Preparation and application of antibody capable of detecting various IMP variants

Abstract

The invention discloses preparation and application of an antibody capable of detecting various IMP variants, and belongs to the field of biological medicine. According to the invention, a conserved epitope sequence of IMP enzyme is obtained according to comparison of amino acid sequences of all IMP variants, and an antibody of a spatial epitope is prepared through structural analysis of the epitope. After a mouse is immunized with IMP immunogen protein, hybridoma cells secreting antibodies are obtained through multi-technology platform screening, specific antibody pairs MK35G9 and MK56G2 which are high in affinity and can be combined with multiple IMP variants are obtained after sequencing, and corresponding CDR is obtained through analysis. According to the present invention, the IMP enzyme detection product development is performed based on the immunochromatography platform, such that advantages of high sensitivity, good specificity, covering of a variety of IMP variants, and rapid and convenient detection are provided, and important help is provided for clinical auxiliary diagnosis and medication guidance.

Description

Technical Field

[0001] The present invention relates to the preparation of an antibody capable of detecting multiple variants of IMP and its application, and belongs to the field of biomedicine. Background Art

[0002] Antimicrobial resistance is an urgent global health and socioeconomic crisis. Antimicrobial resistance threatens all age groups in all regions, with low- and middle-income countries being most affected. It has major impacts on human and animal health, food production and the environment, and threatens the achievement of several Sustainable Development Goals.

[0003] The data reported in the bacterial resistance monitoring reports show that: overall, clinical infections are still dominated by Gram-negative bacteria (70.1%), and the proportion of respiratory specimens has increased to 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; and methicillin-resistant Staphylococcus aureus (MRSA) has also increased.

[0004] As a "refractory opponent" in clinical anti-infection treatment, carbapenem-resistant Enterobacteriaceae (CRE) have become increasingly threatening in recent years. What is even more worrying is the high mortality rate of CRE infection. CRE infection often occurs in patients with serious underlying diseases, immunodeficiency, and / or long-term repeated use of broad-spectrum antibiotics. After infection, the patient's condition is relatively serious - the overall hospital mortality rate is as high as 46.2%.

[0005] Imipenem or IMP-type metallo-β-lactamases (MBLs) have become a common enzyme, especially in Asia, since they were discovered in Japan in the 1990s. There are currently 88 variants of IMP-type enzymes. The most common IMP-type enzyme is a variant of IMP-1. IMP-type MBLs have been detected in more than a dozen species of the Enterobacteriaceae family, with Pseudomonas aeruginosa being the most common carrier of IMP-type enzymes worldwide. Among the 88 IMP-type MBL variants reported worldwide, only 32 variants were found to have susceptibility characteristics. Most bacterial isolates carrying IMP-type MBLs are resistant to carbapenems, especially imipenem and meropenem, followed by third-generation cephalosporins. IMP belongs to class B β-lactamases and has carbapenemase activity.

[0006] A study conducted a computer analysis of the IMP-type MBL gene and a multiple sequence alignment of 88 IMP variants, identifying the conserved sequences of the active site as follows: His95, Phe96, His97, Asp99, Ser100, His157, Cys176, and His215, which are residues in the catalytic site of the lactam ring. The overall analysis showed that the amino acid sequence similarity was 79.3%–96.7%. The IMP enzyme domain prediction results showed that it contains only one domain, the "metal hydrolase-like MBL fold superfamily", covering from amino acid position 23 to position 234. The constructed phylogenetic tree contains 38 variants in group I, 41 variants in group II, and 9 variants in group III.

[0007] Currently, there are limited treatment options for CRE infections, with high mortality and huge economic and social burdens. Early identification of CPGNB can help timely infection prevention and control measures and treatment management, and with the advent of several new antimicrobial drugs, identifying resistance mechanisms has become critical.

[0008] Immunochromatography is a fast and convenient in vitro diagnostic technology that does not require any equipment and is not affected by false positives caused by gene silencing. However, if the expression of the target protein is weak or the key amino acids of the antigen epitope recognized by the antibody used change, false negatives may also occur in immunochromatography. Therefore, it is still urgent for the market to provide a specific antibody with high sensitivity that can bind to multiple variants of the IMP enzyme. Summary of the invention

[0009] In view of the above-mentioned deficiencies in the prior art, the present invention provides an antibody preparation and application capable of detecting multiple variants of IMP, aiming to solve the technical problem that the IMP enzyme has many variants and in the existing immunochromatography, false negatives may occur for multiple variants of the IMP enzyme due to weak expression of the target protein.

[0010] The first technical solution provided by the present invention is an anti-IMP type carbapenemase antibody, which includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are as shown in any one of the groups (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 certain embodiments, the amino acid sequences of the light chain variable region and the heavy chain variable region are as shown in any one of the groups (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 polynucleotide described in the second technical solution.

[0018] In certain embodiments, the recombinant vector uses plasmid pCMV-C-Flag as an 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 certain embodiments, the recombinant cell is hosted by a fungus, a bacterium, an animal cell or a plant cell.

[0021] In certain embodiments, the fungus comprises yeast or mold, and the bacteria comprises Escherichia coli.

[0022] In certain 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-type carbapenemase antibody, wherein the method comprises culturing the recombinant cell 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 the present invention is a biologically labeled or chemically labeled product, wherein the product is an antibody labeled with a marker, and the antibody is derived from any one of the following:

[0025] (1) The antibody described in the first technical solution;

[0026] (2) The culture of the recombinant cells described in the fourth technical solution.

[0027] In some embodiments, the label includes but is not limited to enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, β-galactosidase-anti-β-galactosidase bridge; the biotin includes but is not limited to biotin and its derivatives; the fluorescein includes but is not limited to AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green488, PacificBlue dye, Pacific Orange dye, Texas Red, PerCP dyes; the chemiluminescent type includes but is not limited to isoluminol and its derivatives, acridinium esters and their derivatives, terpyridine ruthenium and its derivatives, etc.; the isotope type includes but is not limited to iodine labeling; the colloidal labeling includes but is not limited to colloidal gold, colloidal carbon, colloidal selenium, etc.

[0028] The seventh technical solution provided by the present invention is a kit, which contains the antibody described in the first technical solution or the biological marker or chemically labeled product described in the sixth technical solution.

[0029] In certain embodiments, the kit is an immunochromatography kit, comprising a test card, wherein the test card comprises: a PVC base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and absorbent paper; the sample pad, the conjugation pad, the nitrocellulose membrane and the absorbent paper are overlapped and pasted on the base plate in sequence; the conjugation pad is sprayed with a tracer marker labeled with an antibody having a light chain variable region amino acid sequence as shown in SEQ ID NO: 12 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 16, a C line and a T line, wherein the C line is fixed with a mouse anti-human IgG antibody, and the T line is fixed with an antibody having a light chain variable region amino acid sequence as shown in SEQ ID NO: 4 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 8.

[0030] The eighth technical solution provided by the present invention is the use 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 certain embodiments, the product comprises a reagent, a kit, a detection chip or a biosensor.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] According to the comparison of the amino acid sequences of various IMP variants, the present invention obtains the conservative antigen epitope sequence of the IMP enzyme, and through the structural analysis of the antigen epitope, it is intended to prepare an antibody of the spatial antigen epitope. After immunizing mice with IMP immunogen protein, hybridoma cells secreting antibodies are screened and obtained by multi-technical platforms, and after sequencing, specific antibody pairs with strong affinity and capable of binding to multiple IMP variants are obtained, such as the light chain variable regions shown in SEQ ID NO:4 and SEQ ID NO:12, respectively; and the heavy chain variable regions shown in SEQ ID NO8 and SEQ ID NO 16, respectively, and the corresponding CDRs are obtained by analysis. The present invention develops IMP enzyme detection products based on the immunochromatography platform, which has the advantages of high sensitivity, good specificity, can cover a variety of IMP variants, and fast and convenient detection, and provides important help for clinical auxiliary diagnosis and guiding medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a sequence comparison diagram of the three groups of the IMP enzyme phylogenetic tree.

[0035] Figure 2 The purification results of the immunogen and antibodies MK35G9 and MK46G2 in the specific embodiment 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following is a detailed description of the present invention in conjunction with the accompanying drawings. Figures 1-2 , the present invention is further described in detail.

[0037] The raw materials used in the examples are:

[0038] 1. pET28a vector 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 with 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar powder. The pH value needs to be adjusted to 7.3±0.1.

[0042] Serum-free DMEM medium was purchased from Gibco;

[0043] 4. The implementation of the BALB / c mouse immunization process was entrusted to Jiangsu Dongkang Biological Co., Ltd.

[0044] Example 1 Preparation of anti-IMP monoclonal antibodies MK35G9 and MK46G2

[0045] Step 1: Plasmid construction

[0046] The present invention selects representative variants of each group according to the three groups of the phylogenetic tree of IMP enzymes, namely, blaIMP-12, blaIMP-19, blaIMP-8, and blaIMP-2 of group I; blaIMP-4, blaIMP-15, blaIMP-26, blaIMP-70, blaIMP-1, and blaIMP-30 of group II; and blaIMP-16 of group III. The sequence comparison is as follows Figure 1 As shown, and marked with different colors according to the amino acid characteristics.

[0047] Comprehensive comparative analysis showed that blaIMP-16 had many mutation sites. Combined with protein structure analysis, most of its mutation sites did not belong to potential antigenic epitopes, and then a relatively conservative sequence segment 46-235AA was selected to prepare an immunogen (Uniprot: A0A2Z2CFN0) using 46-235AA of blaIMP-4, a common variant in China. 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 the nucleotide sequence SEQ ID NO: 18, sent to Sangon Biotechnology for gene synthesis, and constructed into the pET28a vector.

[0053] The expression plasmid constructed above was extracted and transformed into DH5α competent cells and cultured overnight on LB plates. Single clone colonies were picked for expansion and culture, and the bacterial liquid was collected to extract plasmid pET28a-IMP4 (46-235) (according to the instructions of the plasmid extraction kit produced by Sangon Biotech Co., Ltd.), and sent to Universal Biotech for sequencing confirmation.

[0054] Step 2: Immunogen preparation

[0055] The amplified expression plasmid pET28a-IMP4 (46-235) was transformed into the BL21 (DE3) strain and cultured overnight on LB plates. Single clones were picked and cultured to OD600 of 0.6-0.8, and IPTG with a final concentration of 1 mM was added to induce expression. After 4 hours of expression, the bacteria were collected by centrifugation.

[0056] The cells were resuspended in binding buffer (20mM PB pH7.2, 100mM NaCl, 1mM PMSF), ultrasonicated for 30min in an ice bath, and centrifuged at 14000rpm for 20min to collect the supernatant. The Ni-NTA column was pre-equilibrated with binding buffer, and the supernatant solution after the disruption was passed through the Ni-NTA column for binding. A low concentration of imidazole buffer (containing 30, 50, 80, and 100mM imidazole binding buffers, respectively) was passed through the Ni-NTA column to wash the impurities. The target protein was then eluted with a high concentration of imidazole buffer (containing 200mM imidazole binding buffer) and collected separately. The SDS-PAGE electrophoresis band was at the 25kD position, and the purity was >95%.

[0057] Step 3: Animal immunization and cell fusion

[0058] The above target protein antigen was emulsified with Freund's complete adjuvant, and 5 SPF 8-12 week old BALB / c female mice were selected for subcutaneous injection. The first immunization was performed with Freund's complete adjuvant, 50 μg / mouse, total dose 0.5 ml / mouse, intraperitoneal injection; the second immunization was performed 3 weeks later, with Freund's incomplete adjuvant, dose 50 μg / 0.5 ml / mouse; the third injection was performed 10 days later. Before the fourth immunization, blood was collected from the tail vein to detect the change of antibody level in the serum (ELISA method). If the titer did not reach 1:10, 4 If OD is above 1.0, continue immunization. If ELISA has reached the titer, the final immunization can be carried out and serum can be tested after immunization.

[0059] Preparation of myeloma cells: 48 hours before fusion, use 1640 culture medium containing 20% ​​calf serum to proliferate myeloma cells. 24 hours before fusion, change the medium once with the same culture medium, gently remove the original culture medium in the bottle, add a little cold serum-free culture medium, use a pipette to blow the cells evenly in the culture medium, centrifuge and wash, then suspend in serum-free culture medium and incubate at 37°C for use.

[0060] Preparation of spleen cells from immunized mice: Take mice on the third day after the last immunization, bleed from their eye sockets, separate the serum and freeze for later use. Kill the mice by cervical dislocation and soak them in 75% ethanol solution for 3-5 minutes. Remove the spleen aseptically and place it on a 200-mesh steel mesh in a flat dish. Add 10 mL of 1640 culture medium and use a syringe core to gently squeeze the spleen cells through the mesh to prepare a spleen cell suspension. Wash the spleen cells twice with 1640 culture medium. 0.5×10 8 splenocytes.

[0061] Feeder cell preparation: After killing the mouse, disinfecting and fixing the body surface, inject 10mL of culture medium into the abdominal cavity. Fix the syringe with the right hand so that the needle remains in the abdominal cavity. Use the alcohol cotton ball in the left hand to gently massage the abdomen for 1 minute, then aspirate the injected culture medium. Centrifuge at 1000rpm for 10 minutes and discard the supernatant. First, suspend the precipitated cells with 5mL of HAT medium to a concentration of 2×10 8 / mL, add the cells into a 96-well cell culture plate, 0.1mL per well, and place in a CO2 incubator for later use.

[0062] The prepared myeloma cells were mixed with mouse spleen cells at a ratio of 1:5, 20 mL of 1640 culture medium was added, and the supernatant was removed by centrifugation at 1000 rpm. Preheated 50% PEG4000 was immediately added to fuse the cells. After standing for 1 minute, serum-free DMEM medium was added to terminate the fusion. After standing at 37°C for 10 minutes, the cells were centrifuged and the supernatant was removed. The cells were resuspended in HAT medium, distributed into 96-well plates, and cultured in a cell culture incubator for about 10 days until the fused cells covered 20%-50% of the bottom of the well.

[0063] Step 4: Screening and cloning of hybridoma positive clones

[0064] The positive clones were screened by the indirect ELISA method, the antigen collected in step 3 was coated, HRP-labeled rabbit anti-mouse IgG was used as the secondary antibody, the serum collected after the eyeballs of immunized mice were removed was used as the positive control, and the supernatant of myeloma SP2 / 0 cells was used as the negative control. The hybrid clones that tested positive for antibodies were cloned and cultured, and the hybridoma cells were cultured by the limiting dilution method, and repeated until the positive pore rate reached 100%, ensuring that the antibody was produced by a single clone, and then the cells were expanded and cultured. The culture conditions were: 37°C, 220rpm, 5% CO2, and the antibody IgG was prepared.

[0065] When the cell survival rate is less than 50%, the culture medium is taken out and collected into a centrifuge bottle, centrifuged at 12000rpm for 10min, and the supernatant is collected and filtered through a 0.45μm filter to remove impurities. The Protein G affinity chromatography column is balanced with binding buffer, and the antibody is purified in this way. After loading, the impurities are washed with binding buffer and eluted with 0.1M Gly-HCl pH3.0. Finally, the antibody properties are determined by SDS-PAGE electrophoresis, and after confirmation, the antibody is dialyzed into binding buffer, and the sample is sent to General Bio for sequencing. A total of 6 secretory antibodies of IMP enzymes are obtained: MK22A1, MK28D5, MK35G9, MK46C4, MK56G2, and MK77H6.

[0066] Step 5: Antibody pairing and titer testing

[0067] The 6 antibodies obtained were screened for 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 performed using an immunochromatography platform, and a pair of antibodies were finally identified as shown in Table 2.

[0072] Table 2 Identified antibodies

[0073] Serial number Capture Antibody Labeled Antibodies 1 MK35G9 MK56G2

[0074] The antibody titer was detected by ELISA: the IMP enzyme prepared above was diluted with 50 mM Na2CO3 pH9.6 buffer to a final concentration of 1 μg / ml, added to a 96-well plate, coated overnight at 4°C, washed 3 times with 1×PBST, and patted dry; blocked and incubated with PBST containing 2% BSA (purchased from Sigma) for 2 h, washed in the same manner, and the above antibody was diluted with 1×PBS buffer at 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, 1:1280000, and 1:2560000 to obtain antibody test products of different concentrations. The diluted antibody test product was added to the above ELISA plate and incubated at 37°C for 1 hour; then HRP-goat anti-mouse IgM (purchased from Sigma) diluted 1:4000 was added and incubated at 37°C for 1 hour; finally, TMB substrate was added to develop color at room temperature in the dark for 10 minutes, and sulfuric acid was used to terminate the reaction. The OD450nm value was measured by an ELISA instrument, and the titer of each antibody is shown in Table 3.

[0075] Table 3 Antibody titer

[0076] name Potency MK35G9 1：1280000 MK56G2 1：1280000

[0077] Step 6: Recombinant antibody preparation and purification

[0078] The purified antibodies MK35G9 and MK56G2 were sent to Sangon Biotechnology for sequencing, and the antibody variable region sequences were obtained.

[0079] That is, the amino acid sequence of the antibody pair determined by the present 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] Among them, the light chain variable region has a light chain CDR composed of CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of the CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NOs: 1 to 3, respectively; the heavy chain variable region has a heavy chain CDR composed of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NOs: 5 to 7, respectively. The 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] Among them, the light chain variable region has a light chain CDR composed of CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of the CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NOs: 9 to 11, respectively; the heavy chain variable region has a heavy chain CDR composed of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NOs: 13 to 15, respectively. The 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] Through molecular construction methods, the heavy chain variable region of the above-mentioned antibody was grafted onto the nucleotide sequence of the human IgG heavy chain constant region skeleton, and the light chain variable region VL nucleotide sequence was grafted onto the nucleotide sequence of the human IgG light chain constant region skeleton. The heavy chain variable region and light chain variable region of the antibody obtained by sequencing were recombined with the constant region of mouse IgG and cloned into the modified mammalian cell expression vector pCMV-C-Flag for expression and recombination in Expi 293F cells (the vector was purchased from Beyotime, and the human IgG constant region gene was synthesized by Biotech). The process of grafting and constructing eukaryotic expression plasmids and the reagents or parameters used in each process are not limited. As long as the technology in the prior art can achieve the above purpose, it will not be repeated here. Antibody purification was carried out according to the standard operating procedures for protein purification, and the anti-IMP recombinant antibodies MK35G9-R and MK56G2-R were obtained by expression.

[0093] Step 7: Antibody affinity verification

[0094] The present invention utilizes The detection instrument measures the affinity of the above-mentioned pair of antibodies MK35G9-R and MK56G2-R. The above-mentioned IMP enzyme immunogen is fixed at the bottom of the biosensor, which is covered by a biofilm layer. When visible light with a certain bandwidth is incident vertically on the biofilm layer, the light is reflected at the two interfaces of the biofilm layer to form an interference wave of a certain wavelength detected by the spectrometer. The fixed IMP enzyme interacts with the antibodies MK35G9-R or MK56G2-R of different concentrations in the buffer. The thickness of the biolayer increases, and the interference spectrum curve moves in the direction of increasing wavelength. The phase shift is detected and analyzed by the workstation, and the kinetic data of the antibody on the sensor surface can be obtained. The saved data is imported into the data analysis module of the BLI instrument, and the affinity (K D =kd / ka), the results are shown in Table 4.

[0095] Table 4 Affinity test

[0096]

[0097] The results show that the K D The value is 5.04×10 9 M, K of MK56G2-R D The value is 4.81×10 9 M, has stronger affinity.

[0098] Example 2 Preparation of a detection kit for multiple variants of IMP enzyme

[0099] This example uses the antibody pair with high affinity in Example 1, based on the immunochromatography platform, to further develop a detection kit with high sensitivity, good specificity, and the ability to cover multiple variants. The preparation method of the kit is as follows:

[0100] Step 1: Add 200-800 μL of 0.05-0.2M K2CO3 solution dropwise to 100mL colloidal gold solution while stirring. After adding, seal and mix slowly for 2-5 minutes. After mixing, add MK35G9-R antibody dropwise. After adding, seal and mix slowly for 2-5 minutes. Incubate for 30-60 minutes. After incubation, add 20-80mL blocking solution (1-3% BSA) dropwise, mix for 2-5 minutes, let stand and block for 30-60 minutes. After the standing time, continue to seal and mix for 2-5 minutes. After blocking, centrifuge and set the parameters: centrifugal force 4000-6000rpm, 2-8℃, centrifugation time 20-60min. After centrifugation, remove the supernatant to obtain the gold-labeled precipitate, and preserve it with preservation solution to obtain the gold-labeled solution;

[0101] Step 2: Spray the gold label solution onto the glass cellulose membrane and dry it to make a gold label pad;

[0102] Step 3: Add 1-10% sucrose solution to MK56G2-R antibody, mix well to form a test line coating solution, then add 1-10% sucrose solution to goat anti-mouse IgG antibody, mix well to form a quality control line coating solution, streak the quality control line coating solution and the test line coating solution 0.5-2 μL / cm on a nitrocellulose membrane, and obtain a coating plate after drying;

[0103] Step 4: Stick the gold label pad, absorbent paper and sample pad on the PVC base plate in sequence, cut them into 3cm wide test strips and put them into the corresponding card shell.

[0104] Example 3 Comparative Application of Detection Kits for Multiple Variants of IMP Enzyme

[0105] The present embodiment uses clinically confirmed positive blaIMP-4, blaIMP-12, blaIMP-19, blaIMP-8, blaIMP-2, blaIMP-15, blaIMP-26, blaIMP-70, blaIMP-1, blaIMP-30, blaIMP-16 bacterial culture samples, picks 3 loops of bacterial samples with an inoculation loop, and immerses the inoculation loop in the diluent, shakes back and forth, mixes for at least 30 seconds, and ensures that the sample is fully eluted in the diluent tube. Cover the diluent tube cover and oscillate evenly using a vortex oscillator. 4 drops of treated sample treatment solution are vertically added to the sample addition hole of the test card, and the results displayed are observed within 15-20 minutes, and the comparison kit is synchronously detected (the comparison kit 1 and the comparison kit 2 used are all purchased from commercially available products), and the test results are statistically shown in Table 5.

[0106] Table 5 Kit test results

[0107]

[0108] Note: √ means detectable, × means undetectable.

[0109] The kit developed by the present invention can detect all the common IMP enzyme variants tested above, among which the comparison kit has missed detection of blaIMP-30, blaIMP-19, blaIMP-8, and blaIMP-2 variants; the kit of the present invention can make up for the defects of commercially available products, can detect common and frequently occurring variants, can realize the detection of multiple high-incidence variants, can cover the common IMP enzyme resistance in recent years, and the kit has high sensitivity and good specificity, providing important help for clinical medication and diagnosis and treatment.

[0110] Example 4 Performance Summary of the Detection Kit for Multiple Variants of IMP Enzyme

[0111] 1. Minimum detection limit

[0112] A calibrated negative sample was taken to prepare a negative matrix, and the above-mentioned IMP enzyme was diluted in multiple concentration gradients, that is, the dilution concentrations were 1000, 800, 600, 400, 200, 100, and 50 pg / mL. Each concentration gradient was repeatedly tested 20 times using the kit of Example 2, and the test was continuously tested for 3 days. The color development and positivity of the test results of each concentration were observed, and the lowest concentration with a positive detection rate of 95% and consistent color development was taken as the detection limit.

[0113] Table 6 Detection limit test results

[0114]

[0115]

[0116]

[0117] According to the above result judgment method, when the negative sample is used to dilute the IMP-type carbapenemase to a 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 that the IMP-type carbapenemase is not higher than 200 pg / mL.

[0118] 2. Stability

[0119] Positive samples and negative samples with three different antibody concentrations were used as research materials, and three batches of the kit were stored at 2-8°C, 20±2°C, and 30±2°C for 27 months. The test time was 0 months, 4 months, 8 months, 12 months, 16 months, 20 months, 24 months, 26 months, and 27 months to evaluate the stability of the kit. The results are shown in Tables 7 to 9.

[0120] Table 7 Stability test results (test kit stored at 2-8°C)

[0121]

[0122]

[0123] Table 8 Stability test results (test kit stored at 20±2°C)

[0124]

[0125] Table 9 Stability test results (test kit stored at 30±2°C)

[0126]

[0127] As can be seen from Tables 7 to 9, the three batches of test kits were stored at 2-8°C, 20±2°C and 30±2°C for 27 months, and were tested using strongly positive, moderately positive, weakly positive and negative samples at 0th, 4th, 8th, 12th, 16th, 20th, 24th, 26th and 27th months, respectively. The test results were good, indicating that the test kits had excellent stability.

[0128] 3. Cross-reaction

[0129] Six IMP-type carbapenemase weakly positive samples and six negative samples were used as research materials, and other common carbapenemase positive samples were added respectively, and the cross-reaction of the kit was evaluated using the kit of Example 2. The results are shown in Table 10.

[0130] Table 10 Specificity test results

[0131]

[0132] Judging from Table 10, common carbapenemase-positive samples will not cross-react with the kit, and the performance is good.

[0133] 4. Interference experiment

[0134] Negative matrix and IMP enzyme critical positive samples were selected as basic samples and a certain concentration of interfering substances were added to confirm whether a certain concentration of interfering substances would affect the test results.

[0135] Table 11 Interfering substances affect test results

[0136]

[0137]

[0138] The results in Table 10 show that the kit can effectively resist the interference of endogenous and exogenous substances.

[0139] In summary, the kit based on the antibodies prepared by the present invention has high sensitivity and good analytical specificity.

[0140] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An anti-IMP type carbapenemase antibody, characterized in that: The anti-IMP type carbapenemase antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are as shown in any one of the groups (a) to (b): (a) SEQ ID NO: 1~3, SEQ ID NO: 5~7; (b) SEQ ID NO:9~11, SEQ ID NO:13~15.

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:4, SEQ ID NO:8; (d) SEQ ID NO: 12, SEQ ID NO:

16.

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

4. A recombinant vector carrying the gene according to claim 3.

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

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

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

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

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

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

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

12. The kit according to claim 10 or 11, characterized in that The kit is an immunochromatography kit, comprising a test card, wherein the test card comprises: a PVC base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a blotting paper; the sample pad, the conjugation pad, the nitrocellulose membrane and the blotting paper are overlapped and pasted on the base plate in sequence; the conjugation pad is sprayed with a tracer marker labeled with an antibody whose amino acid sequence of a light chain variable region is shown in SEQ ID NO: 12 and whose amino acid sequence of a heavy chain variable region is shown in SEQ ID NO: 16, a C line and a T line, wherein the C line is fixed with a mouse anti-human IgG antibody, and the T line is fixed with an antibody whose amino acid sequence of a light chain variable region is shown in SEQ ID NO: 4 and whose amino acid sequence of a heavy chain variable region is shown in SEQ ID NO:

8.

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

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

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