Antibodies or antigen-binding fragments thereof binding to imp enzymes and uses thereof

Abstract

The application provides an antibody or antigen-binding fragment thereof combined with IMP enzyme and application thereof, and relates to the technical field of monoclonal antibodies. The antibody or antigen-binding fragment thereof combined with IMP enzyme has light chain CDRs as shown in Seq_1-3 respectively, or as shown in Seq_11-13 respectively; and / or heavy chain CDRs as shown in Seq_4-6 respectively, or as shown in Seq_14-16 respectively. The antibody or antigen-binding fragment thereof combined with IMP enzyme has the characteristics of good specificity and high affinity, and alleviates the technical problem that the effect of the monoclonal antibody combined with IMP enzyme in the prior art is poor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of monoclonal antibodies, and in particular to an antibody or antigen-binding fragment thereof binding to IMP enzyme and application thereof. BACKGROUND

[0002] According to the structure and functional group difference of the active site of carbapenemase, it can be divided into metallo-beta-lactamase (MBLs or class B enzyme) and serine-based carbapenemase (class A and D enzyme). IMP (metallo-beta-lactamases resistant to imipenem) enzyme is the first acquired metalloenzyme detected in gram-negative bacteria, which can hydrolyze penicillins, carbapenems and cephalosporins, but cannot hydrolyze amikacin. At present, there are 52 subtypes of IMP in the world, which have been detected in clinically isolated gram-negative bacteria, such as Pseudomonas, Acinetobacter and Enterobacteriaceae bacteria.

[0003] IMP-1 is considered to be the first mobile MBL detected in gram-negative bacteria and encoded by plasmid, which is resistant to carbapenem antibiotics. IMP-1 was found in Pseudomonas aeruginosa isolated in Japan in 1988. Since then, blaIMP-1 has been isolated from Serratia marcescens in Japan. At present, blaIMP-1 has been detected in gram-negative bacteria in more than 15 countries or regions. IMP-2 was first discovered in a strain of Acinetobacter baumannii in Italy in 1997, and then was also detected in a strain of Acinetobacter baumannii and four other gram-negative bacteria in Japan. In China, IMP-1, IMP-4, IMP-8 and IMP-9 are common carbapenemases. In 1998, the first IMP metalloenzyme was reported in Hong Kong, China, which was IMP-4. Soon after, IMP-8 was isolated from a multidrug-resistant Klebsiella pneumoniae in Taiwan, China. In 2007, IMP-1 was first detected in a strain of Enterobacter cloacae in a hospital in Zhejiang Province, China.

[0004] Most IMP enzymes mediated amino acid sequences contain 246 residues, IMP-9, IMP-11, IMP-21 sequences consist of 245 residues. Some alleles of blaIMP gene are single or double point mutants, for example, the difference between IMP-10 and IMP-1 is mainly caused by a single base leading to amino acid change, IMP-3 has 2 amino acid differences compared with IMP-1, in which the amino acid at position 196 is converted from serine (Ser) residue to glycine (Gly) residue, resulting in IMP-3 unable to hydrolyze penicillins, ampicillin, ceftazidime and imipenem; there are also some alleles that are relatively scattered from each other, IMP-9 and IMP-18 are the most distant IMP variants, with 53 different amino acid residues. IMP enzymes have typical metalloenzyme characteristics, i.e. different degrees of hydrolysis activity to penicillins, cephalosporins and carbapenems, but almost no hydrolysis of amikacin, and are not inhibited by β-lactamase inhibitors.

[0005] The modified Hodge test recommended by the American Clinical Laboratory Standardization Institute (Clinical and Laboratory Standards Institute, CLSI) is used as a phenotypic confirmatory test for carbapenemase-producing Enterobacteriaceae. Escherichia coli (ATCC25922) is smeared on MH agar plates to form a lawn (1:10 dilution of 0.5 McFarland turbidity), then ertapenem or meropenem paper is placed in the center, and the isolated bacteria to be tested is streaked from the edge of the paper to the edge of the plate, and incubated overnight. If a clover leaf-shaped inhibition zone appears, it is positive for carbapenemase phenotype. However, the sensitivity and specificity of this method for IMP enzyme detection need further research and evaluation. In recent years, some scholars have developed a method for routine detection of drug resistance phenotype based on the characteristics of some carbapenemases that can be inhibited by metal ion integrators. The main methods reported in the literature are paper disc diffusion method, EPI microdilution method and concentration gradient (E-test method), which are observed by naked eye. The sensitivity and specificity will be affected by subjective factors, enzyme production capacity of the tested bacteria, etc., and there is a possibility of misreading and missing reading, and the time is relatively long. Carba NP test has good sensitivity to most enzymes, and CIM is a method commonly used in laboratories to detect IMP enzymes, which has good sensitivity and specificity, but is affected by the enzyme production capacity of bacteria.

[0006] With the development of molecular biology, especially the advent of polymerase chain reaction technology, detection and analysis of IMP enzyme at the gene level can not only accurately and quickly detect IMP enzyme genes, but also locate drug resistance genes, which is conducive to the study of drug resistance mechanisms and epidemiology of IMP enzyme-producing bacteria. At present, it is believed that molecular biology technology is the gold standard for detecting drug resistance genotypes. However, with the continuous discovery of new types of IMP enzymes, more specific primers for IMP enzyme genes need to be designed. Using the current designed primers may miss new IMP enzyme genes, and there are many factors such as long cycle, test tools, equipment and hardware requirements, which make the technology unable to be widely applied to primary hospitals, have high operation difficulty, complicated steps, and some tests require professional personnel to learn professional courses, and the cost of single detection is high.

[0007] Monoclonal antibodies are highly uniform antibodies produced by a single B cell clone, only targeting specific antigen epitopes. Monoclonal antibodies are usually prepared by hybridoma cells based on cell fusion technology. Sensitized B cells with specific antibody secretion ability and myeloma cells with unlimited reproduction ability are fused into B cell hybridoma. After the cell group is cultured, specific antibodies against a specific antigen epitope, i.e. monoclonal antibodies, can be prepared. The purpose of specific antibody detection is to assist clinical diagnosis, and in some diseases, it is also an indicator for observing efficacy and prognosis. The detection of specific antibodies also has special and important significance in drug resistance and infectious disease epidemiology. Antibody immunology detection has the following advantages: high specificity, specific monoclonal antibodies can be used for single cytokine detection; simple and fast operation, no need to rely on cell lines, so no need to maintain culture, increased operability, easy to promote and convenient for screening; relatively few influencing factors and easy to control, good repeatability, and easy to standardize the method.

[0008] CN112501131A discloses an anti-IMP enzyme hybridoma cell strain, a monoclonal antibody and an application. The monoclonal antibody has the characteristics of high purity titer and strong specificity, and is suitable for being used as an immunodiagnostic reagent for in vitro diagnosis of IMP enzyme. However, the above-mentioned monoclonal antibody is a murine monoclonal antibody. Although murine monoclonal antibodies are the most widely used antibodies, there are still problems of weak affinity and poor specificity. Therefore, it is necessary to provide an improved IMP enzyme antibody to meet the market demand.

[0009] In view of this, the present application is proposed. SUMMARY

[0010] The first object of the present application is to provide an antibody or antigen binding fragment thereof that binds to IMP enzyme, so as to alleviate the technical problem of poor effect of the monoclonal antibody that binds to IMP enzyme in the prior art.

[0011] A second objective of this invention is to provide biomaterials, reagents, kits, and their applications related to antibodies or antigen-binding fragments of the aforementioned IMP enzyme, in order to improve existing methods for detecting IMP.

[0012] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0013] According to one aspect of the present invention, an antibody or antigen-binding fragment thereof that binds to an IMP enzyme is provided, comprising a light chain variable region and / or a heavy chain variable region;

[0014] 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 CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_1, Seq_2, and Seq_3, respectively.

[0015] Alternatively, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_11, Seq_12, and Seq_13, respectively;

[0016] 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 CDR-H1, CDR-H2, and CDR-H3 are shown in Seq_4, Seq_5, and Seq_6, respectively.

[0017] Alternatively, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in Seq_14, Seq_15, and Seq_16, respectively.

[0018] According to one aspect of the present invention, a biomaterial is provided, the biomaterial comprising a nucleic acid fragment, a carrier, or a host cell;

[0019] The nucleic acid fragment is selected from (a1) or (a2): (a1) DNA or RNA encoding the antibody or its antigen-binding fragment; (a2) a nucleic acid fragment complementary to the nucleic acid fragment defined in (a1); the vector includes the nucleic acid fragment; the host cell is transformed by the vector, or the nucleic acid is integrated into the genome of the host cell.

[0020] According to one aspect of the present invention, the present invention also provides a method for producing an antibody or antigen-binding fragment thereof that binds to an IMP enzyme, comprising expressing the antibody or antigen-binding fragment thereof that binds to an IMP enzyme using the aforementioned host cells.

[0021] According to one aspect of the present invention, the present invention also provides a composition comprising the antibody or antigen-binding fragment thereof that binds to the IPM enzyme and a label; wherein the label is coupled to the antibody or antigen-binding fragment thereof; or, the label and the antibody or antigen-binding fragment thereof are packaged separately.

[0022] According to one aspect of the present invention, the present invention also provides a reagent or kit for detecting IMP enzymes, comprising the above-described antibody or its antigen-binding fragment, or the above-described composition.

[0023] According to one aspect of the present invention, the present invention also provides the use of the above-described antibody or antigen-binding fragment thereof that binds to IMP enzyme, biological materials, production methods, compositions, or reagents or kits for detecting IMP enzyme in the detection of IMP enzyme for non-diagnostic and therapeutic purposes or in the detection of IMP enzyme-producing microorganisms for non-diagnostic and therapeutic purposes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention obtains the conserved sequence of the IMP enzyme through NCB sequence alignment and obtains high-purity protein through prokaryotic gene expression. Hybridoma cells are prepared by immunizing New Zealand white rabbits with the IMP antigen protein and obtaining B cells that secrete antibodies binding to IMP. Hybridoma cells capable of secreting highly specific antibodies are selected through screening. Sequencing yields light chain CDRs as shown in Seq_1~3 or Seq_11~13, and heavy chain CDRs as shown in Seq_4~6 or Seq_14~16.

[0026] The CDR region sequences of the antibodies or antigen-binding fragments that bind to IMP enzymes provided by this invention are derived from rabbits and have the ability to specifically bind to different IMP enzyme subtypes. Antibodies or antigen-binding fragments possessing the aforementioned CDR sequences exhibit multiple antigen recognition sites, high specificity, and high affinity, demonstrating superior performance in all aspects. Therefore, they are suitable as immunodiagnostic reagents for in vitro diagnosis of IMP enzymes or IMP-secreting microorganisms, achieving a titer exceeding 1:1,280,000. In particular, the different antibodies formed by the aforementioned light chain CDRs and heavy chain CDRs in certain combinations can pair well and can be applied to immunoassay methods based on the formation of antibody-antigen-antibody complexes, such as as primary and secondary antibodies in double-antibody sandwich ELISA; or as labeled antibodies and antibodies coating the detection area in immunochromatographic detection cards, respectively.

[0027] The IMP-binding antibodies or their antigen-binding fragments provided by this invention, along with reagents or kits prepared from related biological materials, can be used for early typing of drug-resistant strains, guiding clinical medication, and assisting in clinical infection control and treatment. Reagents or kits containing antibodies or their antigen-binding fragments with the aforementioned CDR sequence can rapidly and accurately determine the degree of bacterial resistance in patients by qualitatively or semi-quantitatively detecting IMP enzymes in bacterial samples isolated from patients or positive blood cultures, and can quickly detect the presence of IMP enzymes in bacterial samples, which can be used clinically to guide medication. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 The SDS-PAGE electrophoresis results of antibodies 1 and 2 prepared in the embodiments of the present invention are shown below.

[0030] Figure 2 The results of the affinity activities of antibodies 1 and 2 prepared in the embodiments of the present invention for IMP enzyme were detected by ELISA.

[0031] Figure 3 The reaction results of antibody 1 prepared in this embodiment of the invention with KPC, NDM, VIM, IMP and OXA-48 enzyme;

[0032] Figure 4 The reaction results of antibody 2 prepared in the embodiments of the present invention with KPC, NDM, VIM, IMP and OXA-48 enzyme are shown. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0035] According to one aspect of the present invention, an antibody or antigen-binding fragment thereof that binds to an IMP enzyme is provided, said antibody or antigen-binding fragment comprising at least one light chain variable region or one heavy chain variable region, or both light chain variable regions and heavy chain variable regions.

[0036] Light chain variable region:

[0037] The light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3.

[0038] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown below:

[0039] CDR-L1: TGTSSDIGNNDYVS(Seq_1);

[0040] CDR-L2: DVSRRPS(Seq_2);

[0041] CDR-L3: SSYAGSSNLV (Seq_3).

[0042] The preferred amino acid sequence of the light chain variable region containing the light chain CDR composed of the above-mentioned CDR-L1 (Seq_1), CDR-L2 (Seq_2), and CDR-L3 (Seq_3) is as follows:

[0043] QSVLTQPSSASTSPGSSVKLSCTGTSSDIGNNDYVSWYQQYMGRPPTNIIYDVSRRPSGVSDRFSGSIDRSSNTAFLTVNNVQADDEADYYCSSYAGSSNLVFGGGTKLTV (Seq_7).

[0044] Alternatively, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown below:

[0045] CDR-L1: RASQTVTSYLA (Seq_11);

[0046] CDR-L2: DASNRAT(Seq_12);

[0047] CDR-L3: QQRSDRPPAFT (Seq_13).

[0048] The preferred amino acid sequence of the light chain variable region containing the light chain CDR composed of the above-mentioned CDR-L1 (Seq_11), CDR-L2 (Seq_12), and CDR-L3 (Seq_13) is as follows:

[0049] TQSPASLSLSPGERATLSCRASQTVTSYLAWYQQRAEQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDRPPAFTFPGGTKVEIK (Seq_17).

[0050] In some optional embodiments, the N-terminus of the aforementioned light chain variable region further contains a signal peptide, and the preferred amino acid sequence of the signal peptide in the light chain variable region is as follows:

[0051] MDMRVPAQLLGLLLLWLSGARC(Seq_21).

[0052] Heavy chain variable region:

[0053] The heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3.

[0054] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown below:

[0055] CDR-H1: GYWMH (Seq_4);

[0056] CDR-H2: YINSDGSTNYADSVKG (Seq_5);

[0057] CDR-H3: GGGYSYGPFD (Seq_6).

[0058] The preferred amino acid sequence of the heavy chain variable region containing the heavy chain CDR composed of the above-mentioned CDR-H1 (Seq_4), CDR-H2 (Seq_5), and CDR-H3 (Seq_6) is as follows:

[0059] EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYWMHWVRQAPEKGLVWVAYINSDGSSTNYADSVKGRFTISRDNAKNTLFLQMNSLRAEDTAMYYCARGGGYSYGPFDYWGQGTSVTVSS (Seq_9).

[0060] Alternatively, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in Seq_14, 15, and 16, respectively:

[0061] CDR-H1: TSSFYWG (Seq_14);

[0062] CDR-H2: NIYYSGSITYNPSLTS (Seq_15);

[0063] CDR-H3: RQITFNYNAVSGHDAFDV (Seq_16).

[0064] The preferred amino acid sequence of the heavy chain variable region containing the heavy chain CDR composed of CDR-H1 (Seq_14), CDR-H2 (Seq_15), and CDR-H3 (Seq_16) is as follows:

[0065] DVQLQESGPGLVKPSQTVSLTCTVSGGSISTSSFYWGWIRQFPGNKLEWIGNIYYSGSITYNPSLTSRVTITRDTSKNQFFLEMNSVTAADTAIYYCAGRQITFNYNAVSGHDAFDVWGTGTTVTVSS (Seq_19).

[0066] In some optional embodiments, the N-terminus of the above-mentioned heavy chain variable region further contains a signal peptide, and the preferred amino acid sequence of the signal peptide in the heavy chain variable region is as follows:

[0067] MDWTWRFLFVVAAATGVQS(Seq_22).

[0068] In some preferred embodiments, the antibody or its antigen-binding fragment that binds to the IMP enzyme contains both a light chain variable region and a heavy chain variable region. When both light chain variable regions and heavy chain variable regions are present, the light chain CDR and heavy chain CDR are preferably combined in the following two ways (A) and (B):

[0069] (A) The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, which make up the light chain variable region CDR, are shown in Seq_1, Seq_2, and Seq_3, respectively;

[0070] Meanwhile, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3, which make up the heavy chain variable region CDR, are shown in Seq_4, Seq_5, and Seq_6, respectively.

[0071] In this combination, a preferred embodiment is that the amino acid sequence of the light chain variable region is as shown in Seq_7 or Seq_23, while the amino acid sequence of the heavy chain variable region is as shown in Seq_9 or Seq_24. The light chain variable region shown in Seq_23 is the N-terminus of the amino acid sequence shown in Seq_7 with the signal peptide shown in Seq_21 fused thereto; the heavy chain variable region shown in Seq_24 is the N-terminus of the amino acid sequence shown in Seq_9 with the signal peptide shown in Seq_22 fused thereto.

[0072] (B) The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, which make up the light chain variable region CDR, are shown in Seq_11, Seq_12, and Seq_13, respectively; and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3, which make up the heavy chain variable region CDR, are shown in Seq_14, Seq_15, and Seq_16, respectively.

[0073] In this combination, a preferred embodiment is that the amino acid sequence of the light chain variable region is as shown in Seq_17 or Seq_25, while the amino acid sequence of the heavy chain variable region is as shown in Seq_19 or Seq_26. The light chain variable region shown in Seq_25 is the N-terminus of the amino acid sequence shown in Seq_17 with the signal peptide shown in Seq_21 fused to it; the heavy chain variable region shown in Seq_26 is the N-terminus of the amino acid sequence shown in Seq_19 with the signal peptide shown in Seq_22 fused to it.

[0074] In some optional embodiments, antibody 1 obtained according to the combination method described in (A) above, and antibody 2 obtained according to the combination method described in (B) above, can also form paired antibodies for use in immunoassay methods based on the detection principle of forming antibody-antigen-antibody complexes, such as double-antibody sandwich ELISA or immunochromatographic assay cards. In some optional embodiments, antibody 1 / antibody 2 is coated on a solid-phase carrier as a primary antibody, and the corresponding antibody 2 / antibody 1 is linked to a label as a secondary antibody, such as horseradish peroxidase or alkaline phosphatase; or, antibody 1 / antibody 2 is labeled with a label, and the corresponding antibody 2 / antibody 1 is used as the antibody coated on the detection area of ​​the immunochromatographic assay card. Antibody 1 and antibody 2 may optionally be, independently, complete antibodies or antigen-binding fragments.

[0075] As is well known in the art, the binding specificity and affinity of antibodies are primarily determined by the CDR sequence. Based on mature and well-known existing technologies, the amino acid sequence of non-CDR regions can be easily altered to obtain variants with similar biological activities. The monoclonal antibody variants of this invention, having a CDR sequence identical to the one described above, possess similar biological activity because they have the same CDR sequence as the IMP-binding antibody described in this invention.

[0076] The antigen-binding fragment is an antibody fragment with the same specificity as the parent antibody, and may be, for example, one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibodies, and the smallest antibody recognition unit. In addition to the functional fragments mentioned above, any fragment with an extended half-life is also included.

[0077] These functional fragments typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art, based on the description herein, infer that the antigen-binding fragments of the present invention can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0078] The antigen-binding fragment can also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as an automated peptide synthesizer; or by expressing a gene encoding the above-mentioned functional fragment in a host cell.

[0079] The antibody or antigen-binding fragment of the IMP enzyme provided by this invention, excluding the CDR region, has the remaining sequence sourced from one or more species including, but not limited to, mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, dairy cows, pigs, horses, monkeys, and humans.

[0080] When the antibody against the IMP enzyme is a complete antibody molecule, its antibody type can be, for example, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Given that those skilled in the art are familiar with the CDR region or the variable regions of the light and heavy chains of an antibody, they can use conventional methods in the art to obtain different types of antibodies, such as fusing a variable region gene with a corresponding heavy chain or heavy chain constant region coding gene and expressing the fusion protein in a host cell to obtain different types of antibodies.

[0081] In some preferred embodiments, the antibody comprises a sequence of a constant region of any one of rabbit antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0082] In some preferred embodiments, the amino acid sequence of the light chain constant region of the antibody is shown in Seq_31; the amino acid sequence of the heavy chain constant region of the antibody is shown in Seq_32.

[0083] According to another aspect of the invention, the invention also provides a biomaterial comprising a nucleic acid fragment, a vector, or a host cell.

[0084] Nucleic acid fragments: selected from (a1) or (a2):

[0085] (a1) It encodes an antibody or antigen-binding fragment thereof that binds to the IMP enzyme; in some preferred embodiments, the nucleic acid fragment is DNA containing a DNA fragment encoding a light chain variable region and / or a DNA fragment encoding a heavy chain variable region.

[0086] Preferably, the nucleotide sequence of the DNA fragment encoding the light chain variable region is as shown in Seq_8, Seq_18, Seq_27, or Seq_28; the sequences shown in Seq_27 and Seq_28 are the sequences obtained by linking the sequences shown in Seq_8 and Seq_18 to encode the signal peptide fragment, respectively.

[0087] Preferably, the nucleotide sequence of the DNA fragment encoding the variable region of the heavy chain is as shown in Seq_10, Seq_20, Seq_29 or Seq_30; the sequences shown in Seq_29 and Seq_30 are the sequences obtained by linking the sequences shown in Seq_10 and Seq_20 to the sequences encoding the signal peptide fragment.

[0088] Preferably, the nucleotide sequence of the DNA fragment encoding the light chain constant region is shown in Seq_33.

[0089] Preferably, the nucleotide sequence of the DNA fragment encoding the heavy chain constant region is shown in Seq_34.

[0090] (a2) Nucleic acid fragments that are complementary to the nucleic acid fragments defined in (a1).

[0091] Vector: Includes the nucleic acid fragments described above. The vector may also include portions encoding other constituent elements, such as, but not limited to, encoding regulatory sequences or marker genes. The vector may be, for example, but not limited to, a prokaryotic expression vector, a eukaryotic expression vector, or an insect expression vector.

[0092] Host cell: The host cell is transformed with the above-mentioned vector to enable the above-mentioned vector to be cloned or expressed; or, the host cell genome integrates the nucleic acid fragment to express the antibody or antigen-binding fragment that binds to the IMP enzyme.

[0093] According to one aspect of the present invention, the present invention also provides a method for producing an antibody or antigen-binding fragment thereof that binds to an IMP enzyme, comprising expressing the antibody or antigen-binding fragment thereof using the aforementioned host cells. This method uses raw materials derived from stably expressed cell lines, eliminating the risk of raw material supply issues and offering stable performance and low cost. Furthermore, the product exhibits strong stability, minimal batch-to-batch variation, and is unaffected by cell line degradation.

[0094] According to another aspect of the present invention, the present invention also provides a composition comprising the antibody or its antigen-binding fragment and a label; wherein the label is conjugated to the antibody or its antigen-binding fragment; or, the label and the antibody or its antigen-binding fragment are packaged separately and then conjugated before use. The label includes, but is not limited to, one or more of enzymes, latex particles, fluorescent molecular labels, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal silver, colloidal carbon, biotin, or streptavidin.

[0095] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Antibodies or antigen-binding fragments of alkaline phosphatase or horseradish peroxidase that bind to IMP can be used as secondary antibodies in chemiluminescent enzyme-linked immunosorbent assay (ELISA) methods and kits.

[0096] According to another aspect of the present invention, the present invention also provides a reagent or kit comprising the antibody or antigen-binding fragment thereof that binds to IMP, or the above-described composition. Optional examples include, but are not limited to, kits for conventional detection methods in the art, such as ELISA kits, Western blotting kits, immunohistochemical kits, or immunochromatographic test strips. It is understood that the reagent may also contain reagents conventional in the art, such as, but not limited to, one or more of lyophilization protectants, buffers, and solvents; the kit may also contain reagents or consumables conventional in the art, such as, but not limited to, one or more of buffer solutions, blocking solutions, secondary antibodies, chromogenic substances, labels and reaction substrates, magnetic microparticles, test strips, and their supporting components.

[0097] In some optional embodiments, the kit includes an immunochromatographic assay card with a labeled anti-IMP enzyme monoclonal antibody 1 / antibody 2 embedded in the sample conjugation pad and an anti-IMP enzyme monoclonal antibody 2 / antibody 1 coated on the detection line (T). If the test sample is positive, the IMP enzyme binds to the labeled antibody 1 / antibody 2 to form a complex. Under chromatography, the complex moves forward along the paper strip and is captured by the pre-coated IMP enzyme antibody 2 / antibody 1 when it passes the detection line (T), forming an immune complex and producing a detectable signal value. If the test sample is negative, no immune complex is formed, and no detectable signal value appears at the detection line. The label can be, for example, but not limited to, colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots. Preferably, antibody 1 is embedded in the sample conjugation pad and antibody 2 is coated on the detection line.

[0098] According to one aspect of the present invention, the present invention also provides the use of the above-described antibody or antigen-binding fragment thereof that binds to IMP enzyme, biological materials, production methods, compositions, or reagents or kits for detecting IMP enzyme in the detection of IMP enzyme for non-diagnostic and therapeutic purposes or in the detection of IMP enzyme-producing microorganisms for non-diagnostic and therapeutic purposes.

[0099] Antibodies or antigen-binding fragments of IMP enzymes, or combinations thereof, are used in the preparation of products for detecting IMP enzymes or IMP-producing microorganisms. These products can be used for early typing of drug-resistant strains and to guide clinical medication, assisting in clinical infection control and treatment. They can quickly and accurately determine the degree of bacterial resistance in patients by qualitatively or semi-quantitatively detecting IMP enzymes in bacterial samples isolated from patients or positive blood culture samples, and can be used clinically to guide medication.

[0100] The technical solution and beneficial effects of the present invention will be further explained below with reference to preferred embodiments.

[0101] Example 1 Preparation of Antigen

[0102] IMP enzyme (IMP) was obtained through NCBI (National Center for Biotechnology Information) sequence alignment. 1~35, IMP 37~46, IMP 48~49, IMP 51~56, IMP 58~85, IMP The conserved sequence (88-89) was used. The expression plasmid pET was constructed using standard molecular biology techniques of enzyme digestion and ligation. 28a (+) PM, the recombinant vector was transformed into *E. coli* DH5α competent cells using the CaCl2 heat shock method. Positive clones were screened using LB medium containing 100 μg / mL ampicillin. *E. coli* were cultured routinely, and plasmids were extracted for PCR identification to confirm the presence of the target gene. The extracted expression plasmid pET was then used. 28a (+) After PMAA was transformed into E. coli BL21(DE3) competent cells, it was plated on selective medium for screening to obtain single colonies resistant to 100 μg / mL ampicillin, followed by overnight liquid culture. One mL of the overnight culture was inoculated into 200 mL of LB medium containing 100 μg / mL ampicillin and cultured with shaking until the logarithmic growth phase (OD600 at 0.5). 0.6), add IPTG (1 mmol / L), induce culture at 16℃ for 3h, purify the fermentation broth through nickel column, and obtain high-purity protein through prokaryotic gene expression.

[0103] Example 2 Animal Immunization

[0104] Select New Zealand white rabbits of suitable age and weighing approximately 1.5 kg, and house them in a standard animal facility for 3 days. If no abnormalities are observed, begin immunization: Add 100 μg of IMP enzyme antigen to 0.5 mL of autoclaved physiological saline, mix thoroughly using a micro vortex mixer, add 0.5 mL of Freund's complete adjuvant, and emulsify thoroughly by pushing and pulling with a syringe. Administer the emulsification via multiple subcutaneous injections on the back of the New Zealand white rabbits. Two weeks later, administer booster immunizations, followed by booster immunizations every week for a total of six immunizations. Starting from the third immunization, one week after immunization, collect 200–500 μL of blood from the marginal ear vein of the rabbits. Collect the immunized rabbit blood in sterile test tubes, label them, and send them to the preparation room. Use serum from unimmunized rabbits as a control. Measure the titer and affinity using ELISA. After the final immunization, harvest the spleen for cell fusion to prepare hybridoma cells.

[0105] Example 3 Preparation and screening of hybridoma cells

[0106] The prepared rabbit antiserum was tested for potency. If the potency was qualified, rabbit spleen was used for cell fusion to prepare a monoclonal hybridoma cell line. The method was as follows: Immunized New Zealand rabbits were sacrificed, and the spleen was removed under aseptic conditions. The spleen was washed once with cell culture medium, then crushed and passed through a stainless steel sieve. The resulting cells were centrifuged and washed twice with cell culture medium. SP2 / 0 myeloma cells in the logarithmic growth phase were mixed with spleen cells, washed once with cell culture medium without fetal bovine serum, centrifuged, and the supernatant was discarded. Polyethylene glycol solution was added, and the mixture was incubated at 37°C for 90 seconds. The reaction was terminated with cell culture medium without fetal bovine serum (FBS), followed by centrifugation. Cells were resuspended in HAT selection medium containing 20% ​​FBS and added to 96-well plates, where they were cultured at 37°C and 5.0% CO2. Cells in good growth condition within the 96-well plates were diluted with cell culture medium to 1-3 cells / mL and added to the plates, then cultured in a cell culture incubator at 37°C and 5.0% CO2. Each cell line was numbered, and cell lines showing positive results in the culture supernatant were selected for further expansion to obtain hybridoma cell lines. The obtained hybridoma cells were screened using ELISA. Cell growth was observed on day 5 post-fusion, and the titer of the cell culture supernatant was detected using indirect ELISA on days 10-14. The hybridoma cells with the highest titer were expanded until 100% positivity was achieved, and the resulting hybridoma cell lines were cryopreserved in liquid nitrogen for later use.

[0107] Example 4: Isolation of antibody variable region gene from hybridoma cells using RT-PCR

[0108] After homogenizing the hybridoma cells selected in Example 3, RNA was extracted by adding cell lysis buffer. RNA was precipitated from the aqueous phase with isopropanol, centrifuged, washed to remove impurities, resuspended, and reverse transcribed to obtain cDNA. PCR was performed using specific primers from New Zealand rabbits, with the hybridoma cell cDNA as a template, to amplify the variable regions of the heavy and light chains of the antibody. A 50 μL system contained 5 μL of cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM of specific primers. PCR amplification was performed under the following conditions: pre-denaturation at 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 50 s, 35 cycles; 72℃ for 7 min. The obtained PCR products were identified by 1% agarose gel electrophoresis, the target fragment was recovered, and sequencing was performed. The sequencing results were compared with the IMGT database (http: / / www.imgt.org / IMGT_vquest / vquest). The sequence information of antibody 1 and antibody 2 is shown below:

[0109] Antibody 1:

[0110] Light chain:

[0111] MDMRVPAQLLGLLLLWLSGARCQSVLTQPSSASTSPGSSVKLSCTGTSSDIGNNDYVSWYQQYMGRPPTNIIYDVSRRPSGVSDRFSGSIDRSSNTAFLTVNNVQADDEADYYCSSYAGSSNLVFGGGTKLTV (Seq_23);

[0112] Among them, positions 1-22 are signal peptides; positions 23-44 are FR1; positions 45-58 are CDR-L1; positions 59-73 are FR2; positions 74-80 are CDR-L2; positions 81-114 are FR3; positions 115-124 are CDR-L3; and positions 125-133 are FR4.

[0113] Heavy chain:

[0114] MDWTWRFLFVVAAATGVQSEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYWMHWVRQAPEKGLVWVAYINSDGSSTNYADSVKGRFTISRDNAKNTLFLQMNSLRAEDTAMYYCARGGGYSYGPFDYWGQGTSVTVSS (Seq_24);

[0115] Among them, positions 1-19 are signal peptides; positions 20-49 are FR1; positions 50-54 are CDR-H1; positions 55-68 are FR2; positions 69-85 are CDR-H2; positions 86-117 are FR3; positions 118-127 are CDR-H3; and positions 1-139 are FR4.

[0116] Antibody 2:

[0117] Light chain:

[0118] MDMRVPAQLLGLLLLWLSGARCTQSPASLSLSPGERATLSCRASQTVTSYLAWYQQRAEQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDRPPAFTFGPGTKVEIK(Seq_25);

[0119] Among them, positions 1-22 are signal peptides; positions 23-41 are FR1; positions 42-52 are CDR-L1; positions 53-67 are FR2; positions 68-74 are CDR-L2; positions 75-106 are FR3; positions 107-117 are CDR-L3; and positions 118-127 are FR4.

[0120] Heavy chain:

[0121] MDWTWRFLFVVAAATGVQSDVQLQESGPGLVKPSQTVSLTCTVSGGSISTSSFYWGWIRQFPGNKLEWIGNIYYSGSITYNPSLTSRVTITRDTSKNQFFLEMNSVTAADTAIYYCAGRQITFNYNAVSGHDAFDVWGTGTTVTVSS (Seq_26);

[0122] Among them, positions 1-19 are signal peptides; positions 20-49 are FR1; positions 50-56 are CDR-H1; positions 57-70 are FR2; positions 71-86 are CDR-H2; positions 87-118 are FR3; positions 119-136 are CDR-H3; and positions 137-147 are FR4.

[0123] Example 5: Construction, expression, and purification of monoclonal antibodies

[0124] Homologous recombination arms and signal peptide sequences were added to both ends of the antibody heavy chain variable region gene and the light chain variable region gene, respectively, using homologous recombination primers. The amino acid sequence of the heavy chain variable region signal peptide is shown in Seq_22, and the amino acid sequence of the light chain variable region signal peptide is shown in Seq_21. The expression plasmids containing the rabbit antibody heavy chain IgG1 constant region and the expression plasmid containing the rabbit antibody light chain IgG1 constant region were linearized using double enzyme digestion to generate homologous recombination arms. The amino acid sequence of the light chain constant region is shown in Seq_31, and the nucleotide sequence is shown in Seq_33; the amino acid sequence of the heavy chain constant region is shown in Seq_32, and the nucleotide sequence is shown in Seq_34. The variable region gene fragments with added homologous recombination arms and the linearized plasmids were ligated together by homologous recombination to construct complete light chain expression vectors and heavy chain expression vectors. The recombinant products were transformed into TOP10 E. coli competent cells for plasmid amplification.

[0125] The obtained monoclonal antibody heavy and light chain expression plasmids were added to Opti-Mem transfection medium at a 1:1 ratio. After thorough mixing, PEI transfection reagent (4 times the mass of DNA) was added. After mixing, the mixture was placed at room temperature in the dark for 30 min, and then added to 293T cells. After incubation for 6 h, the transfection system was removed, and FreeStyle™ 293 expression medium was added. The expressed antibody supernatant was purified using the AKTA protein purification system and the affinity purification method (Protein A) to obtain the anti-IMP enzyme monoclonal antibody. The specific steps were as follows: (1) Centrifuge the expressed antibody supernatant at 2500×g at room temperature for 10 min to remove the precipitate; (2) Wash the affinity purification column containing Protein A thoroughly with 10 times the volume of binding buffer; (3) Pass the expression supernatant through the purification column at a flow rate of 5 mL / min; (4) Wash the purification column thoroughly with 20 times the volume of binding buffer; (5) Use 0.1M The purification column was eluted with citrate buffer at pH 3.0-3.5 until the elution peak reached equilibrium. The pH was then adjusted to 7.0 with 1M Tris-HCl buffer at pH 9.0. (6) The purified monoclonal antibody was concentrated using a centrifugal column. PBS was used as the antibody storage buffer. Finally, the concentration of the concentrated antibody was determined using the BSA protein concentration detection method.

[0126] Example 6 Molecular weight determination

[0127] The molecular weight of monoclonal antibodies was identified by SDS-PAGE electrophoresis. 5 μg of sample was loaded into each lane, using a known molecular weight standard series as a reference. Electrophoresis was first performed at 90 V for 20 min, then at 140 V until all the indicator was elute. The gel was then removed, stained with Coomassie Brilliant Blue, and the molecular weight of the biological material was analyzed after staining. The SDS-PAGE electrophoresis image is shown below. Figure 1 As shown.

[0128] Example of effect 1

[0129] The affinity activity (titer) of monoclonal antibodies for IMP enzyme was detected by ELISA. The main steps are as follows: (1) Dilute the IMP enzyme antigen with PBS to 1 ng / μL, add 100 μL to each well of a 96-well microplate, and coat at 37°C for 2 h; (2) Discard the supernatant, wash the plate 3 times with 0.01 M PBST, prepare blocking buffer containing 3% BSA with PBST, add 100 μL to each well, and block at 37°C for 2 h; (3) Discard the supernatant, wash 5 times with PBST, and add the purified and concentrated antibody to the plate. The antibody was serially diluted from 1:1000 to 1:2560000, and 100 μL was added to each well. The mixture was incubated at 37°C for 1 h. (4) The antibody diluent was discarded, and the antibody was washed 6 times with PBST. Goat anti-rabbit IgG-HRP was diluted with 1:5000 blocking buffer, and 100 μL was added to each well. The mixture was incubated at 37°C for 1 h. (5) The secondary antibody diluent was discarded, and the antibody was washed 6 times with PBST. TMB was added, 100 μL / well, and the mixture was incubated at 37°C for 15 min in the dark. (6) 50 μL of 1M dilute sulfuric acid was added to each well to stop the reaction, and the absorbance was measured at 450 nm. The results are as follows. Figure 2 As shown, the screened monoclonal antibodies have a strong binding ability to IMP enzyme, and the titer against IMP enzyme antigen reaches 1:1280000 (OD value > 0.5).

[0130] Comparison of Effect Example 2 with Existing Monoclonal Antibodies

[0131] The affinity activity (titer) of the IMP enzyme was detected using the antibody modified in this invention, the natural antibody, and the published murine monoclonal antibody (purchased from Zhuhai Bomei Biotechnology Co., Ltd.), respectively, with the specific steps being the same as in Example 1. The results are shown in Table 1. Monoclonal antibody 1 and antibody 2 showed increased affinity and enhanced biological activity compared to the prior art.

[0132] Table 1

[0133]

[0134] Example 3: Cross-reaction

[0135] Microplates were coated with KPC, NDM, VIM, IMP, and OXA-48 enzymes, respectively, with a coating volume of 50 ng per well. Monoclonal antibodies were diluted to 10 ng / mL and added to each well (100 μL). The plates were incubated at 37°C for 1 h. After washing, 100 μL of HRP-labeled goat anti-rabbit secondary antibody was added to each well, and the plates were incubated at 37°C for 0.5 h. After washing, TMB was added, and the plates were incubated at 37°C for 15 min before readings were stopped. The results for antibody 1 are shown below. Figure 3 As shown, the results for antibody 2 are as follows: Figure 4 As shown, this indicates that the monoclonal antibody does not cross-react with other types of carbapenemases and has high specificity.

[0136] Example 4: Antibody pairing verification

[0137] Two antibodies were used: Antibody 1 as the capture antibody and Antibody 2 as the labeling antibody (HRP-labeled). The capture antibody was also HRP-labeled as a control. The capture antibody was coated onto an antigen plate. First, serially diluted antigen was added and incubated, followed by washing away unbound antigen. Then, the labeled antibody was added and incubated, followed by washing away unbound labeled antibody. Finally, chromogenic buffer was added for color development. If color development occurred, it indicates that the labeled antibody specifically binds to the antigen, and the capture antibody and labeled antibody are a paired antibody pair. If color development did not occur, it indicates that the labeled antibody could not bind to the antigen and was eluted, and the capture antibody and labeled antibody are not a paired antibody pair. The results are as follows: Figure 4 As shown, this indicates that the pairing of these two antibodies has the best ability to bind to the antigen.

[0138] Table 2

[0139]

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or its antigen-binding fragment that binds to an IMP enzyme, characterized in that, It comprises a light chain variable region and a heavy chain variable region; the antibody or its antigen-binding fragment is selected from (A) or (B): (A) 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 CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO.: 1, SEQ ID NO.: 2, and SEQ ID NO.: 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 CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO.: 4, SEQ ID NO.: 5, and SEQ ID NO.: 6, respectively. (B) 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 CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO.: 11, SEQ ID NO.: 12, and SEQ ID NO.: 13, 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 CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The N-terminus of the variable region of the light chain is connected to a signal peptide; The amino acid sequence of the signal peptide in the light chain variable region is shown in SEQ ID NO.:

21.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The N-terminus of the heavy chain variable region is connected to a signal peptide; The amino acid sequence of the signal peptide in the heavy chain variable region is shown in SEQ ID NO.:

22.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, (A) The amino acid sequence of the light chain variable region is shown in SEQ ID NO.: 7 or SEQ ID NO.: 23, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.: 9 or SEQ ID NO.: 24; (B) The amino acid sequence of the light chain variable region is shown in SEQ ID NO.: 17 or SEQ ID NO.: 25, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.: 19 or SEQ ID NO.:

26.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antibody type is IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antibody or its antigen-binding fragment, excluding the CDR region, has a sequence source species selected from one or more of the following: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antibody contains a sequence of a constant region of any one of the rabbit antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO.: 31; the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO.:

32.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antigen-binding fragment is one or more of F(ab')2, Fab', Fab, Fv, scFv, and dsFv.

10. A biomaterial, characterized in that, It can be a nucleic acid fragment, vector, or host cell; The nucleic acid fragment is (a1): (a1) DNA or RNA encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-9; The vector includes the nucleic acid fragment; The host cell is transformed by the vector.

11. The biomaterial according to claim 10, characterized in that, The nucleotide sequence of the DNA fragment encoding the light chain constant region is shown in SEQ ID NO.: 33; The nucleotide sequence of the DNA fragment encoding the heavy chain constant region is shown in SEQ ID NO.:

34.

12. A method for producing the antibody or antigen-binding fragment thereof that binds to IMP enzyme according to any one of claims 1-9, characterized in that, This includes using the host cell described in claim 10 to express the antibody or antigen-binding fragment thereof that binds to the IMP enzyme.

13. A composition, characterized in that, The composition comprises a marker and an antibody or antigen-binding fragment thereof as described in any one of claims 1-9; the marker is coupled to the antibody or antigen-binding fragment thereof; or the marker and the antibody or antigen-binding fragment thereof are packaged separately.

14. The composition according to claim 13, characterized in that, The label is one or more of the following: enzyme, fluorescent molecular label, quantum dot, fluorescent microsphere, colored microsphere, colloidal gold, colloidal silver, colloidal carbon, biotin, or streptavidin; The enzymes include alkaline phosphatase or horseradish peroxidase.

15. A reagent or kit for detecting IMP enzyme, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-9, or the composition as described in claim 13.

16. The reagent or kit according to claim 15, characterized in that, The kit is an immunochromatographic assay card; In the immunochromatographic assay card, the antibody markers are colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots.

17. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-9, the biomaterial according to claim 10, the production method according to claim 12, the composition according to claim 13, or the reagent or kit for detecting IMP enzyme according to claim 15 in the detection of IMP enzyme for non-diagnostic and therapeutic purposes or in the detection of IMP-producing microorganisms for non-diagnostic and therapeutic purposes.

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