody MK-5D6 and MCR-1 protein was 3.9 × 10⁻⁶. -9 The dissociation constant (KD) of M with MCR-2 protein is 2.5 × 10⁻⁶. -9 M, the dissociation constant (KD) between antibody MK-3C3 and MCR-1 protein is 2.6 × 10⁻⁶. -9 The dissociation constant (KD) of M with MCR-1 protein is 3.1 × 10⁻⁶. -9 M indicates that both antibodies have high affinity for both MCR-1 and MCR-2.

[0056] Pairing validation: Using MK-3C3 as the capture antibody and MK-5D6 as the detection antibody, the test was performed in a sandwich ELISA or immunochromatography. The results showed that the paired combination had a strong signal and low background, making it suitable for sandwich method detection.

[0057] The ELISA assay procedure is as follows: Dilute the capture antibody MK-3C3 to the recommended working concentration (1 μg / mL) with carbonate coating buffer (pH 9.6). Add 100 μL of the diluted capture antibody to each well of the ELISA plate and incubate overnight at 4°C. Discard the liquid in the wells and wash three times with PBST buffer containing 0.05% Tween-20, allowing it to stand for 1 minute each time and then pat dry. Add 200 μL of PBST blocking buffer containing 5% BSA to each well and block at 37°C for 1 hour. After washing three times (as above), add 100 μL of serially diluted target antigen solution per well to the experimental group; add an equal volume of diluent without antigen to the negative control group; add only diluent to the blank group; incubate at 37°C for 1-2 hours. After washing three times, dilute the detection antibody MK-5D6 to the appropriate concentration with diluent. Add 100 μL of diluted MK-5D6 to each well, incubate at 37°C for 1 hour, wash 3 times, add 100 μL of HRP-labeled goat anti-mouse IgG antibody to each well, and incubate at 37°C in the dark for 1 hour. Wash thoroughly 5 times, add 100 μL of freshly prepared TMB substrate solution to each well, and incubate at room temperature in the dark for 10-20 minutes. Stop the reaction by adding 50 μL of 2M H2SO4 stop solution to each well. Immediately read the absorbance of each well at 450 nm using a microplate reader.

[0058] The immunochromatographic test strip procedure is as follows: The detection antibody MK-5D6 is labeled with colloidal gold at a concentration of 20 μg / mL as the labeling complex. The capture antibody MK-3C3 is coated at a concentration of 1.0 mg / mL onto the nitrocellulose membrane at the test line position. Goat anti-mouse IgG antibody is coated at a concentration of 2.0 mg / mL onto the control line position. The sample pad, conjugation pad (containing the labeled MK-5D6), nitrocellulose membrane, and absorbent pad are assembled into the test strip. A sample containing the target antigen is added, and the color development of the test line and control line is observed.

[0059] Using conventional plasmid construction methods, the heavy chain variable region obtained from sequencing and the human IgG heavy chain constant region (Uniprot: P01857), and the light chain variable region and the human IgG light chain constant region (Uniprot: S6BGD6) were ligated and constructed, and then respectively constructed into the pcDNA3.1 plasmid to obtain pcDNA3.1-MK-5D6-H, pcDNA3.1-L, pcDNA3.1-MK-3C3-H, and pcDNA3.1-MK-3C3-L expression plasmids. The heavy chain expression plasmid:light chain expression plasmid molar ratio was 1:1 and transfected into the Expi 293F mammalian cell line for expression.

[0060] The process of constructing eukaryotic expression plasmids and the reagents or parameters used in each process are not limited. Any existing technology that can achieve the above purpose is acceptable and will not be elaborated here. Antibody purification is carried out according to the standard operating procedure for protein purification, and recombinant antibodies are obtained by expression and are still named MK-5D6 and MK-3C3.

[0061] Example 3: Assembly and performance testing of the immunochromatographic reagent kit Preparation of gold-labeled antibodies: A 20mM PB buffer system at pH 6.5 was used to ensure the colloidal gold surface was charged, which was conducive to antibody adsorption. Preliminary experiments determined that a 1:1 volume ratio of MK-5D6 to colloidal gold was optimal, yielding gold-labeled antibodies with good stability after labeling. Under suitable conditions, MK-5D6 and colloidal gold were allowed to fully contact and bind. After the reaction, a blocking agent was typically added to block unoccupied surface sites on the colloidal gold, thereby improving probe stability and reducing non-specific binding. The gold-labeled antibodies were resuspended or adjusted to a suitable spray-coating solution and uniformly sprayed onto glass fiber conjugate pads.

[0062] Sample pad pretreatment: The sample pads are pretreated with a treatment solution containing buffer salts, surfactants, and protein stabilizers to improve sample flowability and reduce matrix interference. After treatment, the pads are dried and stored.

[0063] Streaking procedure: Using a membrane spraying / stretching device, MK-3C3 antibody at a concentration of 1.0 mg / mL is evenly sprayed onto the designated position (as the T line) of the nitrocellulose membrane, and goat anti-mouse IgG is sprayed onto the C line position. After stretching, the membrane is dried.

[0064] Chromatography strip assembly: On a PVC base plate, sequentially attach the sample pad, gold-labeled binding pad, nitrocellulose membrane with T / C lines, and absorbent pad in the flow direction. Cut into 3mm wide test strips, insert into cartridges, and seal. Label with batch number, expiration date, etc., and store.

[0065] Performance testing: Sensitivity: Detection of recombinant MCR-1 and MCR-2 proteins at serial dilutions of 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.02, and 0.01 ng / mL showed a detection limit of 0.1 ng / mL for both. Detection of serial dilutions at 1×10⁻⁶ ng / mL was also performed. 7 1×10 6 1×10 5 1×10 4 1×10 3 The detection limit for MCR-1 and MCR-2 polymyxin-resistant Escherichia coli bacterial suspensions at CFU / mL was 1×10⁻⁶. 4 CFU / mL.

[0066] Specificity: Cultures of *E. coli* containing MCR-1 / MCR-2 / MCR-3 / MCR-4 / MCR-5 / MCR-6 / MCR-7 / MCR-8 / MCR-9 and standard *E. coli* (strains purchased from ATCC) were tested. Results showed that only *E. coli* cultures containing MCR-1 and MCR-2 exhibited chromogenic bands on the T-line, demonstrating good specificity.

[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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. Antibodies against MCR-1 and / or MCR-2 proteins, characterized in that, The anti-MCR-1 and / or MCR-2 protein antibody includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a CDR. L1, CDR L2, CDR The light chain CDR consists of L3, and the heavy chain variable region has CDR. H1, CDR H2, CDR The heavy chain CDR composed of H3, the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR The amino acid sequence of H3 is shown in any one of (A) to (B): (A) SEQ ID NO:4~6, SEQ ID NO:8~10; (B) SEQ ID NO: 12~14, SEQ ID NO: 16~18.

2. The antibody according to claim 1, characterized in that, The amino acid sequences of the light chain variable region and the heavy chain variable region are shown in any one of groups (C) to (D): (C) SEQ ID NO:3, SEQ ID NO:7; (D) SEQ ID NO: 11, SEQ ID NO:

15.

3. A gene encoding the anti-MCR-1 and / or MCR-2 protein antibody as described in claim 1 or 2.

4. A recombinant vector carrying the gene of claim 3.

5. Recombinant cells expressing the anti-MCR-1 and / or MCR-2 protein antibody as described in claim 1 or 2, or containing the gene as described in claim 3, or transformed with the recombinant vector as described in claim 4.

6. A method for preparing antibodies against MCR-1 and / or MCR-2 proteins, characterized in that, The method is to culture the recombinant cells of claim 5 to obtain a culture containing the anti-MCR-1 and / or MCR-2 protein antibodies of claim 1 or 2.

7. 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 anti-MCR-1 and / or MCR-2 protein antibody as described in claim 1 or 2; (2) The culture of the recombinant cells of claim 6.

8. A reagent kit, characterized in that, The kit contains the anti-MCR-1 and / or MCR-2 protein antibodies as described in claim 1 or 2, or the biomarker or chemically labeled product as described in claim 7.

9. The reagent kit according to claim 8, characterized in that, The kit includes an enzyme-linked immunosorbent assay (ELISA) kit and an immunochromatographic assay kit.

10. The reagent kit according to claim 9, characterized in that, The kit includes 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 adhered to the base plate; the conjugate pad is coated with a tracer marker for the detection antibody, a C-line, and a T-line, wherein the C-line is fixed with mouse anti-human IgG antibody, and the T-line is fixed with capture antibody; the detection antibody is an MCR-1 / MCR-2 protein antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO:3 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:7; the capture antibody is an MCR-1 / MCR-2 protein antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO:11 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:

15.

11. The use of the anti-MCR-1 and / or MCR-2 protein antibody of claim 1 or 2, or the gene of claim 3, or the expression recombinant vector of claim 4, or the recombinant cell of claim 5, or the method of claim 6, or the biomarker or chemically labeled product of claim 7 in the preparation of products for detecting MCR-1 and / or MCR-2 proteins.

12. The application according to claim 11, characterized in that, The products include reagents, kits, detection chips, or biosensors.

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