CN115991783A

CN115991783A - VIM enzyme monoclonal antibody and application

Info

Publication number: CN115991783A
Application number: CN202211185709.4A
Authority: CN
Inventors: 冯涛; 刘春龙; 付成华; 粟艳; 周泽奇
Original assignee: Dana Hunan Biotechnology Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dana Hunan Biotechnology Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-04-21

Abstract

The invention relates to the technical field of monoclonal antibodies, in particular to a VIM enzyme monoclonal antibody and application thereof. The VIM enzyme binding molecule provided by the invention has good specificity, high bioactivity, strong stability, small batch-to-batch difference, is not influenced by cell strain degradation, has high affinity with the VIM enzyme, has a titer of more than 1:1280000, and can be used for VIM enzyme detection or preparation of purified products.

Description

VIM enzyme monoclonal antibody and application

Technical Field

The invention relates to the technical field of monoclonal antibodies, in particular to a VIM enzyme monoclonal antibody and application thereof.

Background

Carbapenemases can be classified into metallo-beta-lactamases (MBLs or class B enzymes) and serine-based carbapenemases (class a and D enzymes) based on differences in the structure and functional groups of the carbapenemases, which are one of the important members of MBLs. VIM-2 and VIM-1 were found in Pseudomonas aeruginosa in Europe in 1996 and 1997, respectively, and have been paid attention again to because of the highest similarity (32% amino acid identity) between the novel Derilla-lactamase (NDM-1) and VIM-1, VIM-2 sequences, which were newly reported in 2009. Subsequent reports have been made in enterobacteriaceae, pseudomonas and acinetobacter worldwide. To date, 73 VIM variants have been reported and divided into 3 groups based on phylogenetic analysis: VIM-1, VIM-2 and VIM-7. It hydrolyzes almost all beta-lactam antibiotics (except for single-ring beta-lactam antibiotics such as aztreonam), and studies have shown that blaVIM is usually co-present with one or more aminoglycoside resistance genes and carried by a variety of class I integrants, integrated in transposons, contained on plasmids or chromosomes, and can be spread and prevalent in gram-negative bacteria along with the transfer of integrants or plasmids, resulting in widespread worldwide spread of VIM. In addition, co-existence with other beta-lactamase genes results in the emergence of widely and pan-resistant strains, making the selection of clinical therapeutic drugs very limited. To date, VIM has been detected in at least 23 gram-negative bacilli in more than 40 countries and regions.

Most VIM enzyme detection methods take the hydrolysis of enzyme as a principle, and the sensitivity and the specificity of the VIM enzyme detection method can be influenced by subjective factors, the enzyme production capacity of bacteria to be detected and the like according to visual observation results, so that the possibility of misreading and misreading exists, and the time is long. The sensitivity range of the chromogenic medium has larger fluctuation due to different chromogenic mediums. The improved Hodge test has more prominent detection capability on class A and class D enzymes, has poorer specificity on class B enzymes and can generate false negative. If the strain is capable of producing ESBLs or AmpC enzymes, or a porin deletion, false positive results may occur in both of the above methods. The Carba NP assay is very sensitive to most enzymes and CIM is a method commonly used in the laboratory to detect VIM enzymes, which is highly sensitive and specific but is affected by the ability of the bacteria to produce enzymes. In the molecular biological detection method, the PCR method is generally used as a gold standard for detection, and on the basis of tradition, various novel molecular biological technologies such as real-time fluorescence quantitative PCR, gene chips, loop-mediated isothermal amplification and the like are developed, so that the application field is gradually expanded. Compared with the traditional PCR, the method has higher sensitivity and specificity in the aspect of VIM enzyme detection, has higher advantages in the aspects of detection quantity and detection time, but has higher operation difficulty, complicated steps, special requirements on operators in part of tests, high requirements on hardware such as test tools and equipment by a special platform or laboratory, and higher single detection cost, and part of tests require the operators to learn special courses.

As for the detection method of VIM enzyme, improvement of the existing method or establishment of a new detection method can be used as a research idea. The rapid detection method is established, so that the rapid detection method meets the requirements of high efficiency, accuracy and high flux, is low in cost and easy to implement, has a low detection threshold, is convenient for large-scale popularization, has higher sensitivity and specificity, and is a great challenge for researchers.

Monoclonal antibodies are highly homogeneous antibodies raised by single B cell cloning and directed against only specific epitopes, usually prepared by hybridoma cells, and based on cell fusion technology, sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproductive ability are fused into B cell hybridomas, and after culturing into cell populations, specific antibodies directed against one epitope, i.e. monoclonal antibodies, can be prepared. The purpose of specific antibody detection is to assist clinical diagnosis, and is an index for observing curative effect and prognosis in certain diseases, and the detection of specific antibody has special and important significance in the research of drug resistance and epidemiology of infectious diseases. The immunological detection of antibodies has the following advantages: the specificity is high, and a specific monoclonal antibody is used, so that the method can be used for detecting single cytokines; the operation is simple and quick, and the cell strain is not needed, so that the maintenance culture is not needed, the operability is increased, the popularization is easy, and the general investigation is convenient; the influence factors are relatively few and easy to control, the repeatability is good, and the method is easy to standardize.

The existing VIM detection method has poor specificity and long time consumption, and delays diagnosis and treatment of patients. CN112980803A discloses an anti-VIM enzyme hybridoma cell strain, a monoclonal antibody and application, wherein the monoclonal antibody has the characteristics of high purity titer and strong specificity, and is suitable for being used as an immunodiagnosis reagent for in vitro diagnosis of VIM enzyme. However, the above monoclonal antibodies are murine monoclonal antibodies, and are all monoclonal antibodies obtained directly by animal immunization. Although murine monoclonal antibodies are among the most widely used antibodies, there are still problems of weak affinity and poor specificity.

In view of this, the present invention has been made.

Disclosure of Invention

The present invention aims to provide a VIM enzyme binding molecule which can specifically bind to a plurality of types of VIM enzymes, and which can further improve the sensitivity and specificity of detection when used in VIM enzyme detection and diagnosis.

Another object of the present invention is to provide a monoclonal antibody that specifically binds to VIM enzyme and a detection kit containing the monoclonal antibody.

In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:

in a first aspect, the invention provides a VIM enzyme binding molecule comprising a moiety a or a moiety b that specifically binds to a VIM enzyme;

the module a comprises a first VH domain comprising a first CDR-H1 having the amino acid sequence shown in SEQ ID No.1 (DFWIC), a first CDR-H2 having the amino acid sequence shown in SEQ ID No.2 (CMVPDGSGFGFSASWAKG) and a first CDR-H3 having the amino acid sequence shown in SEQ ID No.3 (YGDVGGPYSFKL);

the module b comprises a second VH domain comprising a second CDR-H1 having the amino acid sequence shown in SEQ ID No.4 (SSSYYWG), a second CDR-H2 having the amino acid sequence shown in SEQ ID No.5 (STYYSGSTYYNPSLKS) and a second CDR-H3 having the amino acid sequence shown in SEQ ID No.6 (HPMVVVTAKFDY).

In an alternative embodiment, the first VH domain has the amino acid sequence shown in SEQ ID No.13 (EVQLVESTGGLVKPGGSLKLSQAASGFTFNNDFWICWFRQAPEKGLEWVACMVPDGSGFGFSASWAKGRATISRDNAKGTLFLQMTSLRSEDTAMYYCARYGDVGGPYSFKLWGQGTTVTVSS);

the second VH domain has the amino acid sequence shown in SEQ ID No.14 (DVQLQESTPGLVKPSQTVSLTCSVTGISITSSSYYWGWIRVFPGNKLEGIGSTYYSGSTYYNPSLKSRTTITRDTSKYQFFLEMNSLTKEDTATYYCARHPMVVVTAKFDYWGQGTTVTVSS).

In an alternative embodiment, the module a further comprises a first VL domain comprising a first CDR-L1 having the amino acid sequence shown in SEQ ID No.7 (RASQSVSSTNLA), a first CDR-L2 having the amino acid sequence shown in SEQ ID No.8 (gasstrat) and a first CDR-L3 having the amino acid sequence shown in SEQ ID No.9 (QYYGTSLWT);

the module b further comprises a second VL domain comprising a second CDR-L1 having the amino acid sequence shown in SEQ ID No.10 (GASQSVSSSYLA), a second CDR-L2 having the amino acid sequence shown in SEQ ID No.11 (DASRAT) and a second CDR-L3 having the amino acid sequence shown in SEQ ID No.12 (QQYGSSPYT).

In alternative embodiments, the first VL domain has the amino acid sequence shown in SEQ ID No.15 (TQDPASLSFSLGETATLSCRASQSVSSTNLAWYLQKAEQVPRALIHGASSRATGVPVQFSGTGSGTDFTGTISSLEPEDAAVYYCQYYGTSLWTFGQGTKVEIK);

the second VL domain has the amino acid sequence shown in SEQ ID No.16 (DIVLTQAPASLGFSLGETATASCGASQSVSSSYLAWYVQKAEQVPRLLIHDASSRATGVPYRFSGTGSGTDFTDTISSLEPEDAAVYYCQQYGSSPYTFGQGTKVEIK).

In alternative embodiments, the VIM enzyme binding molecule is selected from the group consisting of scFv molecules, fv molecules, fab molecules, or intact antibody molecules that specifically bind to the VIM enzyme;

the intact antibody molecule comprises a monoclonal antibody or a cloned antibody.

In alternative embodiments, the monoclonal antibody comprises the following (a) or (B):

(A) Having the first VH domain of the preceding embodiment and the first VL domain of the preceding embodiment;

(B) Has the second VH domain of the previous embodiment and the second VL domain of the previous embodiment.

In a second aspect, the present invention provides the use of a VIM enzyme binding molecule according to any of the preceding embodiments in the preparation of a VIM enzyme antigen detection product, or in an in vitro detection of a VIM enzyme antigen not for the purpose of disease diagnosis or treatment;

the detection method comprises a double antibody sandwich indirect ELISA method or an immunochromatography method.

In alternative embodiments, the label in the immunochromatography is selected from the group consisting of colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots; preferably colloidal gold.

In a third aspect, the present invention provides a kit for detecting a VIM enzyme, comprising a VIM enzyme binding molecule according to the previous embodiment.

In an alternative embodiment, an immunochromatographic assay card comprising the VIM enzyme-binding molecule of any of the preceding embodiments and a label is included in the kit.

Preferably, the label is selected from the group consisting of colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots; further preferably colloidal gold.

The anti-VIM enzyme monoclonal antibody constructed by the invention has strong specific binding capacity to different subtypes of VIM enzyme, has important clinical application value for establishing a sensitive, rapid and specific VIM detection diagnosis method, and has wide application prospect in the fields of in-vitro diagnosis and clinical treatment.

The anti-VIM enzyme monoclonal antibody provided by the invention has the characteristics of multiple antigen recognition sites, good specificity, high affinity and the like, and has better performance in all aspects, so that the anti-VIM enzyme monoclonal antibody is suitable for being used as an immunodiagnosis reagent for in-vitro diagnosis of VIM enzyme, and the titer is more than 1:1280000; the prepared colloidal gold kit can be used for early typing of drug-resistant strains, guiding clinical medication and assisting in clinical infection control and treatment.

The invention establishes an immunochromatography detection system by using the monoclonal antibody for resisting the VIM enzyme, and the method can obtain results from the cultured strain within 15 minutes, has higher sensitivity and specificity, is simple and quick to operate, does not need any instrument and equipment, does not need professional personnel to operate, is convenient for clinical popularization, and can rapidly, conveniently and accurately detect the VIM enzyme.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of electrophoresis of two sets of monoclonal antibodies obtained in example 6 of the present invention;

FIG. 2 shows the results of two sets of monoclonal antibody titer determinations obtained in example 7 of the present invention;

FIG. 3 shows the results of a cross-reaction experiment with a first monoclonal antibody according to example 9 of the present invention;

FIG. 4 shows the results of a cross-reaction experiment for a second monoclonal antibody in example 9 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In a certain embodiment, the invention provides in a first aspect a VIM enzyme binding molecule comprising a module a or a module b that specifically binds to a VIM enzyme;

The scFV molecule, fv molecule, fab molecule and whole antibody molecule can be obtained by artificial synthesis, the whole antibody molecule can be obtained by constructing hybridoma cells, expressing and secreting, and the selection of specific compositions such as connecting peptide or constant chain required for constructing scFV molecule, fv molecule, fab molecule and whole antibody molecule can be routinely adjusted by those skilled in the art according to actual needs.

It should be noted that, the in vitro detection of the VIM enzyme antigen not aiming at disease diagnosis or treatment comprises the rapid and accurate judgment of the bacterial drug resistance degree of a patient by qualitatively or semi-quantitatively detecting the VIM enzyme in a bacterial sample or a positive blood culture sample separated from the patient, thereby determining the drug effect and providing a basis for drug development.

In some alternative embodiments, the kit comprises an immunochromatographic assay card, a first or second monoclonal antibody against the VIM enzyme labeled with a label is embedded on a sample binding pad, and a second or first monoclonal antibody against the VIM enzyme is coated on the detection line (T). If the detection sample is positive, the VIM enzyme is combined with the first or second monoclonal antibody marked by the marker to form a complex, the complex moves forward along the paper strip under the action of chromatography and is captured by the second or first monoclonal antibody of the VIM enzyme pre-coated when passing through the detection line (T), so as to form an immune complex, and a detectable signal value appears; if the test sample is negative, no immune complex is formed, and no detectable signal value appears at the test line. The label may be, for example, but not limited to, colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots. Preferably, the first monoclonal antibody is embedded in the sample binding pad and the second monoclonal antibody is coated in the detection line.

Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.

Example 1: preparation of antigens

The invention is based on NCBI (National Center for Biotechnology Information, national Biotechnology information in the United states)Center) sequence alignment to obtain the conserved sequences of VIM enzymes (VIM-1-20, VIM-23-73). Adopts the conventional digestion and connection technology in the molecular biology field to construct the expression plasmid pET-28a (+) -PM, adopts CaCl ₂ The recombinant vector was transformed into E.coli DH5a competence by heat shock. Positive clones were selected using LB medium containing 100. Mu.g/mL ampicillin. E.coli is cultivated conventionally, plasmids are extracted for PCR identification, and existence of target genes is determined. After transformation of the extracted expression plasmid pET-28a (+) -PMAA into competent cells of E.coli BL21 (DE 3), the cells were spread on a selection medium, screened for single colonies resistant to 100. Mu.g/mL ampicillin, and liquid cultured overnight. 1mL of overnight culture is inoculated into 200mL of LB culture medium containing 100 mug/mL of ampicillin, shake culture is carried out until logarithmic phase (OD 600 is 0.5-0.6), IPTG (1 mmol/L) is added, induction culture is carried out for 3h at 16 ℃, fermentation broth is purified by nickel column, and high purity protein is obtained through prokaryotic gene expression.

Example 2: immunization of animals

Selecting New Zealand white rabbits with proper age and weight of about 1.5 kg, and raising in a standard animal house for 3 days, and starting immunization if no abnormal condition exists: adding 100 mug of VIM enzyme antigen into 0.5mL of physiological saline sterilized under high pressure, fully and uniformly mixing the mixture by a miniature vortex oscillator, adding 0.5mL of Freund's complete adjuvant, pushing and pulling the mixture by a syringe to fully mix and emulsify the mixture, and carrying out back subcutaneous multipoint injection immunization on New Zealand big ear rabbits; boosting is carried out after two weeks, boosting is carried out once every other week, six times of boosting is carried out, and 200-500 mu L of white rabbit ear edge venous blood is taken after one week of immunization from the third immunization, and the potency and affinity are measured; after the last immunization, spleen was taken for cell fusion for preparing hybridoma cells.

Example 3: preparation and selection of hybridoma cells

And (3) performing titer detection on the prepared rabbit antiserum, and performing cell fusion by using a rabbit spleen under the condition of qualification to prepare a monoclonal hybridoma cell strain, wherein the method comprises the following steps of: killing immunized New Zealand big ear rabbits, taking out spleen under aseptic condition, cleaning with cell culture solution for 1 time, grinding, sieving with stainless steel screen to obtainIs centrifuged and washed 2 times with cell culture solution; mixing SP2/0 myeloma cells in logarithmic growth phase with spleen cells, washing with cell culture solution without fetal calf serum, centrifuging, discarding supernatant, adding polyethylene glycol solution, and treating at 37deg.C for about 90 s; stopping the reaction with a cell culture solution containing no fetal bovine serum, centrifuging, resuspending the cells with HAT selection medium containing 20% fetal bovine serum, adding the cells into a 96-well plate, and adding 5.0% CO at 37deg.C ₂ Culturing in medium; diluting cells with good growth state in a 96-well plate to 1-3 cells/mL by using a cell culture solution, adding the cells into the 96-well plate, and placing the cells into a cell culture box at 37 ℃ and 5.0% CO ₂ Culturing under the condition, numbering each cell strain, and selecting the cell strain positive to the supernatant of the culture solution for expansion culture to finally obtain the hybridoma cell strain. Screening the obtained hybridoma cells by adopting an ELISA method, observing the growth condition of the cells on the 5 th day after fusion, detecting the titer of a cell culture supernatant by adopting an indirect ELISA method on the 10 th to 14 th days, performing expanded culture on positive hybridoma cells with the strongest titer until the cell positive rate reaches 100%, and obtaining hybridoma cell strains by strain fixation, and freezing in liquid nitrogen for later use.

Example 4: isolation of antibody variable region genes from hybridoma cells Using RT-PCR

Homogenizing hybridoma cells, adding cell lysate to extract RNA, precipitating RNA from an aqueous phase layer by isopropanol, centrifuging, washing the precipitated RNA to remove impurities, and carrying out reverse transcription after re-suspension to obtain cDNA; PCR was performed using specific primers for New Zealand rabbits, using hybridoma cell cDNA as a template, and amplifying heavy and light chain variable region genes of the antibody, wherein a 50. Mu.L system contains 5. Mu.L cDNA, hotStarTaq Plus enzyme, dNTPs and 0.5. Mu.M specific primers, and PCR amplification was performed under the following conditions: pre-denaturation at 94℃for 5min;94℃for 30s,55℃for 30s,72℃for 50s,35 cycles; 7min at 72 ℃; the obtained PCR products are identified by 1% agarose gel electrophoresis, target fragments are recovered, the target fragments are subjected to sample feeding and sequencing, and sequencing results are compared with an IMGT database (http:// www.imgt.org/IMGT_vquest/vquest) to obtain two groups of antibody variable region gene fragments, wherein a first monoclonal antibody has a first VH domain (the amino acid sequence of which is shown as SEQ ID No. 13) and a first VL domain (the amino acid sequence of which is shown as SEQ ID No. 14), and a second monoclonal antibody has a second VH domain (the amino acid sequence of which is shown as SEQ ID No. 15) and a second VL domain (the amino acid sequence of which is shown as SEQ ID No. 16).

Example 5: construction, expression and purification of monoclonal antibodies

Homologous recombination primers are used for respectively adding homologous recombination arms and signal peptide nucleic acid fragments at two ends of two groups of antibody heavy chain variable region genes and two ends of light chain variable region genes, wherein the amino acid sequence of the signal peptide corresponding to the heavy chain variable region is shown as SEQ ID No.17 (MDWTWRFLFVVAAATGVQS), and the amino acid sequence of the signal peptide corresponding to the light chain variable region is shown as SEQ ID No.18 (MDMRVPAQLLGLLLLWLSGARC). The obtained nucleotide fragment encoding the heavy chain variable region (containing signal peptide) of the first monoclonal antibody is shown as SEQ ID No.19, the nucleotide fragment encoding the light chain variable region (containing signal peptide) of the first monoclonal antibody is shown as SEQ ID No.20, the nucleotide fragment encoding the heavy chain variable region (containing signal peptide) of the second monoclonal antibody is shown as SEQ ID No.21, and the nucleotide fragment encoding the light chain variable region (containing signal peptide) of the second monoclonal antibody is shown as SEQ ID No. 22.

Then, using double enzymes to linearize expression plasmids containing the heavy and light chain IgG1 constant regions of the rabbit antibody, producing homologous recombination arms; the variable region gene fragment added with the homologous recombination arm and the linearized plasmid are connected in a homologous recombination mode to form a complete expression vector, the recombinant product is transformed into TOP10 escherichia coli competence, and the plasmid is amplified.

Respectively adding the obtained two groups of monoclonal antibody heavy and light chain expression plasmids into an Opti-Mem transfection medium according to the proportion of 1:1, fully mixing, adding a transfection reagent PEI with the mass of 4 times of DNA, mixing, standing at room temperature for 30min in a dark place, and then adding into 293T cells; after 6h incubation, the transfection system is removed, freeStyle 293 expression medium is added, an AKTA Protein purification system is used, and an affinity purification (Protein A) method is adopted to purify the expressed antibody supernatant, so as to obtain the monoclonal antibody of the IMP enzyme, which comprises the following specific steps: (1) Centrifuging the expressed antibody supernatant at 2500 Xg at room temperature for 10min, and removing precipitate; (2) Washing the affinity purification column filled with Protein A with 10 times of Binding Buffer; (3) Passing the expression supernatant through a purification column at a flow rate of 5 mL/min; (4) The purification column was washed thoroughly with 20 volumes of Binding Buffer (Binding Buffer); (5) Eluting the purification column with 0.1M ph=3.0-3.5 citric acid buffer until the elution peak falls to equilibrium, and adjusting the pH to 7.0 with 1M ph=9.0 Tris-HCl buffer; (6) The purified monoclonal antibody was concentrated using a concentration column, PBS was used as a buffer for antibody preservation, and finally the concentration of the concentrated antibody was measured by a BSA protein concentration detection method.

Example 6: determination of molecular weight

Molecular weight of the monoclonal antibody is identified by SDS-PAGE electrophoresis, the sample loading amount of each electrophoresis channel is 5 mug, meanwhile, a standard series of known molecular weight is used as a reference, electrophoresis is carried out for 20min at 90V, electrophoresis is carried out at 140V until the indicator completely runs out, gel is taken off, coomassie brilliant blue is used for dyeing, molecular weight of biological raw materials is analyzed by gel after dyeing, SDS-PAGE electrophoresis is shown in figure 1, and Marker, first monoclonal antibody and second monoclonal antibody are sequentially arranged from left lane to right lane.

Example 7: potency determination

The ELISA method is adopted to detect the affinity activity (titer) of the monoclonal antibody to the VIM enzyme, and the main steps are as follows: (1) The VIM enzyme antigen is diluted to 1 ng/. Mu.L by PBS, 100. Mu.L of the VIM enzyme antigen is added into a 96-well ELISA plate, and the VIM enzyme antigen is coated for 2 hours at 37 ℃; (2) The supernatant was discarded, the plate was washed 3 times with 0.01M PBST, blocking solution containing 3% BSA was prepared with PBST, 100. Mu.L was added to each well, and blocking was performed at 37℃for 2 hours; (3) The supernatant was discarded, washed 5 times with PBST, the purified and concentrated antibody was subjected to gradient dilution at a concentration of 1:2560000 from 1:1000-fold dilution, wells were added with 100. Mu.L each well, and incubated for 1h at 37 ℃; (4) Discarding the antibody diluent, washing with PBST for 6 times, diluting goat anti-rabbit IgG-HRP with 1:5000 blocking solution, adding 100 μl of the mixture into each well, and incubating at 37deg.C for 1 hr; (5) Discarding secondary anti-dilution, cleaning with PBST for 6 times, adding TMB,100 μl/hole, and standing at 37deg.C for 15min in dark; (6) The reaction was stopped by adding 50. Mu.L of 1M dilute sulfuric acid to each well, and the absorbance was measured at 450 nm. As a result, as shown in FIG. 2, two groups of monoclonal antibodies (wherein # 1 and # 2 correspond to the first monoclonal antibody and the second monoclonal antibody, respectively) were screened for strong binding to the VIM enzyme, and titers to the VIM enzyme antigen reached 1:1280000 (OD > 0.5).

Example 8: comparison with the monoclonal antibodies of the prior art

The affinity activity (titer) for VIM enzyme was measured by ELISA using the monoclonal antibody prepared in example 5 of the present invention, the natural antibody (rabbit serum) and the published mouse-derived anti-VIM enzyme monoclonal antibody (purchased from Zhuhai Bomei Biotechnology Co., ltd.) respectively, and the procedure was as in example 7. The results are shown in Table 1, which demonstrate that the monoclonal antibodies of the invention have increased affinity and enhanced biological activity over the prior art.

TABLE 1 comparison of titers of monoclonal antibodies obtained by the present invention with existing monoclonal antibodies

Example 9: cross reaction

Coating the ELISA plates with KPC, NDM, VIM, IMP and OXA-48 enzymes respectively, wherein the coating amount of each hole is 50ng; respectively diluting the two groups of monoclonal antibodies obtained in the example 5 to 10ng/mL, adding the diluted monoclonal antibodies into each ELISA plate, adding 100 mu L of monoclonal antibodies into each hole, and incubating at 37 ℃ for 1h; after washing, adding HRP-labeled goat anti-rabbit secondary antibody, adding 100 mu L of the secondary antibody into each hole, and incubating for 0.5h at 37 ℃; after washing TMB was added and incubated at 37℃for 15min, the reading was terminated. The results are shown in fig. 3 and 4, fig. 3 shows the cross reaction results of the first monoclonal antibody, and fig. 4 shows the cross reaction results of the second monoclonal antibody, and it can be seen from fig. 3 and 4 that the two groups of monoclonal antibodies provided by the invention do not cross react with other carbapenemases, and have strong specificity.

Example 10: antibody pairing validation

The first monoclonal antibody prepared in example 5 was used as a capture antibody, the second monoclonal antibody was used as a labeled antibody (HRP enzyme labeled), the capture antibody was also HRP enzyme labeled, the capture antibody was coated on an antigen plate as a control group, the antigen diluted in a double ratio was added first, the unbound antigen was washed off after incubation, the labeled antibody was added, the unbound labeled antibody was washed off after incubation, and finally the color development was performed by adding a color development solution. If color development is possible, it is indicated that the labeled antibody specifically binds to the antigen, and the capture antibody and the labeled antibody are a pair of paired antibodies. If color development is not possible, it means that the labeled antibody cannot bind to the antigen and is eluted, and the capture antibody and the labeled antibody are not paired antibodies, and the results are shown below, which indicate that the paired antibodies have the best ability to bind to the antigen.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

A VIM enzyme binding molecule comprising a moiety a or a moiety b that specifically binds to a VIM enzyme;

the module a comprises a first VH domain comprising a first CDR-H1 having the amino acid sequence shown in SEQ ID No.1, a first CDR-H2 having the amino acid sequence shown in SEQ ID No.2 and a first CDR-H3 having the amino acid sequence shown in SEQ ID No. 3;

the module b comprises a second VH domain comprising a second CDR-H1 having the amino acid sequence shown in SEQ ID No.4, a second CDR-H2 having the amino acid sequence shown in SEQ ID No.5 and a second CDR-H3 having the amino acid sequence shown in SEQ ID No. 6.
2. The VIM enzyme binding molecule of claim 1, wherein said first VH domain has the amino acid sequence shown in SEQ ID No. 13;

the second VH domain has the amino acid sequence shown in SEQ ID No. 14.
3. The VIM enzyme binding molecule of claim 1, wherein said module a further comprises a first VL domain comprising a first CDR-L1 having the amino acid sequence of SEQ ID No.7, a first CDR-L2 having the amino acid sequence of SEQ ID No.8, and a first CDR-L3 having the amino acid sequence of SEQ ID No. 9;

the module b further comprises a second VL domain comprising a second CDR-L1 having the amino acid sequence shown in SEQ ID No.10, a second CDR-L2 having the amino acid sequence shown in SEQ ID No.11 and a second CDR-L3 having the amino acid sequence shown in SEQ ID No. 12.
4. The VIM enzyme binding molecule of claim 3, wherein said first VL domain has the amino acid sequence shown in SEQ ID No. 15;

the second VL domain has the amino acid sequence shown in SEQ ID No. 16.
5. The VIM enzyme binding molecule of any one of claims 1 to 4, wherein said VIM enzyme binding molecule is selected from the group consisting of scFv molecules, fv molecules, fab molecules, and intact antibody molecules that specifically bind to a VIM enzyme;

the intact antibody molecule comprises a monoclonal antibody or a cloned antibody.
6. The VIM enzyme binding molecule of claim 5, wherein the monoclonal antibody comprises the following (a) or (B):

(A) Having a first VH domain of claim 2 and a first VL domain of claim 4;

(B) Having a second VH domain of claim 2 and a second VL domain of claim 4.
7. Use of a VIM enzyme binding molecule of any one of claims 1 to 6 in the preparation of a VIM enzyme antigen detection product, or in an in vitro detection of a VIM enzyme antigen not for the purpose of disease diagnosis or treatment;

the detection method comprises a double antibody sandwich indirect ELISA method or an immunochromatography method.
8. The use according to claim 7, wherein the label in the immunochromatography is selected from the group consisting of colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots; preferably colloidal gold.
9. A kit for detecting a VIM enzyme, comprising the VIM enzyme binding molecule of any one of claims 1 to 6.
10. The kit of claim 9, wherein the kit comprises an immunochromatographic assay card comprising the VIM enzyme-binding molecule of any one of claims 1 to 6 and a label;

preferably, the label is selected from the group consisting of colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots; further preferably colloidal gold.