Preparation of antibodies capable of detecting multiple variants of imp and uses thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU MEDOMICS MEDICAL TECHNOLOGY CO LTD
- Filing Date
- 2024-12-30
- Publication Date
- 2026-06-23
Smart Images
Figure CN119529101B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the preparation and application of an antibody capable of detecting multiple variants of IMP, belonging to the field of biomedicine. Background Technology
[0002] Antimicrobial resistance is an urgent global health and socioeconomic crisis. It threatens all regions and all age groups, with low- and middle-income countries being the most affected. It has significant impacts on human and animal health, food production, and the environment, and threatens the achievement of several Sustainable Development Goals.
[0003] Data from bacterial resistance surveillance reports show that: overall, clinical infections are still dominated by Gram-negative bacteria (70.1%), with respiratory specimens accounting for 42.2%; the detection rate of carbapenem-resistant Gram-negative bacteria (CRO) remains high, and carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are all on the rise; while methicillin-resistant Staphylococcus aureus (MRSA) is also on the rise.
[0004] Carbapenem-resistant Enterobacteriaceae (CRE) have become increasingly prominent as a "difficult-to-treat adversary" in clinical anti-infective therapy in recent years. Even more worrying is the high mortality rate of CRE infection. CRE infection often occurs in patients with severe underlying diseases, immunodeficiency, and / or long-term repeated use of broad-spectrum antibiotics, and patients with infection tend to have more severe conditions—the overall in-hospital mortality rate is as high as 46.2%.
[0005] Imipenem, or metallo-β-lactamase (MBL), has become a common enzyme, especially in Asia, since its discovery in Japan in the 1990s. There are currently 88 variants of IMP-type enzymes. The most common variant is IMP-1. IMP-type MBL has been detected in more than a dozen species of Enterobacteriaceae, with *Pseudomonas aeruginosa* being the most common IMP-type enzyme carrier globally. Of the 88 IMP-type MBL variants reported worldwide, only 32 variants have been found to possess susceptibility characteristics. Most bacterial isolates carrying IMP-type MBLs are resistant to carbapenems, particularly imipenem and meropenem, followed by third-generation cephalosporins. IMP belongs to the class B β-lactamases and possesses carbapenemase activity.
[0006] A study performed computer analysis on the IMP-type MBL gene, and multiple sequence alignment of 88 IMP variants identified the conserved sequences of the active site as follows: His95, Phe96, His97, Asp99, Ser100, His157, Cys176, and His215. These sequences are residues at the lactam ring catalytic site. Overall analysis showed amino acid sequence similarity of 79.3%–96.7%. IMP enzyme domain prediction indicated that it contains only one domain, namely the "metallohydrolase-like MBL fold superfamily," covering amino acid positions 23 to 234. The constructed phylogenetic tree contains 38 variants in Group I, 41 variants in Group II, and 9 variants in Group III.
[0007] Currently, treatment options for CRE infection are limited, with high mortality rates and a significant economic and social burden. Early identification of CPGNB helps in timely infection prevention and control measures and treatment management. With the advent of several new antimicrobial drugs, identifying resistance mechanisms has become crucial.
[0008] Immunochromatography is a rapid and convenient in vitro diagnostic technique that requires no instruments and is unaffected by false positives from gene silencing. However, false negatives can occur if the target protein is poorly expressed or if the key amino acids of the antigen epitope recognized by the antibody are altered. Therefore, providing a highly sensitive, specific antibody that can bind to multiple variants of the IMP enzyme remains a pressing market need. Summary of the Invention
[0009] To address the shortcomings of the existing technology, this invention provides an antibody preparation method and its application for detecting multiple IMP variants. The aim is to solve the technical problem that IMP enzymes have many variants, and that the weak expression of the target protein in existing immunochromatography can lead to false negatives for multiple IMP enzyme variants.
[0010] The first technical solution provided by this invention is an anti-IMP carbapenemase antibody, which includes 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, and CDR-L3, and the heavy chain variable region has a heavy chain CDR composed of CDR-H1, CDR-H2, and CDR-H3. The amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are shown in any one of (a) to (b).
[0011] (a) SEQ ID NO: 1~3, SEQ ID NO: 5~7;
[0012] (b) SEQ ID NO:9~11, SEQ ID NO:13~15.
[0013] In some embodiments, the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in any one of (c) to (d):
[0014] (c) SEQ ID NO:4, SEQ ID NO:8;
[0015] (d) SEQ ID NO: 12, SEQ ID NO: 16.
[0016] The second technical solution provided by the present invention is a gene encoding the antibody described in the first technical solution.
[0017] The third technical solution provided by the present invention is a recombinant vector carrying the polynucleotides described in the second technical solution.
[0018] In some embodiments, the recombinant vector uses plasmid pCMV-C-Flag as the expression vector.
[0019] The fourth technical solution provided by the present invention is a recombinant cell expressing the antibody described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.
[0020] In some embodiments, the recombinant cells use fungi, bacteria, animal cells, or plant cells as hosts.
[0021] In some embodiments, the fungus includes yeast or mold, and the bacteria includes Escherichia coli.
[0022] In some embodiments, the animal cells include, but are not limited to, 293 cells.
[0023] The fifth technical solution provided by the present invention is a method for preparing an anti-IMP carbapenemase antibody, wherein the method involves culturing the recombinant cells described in the fourth technical solution to obtain a culture containing the antibody described in the first technical solution.
[0024] 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:
[0025] (1) The antibody described in the first technical solution;
[0026] (2) The culture of recombinant cells described in the fourth technical solution.
[0027] 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, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, and Texas... Red and PerCP dyes; the chemiluminescent types include, but are not limited to, isoluminol and its derivatives, acridine esters and their derivatives, ruthenium terpyridine and its derivatives, etc.; the isotope types include, but are not limited to, iodine labeling; the colloidal labels include, but are not limited to, colloidal gold, colloidal carbon, colloidal selenium, etc.
[0028] 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.
[0029] 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 light chain variable region amino acid sequence shown in SEQ ID NO:12 and the heavy chain variable region amino acid sequence shown in SEQ ID NO:16.
[0030] The eighth technical solution provided by the present invention is the application of the 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 a product for detecting IMP-type carbapenemase.
[0031] In some embodiments, the product includes reagents, kits, detection chips, or biosensors.
[0032] Compared with the prior art, the present invention has the following beneficial effects:
[0033] This invention obtains the conserved antigenic epitope sequences of IMP enzymes by comparing the amino acid sequences of various IMP variants, and plans to prepare antibodies against spatial antigenic epitopes through structural analysis of the epitopes. After immunizing mice with IMP immunogen, hybridoma cells secreting antibodies are obtained through screening using multiple technology platforms. Sequencing yields specific antibody pairs with strong affinity that can bind to multiple IMP variants, specifically the light chain variable regions shown in SEQ ID NO:4 and SEQ ID NO:12, and the heavy chain variable regions shown in SEQ ID NO:8 and SEQ ID NO:16, respectively. The corresponding CDRs are also analyzed. This invention, based on an immunochromatographic platform, develops IMP enzyme detection products with advantages such as high sensitivity, good specificity, coverage of multiple IMP variants, and rapid and convenient detection, providing important assistance for clinical auxiliary diagnosis and medication guidance. Attached Figure Description
[0034] Figure 1 This is a comparison diagram of three grouped sequences in the IMP enzyme phylogenetic tree.
[0035] Figure 2 The purification results of immunogen and antibodies MK35G9 and MK46G2 in specific embodiment 1 of the present invention. Detailed Implementation
[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, specific embodiments are described below, with reference to the appendix. Figures 1-2 The present invention will be further described in detail below.
[0037] Raw materials used in the examples:
[0038] 1. The pET28a carrier was purchased from Takara.
[0039] 2. Escherichia coli DH5α and Escherichia coli BL21(DE3) were purchased from Takara.
[0040] 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.
[0041] 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.
[0042] Serum-free DMEM medium was purchased from Gibco;
[0043] 4. The immunization process for BALB / c mice was commissioned to Jiangsu Dongkang Biotechnology Co., Ltd.
[0044] Example 1: Preparation of anti-IMP monoclonal antibodies MK35G9 and MK46G2
[0045] Step 1: Plasmid Construction
[0046] Based on the three groups of the phylogenetic tree of IMP enzymes, this invention selects representative variants from each group: Group I: blaIMP-12, blaIMP-19, blaIMP-8, blaIMP-2; Group II: blaIMP-4, blaIMP-15, blaIMP-26, blaIMP-70, blaIMP-1, blaIMP-30; Group III: blaIMP-16. Sequence comparisons are as follows: Figure 1 As shown, the amino acids are labeled with different colors according to their characteristics.
[0047] Comprehensive comparative analysis revealed that blaIMP-16 has a large number of mutation sites. Combined with protein structure analysis, most of its mutation sites do not belong to potential antigenic epitopes. Therefore, the relatively conserved sequence segment 46-235AA was selected, and the immunogen (Uniprot: A0A2Z2CFN0) was prepared using the 46-235AA of the common Chinese variant blaIMP-4. The amino acid sequence is shown in SEQ ID NO:17.
[0048] SEQ ID NO:17:
[0049] WGVVTKHGLVFLVNTDAYLIDTPFAAKDTEKLVNWFVERGYKIKGSISSHFHSDSSGGIEWLNSQSIPTYASELTNELLKKNGKVQAKNSFSGVSYWLLKNKIEIFYPGPGHTQDNVVVWLPEKKILFGGCFVKPYGLGNLDDANVEAWPHSAEILMSRYGNAKLVVPSHSDVGDASLLKLTWEQAVKGL
[0050] SEQ ID NO:18:
[0051] tggggcgtggtgaccaaacatggcctggtgtttctggtgaacaccgatgcgtatctgattgataccccgtttgcggcgaaagataccgaaaaactggtgaactggtttgtggaacgcggctataaaattaaaggcagcatta gcagccattttcatagcgatagcagcggcggcattgaatggctgaacagccagagcattccgacctatgcgagcgaactgaccaacgaactgctgaaaaaaaacggcaaagtgcaggcgaaaaacagctttagcggcgtgagc tattggctgctgaaaaacaaaattgaaattttttatccgggcccgggccatacccaggataacgtggtggtgtggctgccggaaaaaaaaattctgtttggcggctgctttgtgaaaccgtatggcctgggcaacctggatg atgcgaacgtggaagcgtggccgcatagcgcggaaattctgatgagccgctatggcaacgcgaaactggtggtgccgagccatagcgatgtgggcgatgcgagcctgctgaaactgacctgggaacaggcggtgaaaggcctg
[0052] According to the E. coil coding principle, the above amino acid sequence was converted into a nucleotide sequence SEQ ID NO:18, sent to Sangon Biotech to complete gene synthesis, and constructed into the pET28a vector.
[0053] The constructed expression plasmid was extracted and transformed into DH5α competent cells and cultured overnight on LB plates. Single colonies were picked and expanded, and the bacterial culture was collected to extract the plasmid pET28a-IMP4(46-235) (according to the instructions of the plasmid extraction kit produced by Sangon Biotech). The plasmid was sent to General Biotech for sequencing confirmation.
[0054] Step 2: Immunogen Preparation
[0055] The amplified expression plasmid pET28a-IMP4(46-235) was transformed into BL21(DE3) strain and cultured overnight on LB plates. Single colonies were picked and expanded to an OD600 of 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.
[0056] Bacterial cells were resuspended in binding buffer (20 mM PB pH 7.2, 100 mM NaCl, 1 mM PMSF), sonicated on ice for 30 min, and centrifuged at 14000 rpm for 20 min to collect the supernatant. The Ni-NTA column was pre-equilibrated with binding buffer. The disrupted supernatant was passed through the Ni-NTA column for binding. Low-concentration imidazole buffers (containing 30, 50, 80, and 100 mM imidazole, respectively) were used to wash away contaminating proteins. The target protein was then eluted with high-concentration imidazole buffer (containing 200 mM imidazole). The proteins were collected in separate tubes, and SDS-PAGE electrophoresis showed bands at 25 kDa, with a purity >95%.
[0057] Step 3: Animal Immunization and Cell Fusion
[0058] The target protein antigen was emulsified with Freund's complete adjuvant. Five SPF-grade 8-12 week old female BALB / c mice were selected and administered the first immunization subcutaneously, using Freund's complete adjuvant at 50 μg / mouse, for a total dose of 0.5 ml / mouse, via intraperitoneal injection. A second immunization was administered 3 weeks later using Freund's incomplete adjuvant at a dose of 50 μg / 0.5 ml / mouse. A third immunization was administered 10 days later. Before the fourth immunization, blood was collected from the tail vein to detect changes in serum antibody levels (ELISA method). If the titer did not reach 1:10... 4 If the OD value is above 1.0, continue immunization. If the ELISA titer has been reached, a final immunization can be performed, and serum samples should be taken for testing after immunization.
[0059] Preparation of myeloma cells: 48 hours before fusion, myeloma cells were proliferated and cultured in 1640 medium containing 20% fetal bovine serum. 24 hours before fusion, the medium was changed again, the original medium was gently removed, a small amount of cold serum-free medium was added, and the cells were evenly distributed in the medium by pipetting. After centrifugation and washing, the cells were resuspended in serum-free medium and stored at 37°C for later use.
[0060] Preparation of spleen cells from immunized mice: Mice were taken three days after the last immunization, and blood was expelled from the orbital cavity. Serum was separated and frozen for later use. Mice were euthanized by cervical dislocation and immersed in 75% ethanol solution for 3-5 minutes. The spleen was aseptically removed and placed on a 200-mesh steel mesh in a petri dish. 10 mL of 1640 culture medium was added, and the spleen cells were gently squeezed through the mesh using a syringe core to prepare a spleen cell suspension. The spleen cells were washed twice with 1640 culture medium. 0.5 × 10⁶ spleen cells were obtained from each mouse. 8 One spleen cell.
[0061] Cell preparation: After euthanizing, disinfecting, and fixing the mice, inject 10 mL of culture medium into the peritoneal cavity. Hold the syringe firmly with your right hand, leaving the needle in the peritoneal cavity. Gently massage the abdomen with an alcohol swab in your left hand for 1 minute, then aspirate the injected culture medium. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the pelleted cells in 5 mL of HAT medium until the concentration reaches 2 × 10⁻⁶ cells / mL. 8 Add 0.1 mL of cells to each well of a 96-well cell culture plate and incubate in a CO2 incubator.
[0062] Prepared myeloma cells and mouse spleen cells were mixed at a ratio of 1:5, and 20 mL of 1640 culture medium was added. The mixture was centrifuged at 1000 rpm to remove the supernatant. Preheated 50% PEG4000 fusion cells were added immediately. After incubation for 1 minute, serum-free DMEM medium was added to terminate the fusion. The mixture was incubated at 37°C for 10 minutes, then centrifuged and the supernatant was removed. 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 fusion cells covered 20%-50% of the bottom of the well.
[0063] Step 4: Screening and Cloning of Hybridoma-Positive Clones
[0064] Positive clones were screened using an indirect ELISA method. The antigen collected in step three was coated with the antibody, and HRP-labeled rabbit anti-mouse IgG was used as the secondary antibody. Serum collected after enucleation of immunized mice served as a positive control, and the supernatant from cultured SP2 / 0 myeloma cells served as a negative control. Antibody-positive hybrid clones were subjected to clonal culture using a limiting dilution method, repeated until 100% positive well rate was achieved to ensure antibody production from a single clone. The cells were then expanded for culture. Culture conditions were: 37℃, 220 rpm, 5% CO2, to prepare IgG antibodies.
[0065] When cell viability was less than 50%, the culture medium was removed and collected into a centrifuge bottle. The supernatant was collected after centrifugation at 12000 rpm for 10 min and filtered through a 0.45 μm filter to remove impurities. Protein G affinity chromatography columns were 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 Gly-HCl pH 3.0. Finally, the antibody properties were determined by SDS-PAGE electrophoresis. After confirmation, the antibody was dialyzed into binding buffer, and samples were sent to General Biotechnology for sequencing. Six IMP enzyme secretion antibodies were obtained: MK22A1, MK28D5, MK35G9, MK46C4, MK56G2, and MK77H6.
[0066] Step 5: Antibody pairing and titer testing
[0067] The six antibodies obtained were screened by pairing using the ELISA double-antibody sandwich method, and the pairing results are shown in Table 1.
[0068] Table 1 Antibody pairing
[0069]
[0070]
[0071] Further screening was conducted using an immunochromatographic platform, and a pair of antibodies was finally identified as shown in Table 2.
[0072] Table 2 Identification of Antibodies
[0073] Serial number Capture antibody Labeling antibody 1 MK35G9 MK56G2
[0074] The antibody titer was detected using ELISA: The prepared IMP enzyme 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. The plate was then blocked with PBST containing 2% BSA (purchased from Sigma) for 2 h, washed in the same manner, and the antibody was 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 antibody test samples of different concentrations. The diluted antibody 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 3.
[0075] Table 3 Antibody titer
[0076] Name Titer MK35G9 1：1280000 MK56G2 1：1280000
[0077] Step Six: Recombinant Antibody Preparation and Purification
[0078] The purified antibodies MK35G9 and MK56G2 were sent to Sangon Biotech for sequencing to obtain the antibody variable region sequence.
[0079] The amino acid sequence of the antibody pair determined in this invention is as follows:
[0080] Antibody MK35G9: The amino acid sequence of the light chain variable region is shown in SEQ ID NO:4; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8;
[0081] SEQ ID NO:4:
[0082] DIKMTQSPSSMYASLGERATITC KVSSDLNRYIN WFQQKPGKSPKTAIY RINHLVDGV PSKLSGSGSGQDYSLTISSYELEDLGIYY NIQFDEYPGT FGGGTKLEITR
[0083] SEQ ID NO:8:
[0084] QVQLQQSGAELVKPGASVKLSCKASGYTVM NIMFWYLK QRPGQGLECIG ELNGETGPTNFNEKMRT KATLTVDKSSSTAFMQLSSLTSESLIYYCT NNWPAYFPWY WGQGTLVTVSA
[0085] The light chain variable region comprises a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO:1–3, respectively. The heavy chain variable region comprises a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO:5–7, respectively. Specific sequence information is as follows: SEQ ID NO:1: KVSSDLNRYIN; SEQ ID NO:2: RINHLVDGV; SEQ ID NO:3: NIQFDEYPGT; SEQ ID NO:5: NIMFWYLK; SEQ ID NO:6: ELNGETGPTNFNEKMRT; SEQ ID NO:7: NNWPAYFPWY.
[0086] Antibody MK56G2: The amino acid sequence of the light chain variable region is shown in SEQ ID NO:12; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:16;
[0087] SEQ ID NO:12:
[0088] DIKMTQSPSSMYASLGERVTITC HASQDLNNFLT WFQQKPGKSPKTLIY KINRLVEPG PSHFSGSGSGQDYSLTISSLEYEDYGVYYC LAYEDFPYT FGGGTRLEIK
[0089] SEQ ID NO:16:
[0090] QVQLQQSGAELVKPGASVKLSCTASGNIK DNLTYIHYIR QKPEQGIEWLG RIDPALDYANWDPRFNG KATILTDTSSSTAYLHLSSLTSEDTAVYYCAR RGGYYAFDMY WGQGTSVIVSSA
[0091] The light chain variable region comprises a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO:9-11, respectively; the heavy chain variable region comprises a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO:13-15, respectively. Specific sequence information is as follows: SEQ ID NO:9: HASQDLNNFLT. SEQ ID NO:10: KINRLVEPG. SEQ ID NO:11: LAYEDFPYT. SEQ ID NO:13: DNLTYIHYIR. SEQ ID NO:14: RIDPALDYANWDPRFNG. SEQ ID NO:15: RGGYYAFDMY.
[0092] Using molecular construction methods, the heavy chain variable region of the above-mentioned 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 by Sangon Biotech). 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 standard operating procedures for protein purification, and the anti-IMP recombinant antibodies MK35G9-R and MK56G2-R were obtained.
[0093] Step 7: Antibody Affinity Verification
[0094] This invention utilizes The detection instrument measures the affinity of the aforementioned MK35G9-R and MK56G2-R antibody pair. The biosensor is immobilized with the aforementioned IMP enzyme immunogen, which is covered by a biofilm layer. When visible light with a certain bandwidth is incident perpendicularly on the biofilm layer, the light is reflected at the two interfaces of the biofilm layer, forming an interference wave of a specific wavelength that is detected by the spectrometer. The immobilized IMP enzyme interacts with different concentrations of antibody MK35G9-R or MK56G2-R in the buffer solution. As the biofilm thickness increases, the interference spectrum shifts towards increasing wavelength. This phase shift is detected and analyzed by the workstation, yielding the kinetic data of the antibody on the sensor surface. The saved data is imported into the BLI instrument's built-in data analysis module, and the affinity (K0) is calculated by fitting a kinetic model. D =kd / ka), the results are shown in Table 4.
[0095] Table 4 Affinity Test
[0096]
[0097] The results show that the K of MK35G9-R D The value is 5.04 × 10 9 M, K of MK56G2-R D The value is 4.81 × 10 9 M has a strong affinity.
[0098] Example 2: Preparation of a detection kit for multiple IMP enzyme variants
[0099] This embodiment uses the antibody pair with high affinity from Example 1, and based on the immunochromatographic platform, further develops a detection kit with high sensitivity, good specificity, and coverage of multiple variants. The preparation method of the kit is as follows:
[0100] Step 1: While stirring, add 200-800 μL of 0.05-0.2 M K₂CO₃ solution dropwise to 100 mL of colloidal gold solution. After addition, seal the container and mix slowly for 2-5 minutes. Then, add MK35G9-R antibody 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.
[0101] Step 2: Spray the gold labeling solution onto the glass cellulose membrane and dry it to make a gold labeling pad;
[0102] Step 3: Add 1-10% sucrose solution to MK56G2-R antibody, 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 control line coating solution, and apply 0.5-2 μL / cm of the control line coating solution and the detection line coating solution onto a nitrocellulose membrane, dry to obtain the coated plate;
[0103] 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.
[0104] Example 3: Comparative Application of Detection Kits for Multiple IMP Enzyme Variants
[0105] This embodiment uses clinically confirmed positive bacterial culture samples of blaIMP-4, blaIMP-12, blaIMP-19, blaIMP-8, blaIMP-2, blaIMP-15, blaIMP-26, blaIMP-70, blaIMP-1, blaIMP-30, and blaIMP-16. Three loops of bacterial sample are picked up using an inoculation loop, and the loop is immersed in the diluent. The loop is shaken back and forth for at least 30 seconds to ensure complete elution of the sample into the diluent tube. The diluent tube is capped and vortexed until homogeneous. Four drops of the treated sample solution are vertically added to the sample well of the test card. The results are observed within 15-20 minutes. Simultaneous detection is performed using a comparison kit (comparison kit 1 and comparison kit 2 were both purchased commercially available products). The results are statistically shown in Table 5.
[0106] Table 5. Test results of the reagent kit
[0107]
[0108] Note: √ indicates detectable, × indicates undetectable.
[0109] The kit developed in this invention can detect all common IMP enzyme variants mentioned above. However, the comparative kit has missed detections of variants blaIMP-30, blaIMP-19, blaIMP-8, and blaIMP-2. The kit of this invention can overcome the deficiencies of commercially available products, detect all common and frequently occurring variants, and can detect a variety of high-incidence variants. It can cover common IMP enzyme resistance in recent years. Furthermore, the kit has high sensitivity and good specificity, providing important assistance for clinical medication and diagnosis.
[0110] Example 4: Performance Overview of the Detection Kit for Multiple Variants of IMP Enzyme
[0111] 1. Limit of detection
[0112] Take the labeled negative samples and prepare a negative matrix. Dilute the above IMP enzyme in multiple concentration gradients, namely 1000, 800, 600, 400, 200, 100, and 50 pg / mL. Repeat the test 20 times for each concentration gradient using the kit in Example 2. Test continuously for 3 days and observe the color development and positive status of each concentration test result. The lowest concentration with a positive detection rate of 95% and consistent color development is taken as the limit of detection.
[0113] Table 6 Detection Limit Results
[0114]
[0115]
[0116]
[0117] According to the above result judgment method, when the negative sample is diluted to the detection limit concentration of 200 pg / mL, the positive detection rate is slightly higher than 95%. The detection limit of the kit of the present invention is no higher than 200 pg / mL for IMP carbapenemase.
[0118] 2. Stability
[0119] Using positive and negative samples with three different antibody concentrations as research materials, three batches of the kit 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 assess the stability of the kit. The results are shown in Tables 7–9.
[0120] Table 7. Stability test results (reagent tested and stored at 2–8℃)
[0121]
[0122]
[0123] Table 8. Stability test results (tested with the kit stored at 20±2℃)
[0124]
[0125] Table 9. Stability test results (tested using the kit at 30±2℃)
[0126]
[0127] As can be seen from Tables 7-9, 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 have excellent stability.
[0128] 3. Cross-reaction
[0129] Six weakly positive IMP 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 10.
[0130] Table 10 Specificity Test Results
[0131]
[0132] Based on Table 10, it was determined that carbapenemase-positive samples of common enzyme types did not exhibit cross-reactivity with the kit, indicating good performance.
[0133] 4. Interference Experiment
[0134] Negative matrix and IMP enzyme 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.
[0135] Table 11 Detection Results of Interfering Substances
[0136]
[0137]
[0138] The results in Table 10 demonstrate that the kit can effectively resist interference from both endogenous and exogenous substances.
[0139] In summary, the kit based on the antibody prepared according to the present invention has high sensitivity and good analytical specificity.
[0140] 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. Anti-IMP type carbapenemase antibody, characterized in that, The anti-IMP carbapenemase 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 L3, the heavy chain variable region has CDR H1, CDR H2, CDR H3, the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR The amino acid sequences of H3 are shown in SEQ ID NO:1~3 and SEQ ID NO:5~7, respectively; or the CDR... L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR The amino acid sequences of H3 are shown in SEQ ID NO:9~11 and SEQ ID NO:13~15, respectively.
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 SEQ ID NO:4 and SEQ ID NO:8, respectively; or the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID NO:12 and SEQ ID NO:16, respectively.
3. A gene encoding the antibody of claim 1 or 2.
4. A recombinant vector carrying the gene of claim 3.
5. Recombinant cells expressing the antibody of claim 1 or 2, or containing the gene of claim 3, or transformed with the recombinant vector of claim 4.
6. The recombinant cell according to claim 5, characterized in that, The recombinant cells are bacteria, fungi, animal cells, or plant cells.
7. The recombinant cell according to claim 6, characterized in that, The fungus is selected from yeast or mold, and the bacteria is Escherichia coli.
8. A method for preparing an anti-IMP type carbapenemase antibody, characterized in that, The method is to culture the recombinant cells according to any one of claims 5 to 7 to obtain a culture containing the antibody according to claim 1 or 2.
9. 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 is derived from the antibody described in claim 1 or 2.
10. The product according to claim 9, characterized in that, The labeling agents are selected from enzymes, biotin, luciferins, chemiluminescent agents, isotopes, colloids, latex microspheres, or magnetic beads; the enzymes are selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridges, alkaline phosphatase-antialkaline phosphatase bridges, or β-galactosidase-antiβ-galactosidase bridges; the biotin is biotin; the luciferins are selected from 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, or PerCP dye; the chemiluminescent type is selected from isoluminol, acridine ester, or ruthenium terpyridine; the isotope type is iodine labeling; the colloidal type is selected from colloidal gold, colloidal carbon, or colloidal selenium.
11. 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 9 or 10.
12. The kit according to claim 11, characterized in that, The kits are selected from enzyme-linked immunosorbent assay (ELISA) kits and immunofluorescence kits.
13. The kit according to claim 11 or 12, 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, the light chain variable region amino acid sequence shown in SEQ ID NO:12 and the heavy chain variable region amino acid sequence shown in SEQ ID NO:
16.
14. The use of the antibody of claim 1 or 2, or the gene of claim 3, or the recombinant vector of claim 4, or the recombinant cell of any one of claims 5 to 7, or the biomarker or chemically labeled product of claim 9 or 10 in the preparation of a product for detecting IMP-type carbapenemase.
15. The application according to claim 14, characterized in that, The products are selected from reagents, kits, detection chips, or biosensors.