OXA-48 enzyme monoclonal antibody and its application

Abstract

This invention relates to the field of monoclonal antibody technology, and in particular to OXA-48 enzyme monoclonal antibodies and their applications. The OXA-48 enzyme binding molecules provided by this invention exhibit high specificity, high biological activity, strong stability, small batch-to-batch variation, are unaffected by cell line degeneration, have high affinity for OXA-48 enzyme, and a titer exceeding 1:1,280,000, making them suitable for the detection or purification of OXA-48 enzyme products.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, and in particular to OXA-48 enzyme monoclonal antibody and its applications. Background Technology

[0002] In 1993, Paton et al. reported the first β-lactamase with carbapenemase activity, purified from multidrug-resistant Acinetobacter baumannii isolated from a patient in Edinburgh, Scotland in 1985, and named it ARI21. In 2000, Donald et al. sequenced the amino acid sequence of the ARI21 enzyme and named it OXA223. Between 2000 and 2004, six new class D carbapenemases were discovered worldwide. Subsequently, seven more class D enzymes were found in carbapenem-resistant strains in many countries. To date, at least 45 of the 121 OXA-type enzymes possess carbapenemase activity.

[0003] Typically, class D carbapenemases exhibit weak carbapenem hydrolytic activity. Resistance to carbapenems in isolates producing these enzymes may be a result of concurrent resistance mechanisms, such as loss of outer membrane porins, enhanced pumping activity, and alterations in penicillin-binding protein (PBP). Mature class D carbapenemases contain 243–260 amino acid residues, with a molecular weight ranging from 23 to 35.5 kDa and a pI of 5.1 to >9.0. The hydrolytic rate of these enzymes towards imipenem is 1%–3% that of penicillin, and the hydrolytic rate towards oxacillin is twice that of penicillin. Their hydrolytic activity against third-generation cephalosporins is very weak; their activity can be inhibited by tazobactam and clavulanic acid. In vitro antibiotic susceptibility studies on Escherichia coli transformation conjugates or transformants carrying recombinant plasmids encoding class D carbapenemases revealed high levels of resistance to aminopenicillins and carboxypenicillins, and variable susceptibility to ureacylpenicillins (only OXA240 and OXA248 were resistant to piperacillin). They were sensitive to cephalosporins, including narrow-spectrum cephalosporins, oximinocephalosporins and 72α2-methoxycephalosporins, oxocephalosporins, and monoamides (only OXA248 was resistant to cefotaxime). Except for OXA223, which showed an 8-fold decrease in the MIC of piperacillin in the presence of tazobactam, the other strains were not sensitive to the inhibitory effects of clavulanic acid and tazobactam.

[0004] OXA-48 has demonstrated increasingly important carbapenemase activity in carbapenem resistance, with reports of its presence in Enterobacteriaceae worldwide increasing annually. OXA-48 was first discovered in *Klebsiella pneumoniae* in Turkey in 2001. This strain was resistant to almost all β-lactam antibiotics, including penicillins, cephalosporins, monocyclic lactams, and carbapenems. Further analysis of this *Klebsiella pneumoniae* revealed co-expression of SHV-2a, TEM-1, and OXA-47, and the absence of extracellular membrane proteins, leading to its high resistance to various antibiotics. In the following years, outbreaks or epidemics of OXA-48 strains occurred in various regions of Turkey. Besides Turkey, OXA-48 has been reported in Belgium, Lebanon, Tunisia, Germany, Morocco, France, and other countries. To date, OXA-48 has only been reported in Enterobacteriaceae, including Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Enterobacter cloacae, and Providencia reticularis. It exhibits a sporadic or outbreak trend worldwide. Among class D enzymes, OXA-48 is highly hydrolyzed by penicillins but has weak activity against carbapenems. It cannot be inhibited by inhibitors such as clavulanic acid and tazobactam, but is sensitive to extended-spectrum cephalosporins. Currently reported OXA-48 enzymes include OXA-48, OXA-162, OXA-181, OXA-204, OXA-232, OXA-244, OXA-245, OXA-247, OXA-436, OXA-484, and OXA-519. The rapid spread of OXA-48-producing Enterobacteriaceae in different ecosystems poses a significant challenge to clinical practice.

[0005] Strains producing the OXA-48 enzyme show decreased susceptibility to imipenem and meropenem, with their MICs remaining within the sensitive range, but exhibit moderate sensitivity or resistance to ertapenem. Therefore, ertapenem has higher detection sensitivity for Enterobacteriaceae strains carrying OXA-48 and can be used as a better indicator for OXA-48 detection. The modified Hodge test is the OXA-48 enzyme phenotypic detection method recommended by the Clinical and Laboratory Standards Institute (CLSI). The modified Hodge test should be performed when strains are resistant to third- or fourth-generation cephalosporins, with ertapenem MIC ≥ 2 μg / ml and imipenem and meropenem MICs of 2–4 μg / ml. A positive result is indicated by enhanced growth of the test bacteria within the imipenem inhibition zone; however, false positive reports of this modified Hodge test have been increasing recently. Polymerase chain reaction (PCR) is the gold standard for detecting whether a bacterial strain carries the blaOXA-48 gene. Building upon traditional PCR, various novel molecular biology techniques, such as real-time quantitative PCR, gene chips, and loop-mediated isothermal amplification, have been developed, expanding their applications. Compared to traditional PCR, these methods generally offer higher sensitivity and specificity for OXA-48 enzyme detection, while also providing advantages in terms of detection quantity and time. However, they are more complex to operate, involve more complicated procedures, require specialized personnel for some experiments, and are often performed in specialized platforms or laboratories. The hardware requirements for experimental equipment are high, and some experiments require operators to undergo specialized training, resulting in higher costs per test.

[0006] Given the increasingly serious problem of antibiotic abuse, the emergence of OXA-48 should be a cause for concern. It is worthwhile to establish a rapid, accurate, and easily standardized method for detecting OXA-48 bacteria, so as to strengthen the monitoring of OXA-48 bacteria, reveal its epidemiological characteristics, and take correct and effective management measures to control the infection and its spread.

[0007] Monoclonal antibodies are highly homogeneous antibodies produced from a single B cell clone, targeting only a specific antigenic epitope. They are typically prepared using hybridoma cells. Based on cell fusion technology, sensitized B cells capable of secreting specific antibodies are fused with myeloma cells capable of unlimited proliferation to form a B-cell hybridoma. After culturing the cell population, a specific antibody against a single antigenic epitope, i.e., a monoclonal antibody, can be prepared. The primary purpose of specific antibody detection is to assist in clinical diagnosis. In some diseases, it is also an indicator for observing treatment efficacy and prognosis. Specific antibody detection also has special and important significance in drug resistance and infectious disease epidemiological surveys. Antibody immunological detection has the following advantages: high specificity (using specific monoclonal antibodies, it can be used to detect single cytokines); simple and rapid operation (not dependent on cell lines, therefore no maintenance culture is required, increasing operability, easy to promote and facilitate screening); relatively few influencing factors that are easy to control; good reproducibility; and easy standardization of the method.

[0008] Existing OXA-48 detection methods suffer from poor specificity and long processing times, delaying patient diagnosis and treatment. CN112500489A discloses an anti-OXA-23 enzyme hybridoma cell line, a monoclonal antibody, and its applications; CN114317454A discloses a mouse anti-OXA-48 enzyme hybridoma cell line, a monoclonal antibody, and its applications. Both of these monoclonal antibodies possess high purity, high titer, and strong specificity, making them suitable as immunodiagnostic reagents for in vitro diagnosis of OXA-23 and OXA-48 enzymes. However, these monoclonal antibodies are mouse-derived and obtained directly through animal immunization. Although mouse-derived monoclonal antibodies are among the most widely used antibodies, they still suffer from weak affinity and poor specificity.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] The purpose of this invention is to provide an OXA-48 enzyme binding molecule that can specifically bind to different subtypes of the OXA-48 enzyme, and to use it in the detection and diagnosis of OXA-48 enzyme, thereby further improving the sensitivity and specificity of detection.

[0011] Another object of the present invention is to provide a kit containing an OXA-48 enzyme-specific binding molecule for use in the detection of OXA-48 enzyme.

[0012] To solve the above-mentioned technical problems and achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] In a first aspect, the present invention provides an OXA-48 enzyme-binding molecule, comprising module a or module b that specifically binds to the OXA-48 enzyme;

[0014] The module a includes a first VH domain, which includes a first CDR-H1 having the amino acid sequence shown in SEQ ID No. 1 (DYSMT), a first CDR-H2 having the amino acid sequence shown in SEQ ID No. 2 (LIKRNAYGGTTEYAASVRG), and a first CDR-H3 having the amino acid sequence shown in SEQ ID No. 3 (GYSGYDWEGLDAFDV).

[0015] The module b includes a second VH domain, which includes a second CDR-H1 having the amino acid sequence shown in SEQ ID No. 4 (GYWMH), a second CDR-H2 having the amino acid sequence shown in SEQ ID No. 5 (YINSDGSSTNYADSVKG), and a second CDR-H3 having the amino acid sequence shown in SEQ ID No. 6 (GGGYSYGPFD).

[0016] In an optional embodiment, the first VH domain has the amino acid sequence shown in SEQ ID No. 13 (EVKLVESGGGLVQPGRSLRLSCATSGFTFADYSMTWVRQPPGKGLEWVGLIKRNAYGGTTEYAASVRGRFTISRDNSQSIAYLQMNTLRTEDTPAYYCLRGYSGYDWEGLDAFDVWGQGTMVTVSS); the second VH domain has the amino acid sequence shown in SEQ ID No. 14 (EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYWMHWVRQAPEKGLVWVAYINSDGSSTNYADSVKGRFTISRDNAKNTLFLQMNSLRAEDTAMYYCARGGGYSYGPFDYWGQGTSVTVSS).

[0017] In an optional embodiment, module a further includes a first VL domain, the first VL domain including a first CDR-L1 having the amino acid sequence shown in SEQ ID No. 7 (KSGQSVLFTSNNKNYLA), a first CDR-L2 having the amino acid sequence shown in SEQ ID No. 8 (WASTRES), and a first CDR-L3 having the amino acid sequence shown in SEQ ID No. 9 (QQYYSPGPFT);

[0018] The module b further includes a second VL domain, which includes a second CDR-L1 having the amino acid sequence shown in SEQ ID No. 10 (TGTSSDIGNNDYVS), a second CDR-L2 having the amino acid sequence shown in SEQ ID No. 11 (DVSRRPS), and a second CDR-L3 having the amino acid sequence shown in SEQ ID No. 12 (SSYAGSSNLV).

[0019] In an optional embodiment, the first VL domain has the amino acid sequence shown in SEQ ID No. 15 (DIVMSQSPSSLAVSLGEKVTINCKSGQSVLFTSNNKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLAISSVQAEDVAVYYCQQYYSPGPFTFGQGTKVEIK); the second VL domain has the amino acid sequence shown in SEQ ID No. 16 (QSVLTQPSSASTSPGSSVKLSCTGTSSDIGNNDYVSWYQQYMGRPPTNIIYDVSRRPSGVSDRFSGSIDRSSNTAFLTVNNVQADDEADYYCSSYAGSSNLVFGGGTKLTV).

[0020] In an optional embodiment, the OXA-48 enzyme-binding molecule is selected from scFv molecules, Fv molecules, Fab molecules, or intact antibody molecules that specifically bind to the OXA-48 enzyme.

[0021] The complete antibody molecule includes monoclonal antibodies or clonal antibodies.

[0022] In an optional embodiment, the monoclonal antibody comprises either (A) or (B):

[0023] (A) It has the first VH structural domain and the first VL structural domain as described in the foregoing embodiments;

[0024] (B) It has the second VH structural domain described in the foregoing embodiments and the second VL structural domain described in the foregoing embodiments.

[0025] Secondly, the present invention provides the application of the OXA-48 enzyme binding molecule described in any of the foregoing embodiments in the preparation of OXA-48 enzyme antigen detection products, or in the in vitro detection of OXA-48 enzyme antigen for purposes other than disease diagnosis or treatment.

[0026] The detection methods include double-antibody sandwich indirect ELISA or immunochromatography.

[0027] In an optional embodiment, the label in the immunochromatographic method is selected from colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots; preferably colloidal gold.

[0028] Thirdly, the present invention provides a kit for detecting OXA-48 enzyme, the kit containing the OXA-48 enzyme binding molecule described in the foregoing embodiments.

[0029] In an optional embodiment, the kit includes an immunochromatographic assay card containing the OXA-48 enzyme-binding molecule and marker described in any of the foregoing embodiments.

[0030] Preferably, the marker is selected from colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots; more preferably, it is colloidal gold.

[0031] This invention utilizes proteomics to provide an OXA-48 enzyme-binding molecule based on antigen specificity. In particular, monoclonal antibodies containing this OXA-48 enzyme-binding molecule 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 the OXA-48 enzyme, achieving a titer exceeding 1:1,280,000. Furthermore, in vitro detection kits prepared using these monoclonal antibodies can be used for early typing of drug-resistant strains, guiding clinical medication and assisting in clinical infection control and treatment.

[0032] This invention utilizes a monoclonal antibody against the OXA-48 enzyme to establish a colloidal gold immunochromatographic detection system. This method can obtain results from cultured strains within 15 minutes, exhibiting high sensitivity and specificity. The operation is simple and rapid, requiring no instruments or specialized personnel, making it suitable for clinical application. It enables rapid, convenient, and accurate detection of the OXA-48 enzyme. The raw materials are derived from stably expressing cell lines, eliminating the risk of raw material supply issues, and the system is stable and low-cost. Attached Figure Description

[0033] 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.

[0034] Figure 1 The electrophoresis results of two groups of monoclonal antibodies were obtained in Example 6 of this invention;

[0035] Figure 2 These are the titer determination results of two groups of monoclonal antibodies obtained in Example 7 of this invention;

[0036] Figure 3 The results of the cross-reactivity experiment of the first monoclonal antibody in Example 9 of this invention;

[0037] Figure 4 This is the result of the cross-reaction experiment of the second monoclonal antibody in Example 9 of the present invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0039] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0040] In one specific embodiment, in a first aspect, the present invention provides a module (a1) that specifically binds to the OXA-48 enzyme, the module (a1) comprising a first VH domain, the 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.

[0041] In another specific embodiment, the present invention provides a module (a2) that specifically binds to the OXA-48 enzyme, said module (a2) comprising a first VH domain and a first VL domain. The first VH domain comprises 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 first VL domain comprises a first CDR-L1 having the amino acid sequence shown in SEQ ID No. 7, a first CDR-L2 having the amino acid sequence shown in SEQ ID No. 8, and a first CDR-L3 having the amino acid sequence shown in SEQ ID No. 9.

[0042] In another specific embodiment, the present invention provides a module (b1) that specifically binds to the OXA-48 enzyme, the module (b1) comprising a second VH domain, the 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.

[0043] In another specific embodiment, the present invention provides a module (b2) that specifically binds to the OXA-48 enzyme, said module (b2) comprising a second VH domain and a second VL domain. The second VH domain comprises 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. The second VL domain comprises 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.

[0044] In conjunction with the amino acid sequences containing CDR in the modules described in the first aspect, in a specific embodiment, the present invention provides the following modules:

[0045] Module (a11) includes a first VH domain having the amino acid sequence shown in SEQ ID No. 13.

[0046] Module (a12) includes a first VH domain and a first VL domain, the first VH domain having the amino acid sequence shown in SEQ ID No. 13 and the first VL domain having the amino acid sequence shown in SEQ ID No. 15.

[0047] Module (b11) includes a second VH domain having the amino acid sequence shown in SEQ ID No. 14.

[0048] Module (b21) includes a second VH domain and a second VL domain, the second VH domain having the amino acid sequence shown in SEQ ID No. 14 and the second VL domain having the amino acid sequence shown in SEQ ID No. 16.

[0049] In conjunction with the modules provided in the second aspect, in the third aspect, the present invention provides a first scFV molecule and a second scFV molecule. The first scFV molecule includes a first VH domain and a first VL domain connected by an elastic linker; the second scFV molecule includes a second VH domain and a second VL domain connected by an elastic linker. The amino acid residue composition and length of the linker can be adjusted by those skilled in the art according to actual needs through conventional means, including but not limited to being composed of glycine (Gly) and serine (Ser) with a length of 15 to 25 amino acid residues.

[0050] Fourthly, the present invention provides a first Fv molecule and a second Fv molecule, wherein the first Fv molecule includes a first VH domain and a first VL domain linked by a short peptide; and the second Fv molecule includes a second VH domain and a second VL domain linked by a short peptide. The short peptides can be obtained by those skilled in the art through conventional means according to actual needs, and include, but are not limited to, short peptide chains composed of 3 to 9 amino acid residues.

[0051] Fifthly, the present invention provides a first Fab molecule and a second Fab molecule. The first Fab molecule includes a first VH domain, a first VL domain, a light chain constant region (CL), and a heavy chain constant region (CH1). The second Fab molecule includes a second VH domain, a second VL domain, a light chain constant region (CL), and a heavy chain constant region (CH1). Those skilled in the art can select CH1 and CL according to actual needs. For example, CH1 is selected from any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, and CL is selected from the κ chain or the λ chain. Furthermore, those skilled in the art can adjust the sequences and modifications of the selected CH1 and CL using conventional methods according to actual needs.

[0052] Sixthly, the present invention also provides a complete antibody molecule based on the foregoing embodiments. The complete antibody molecule includes two identical heavy chains and two identical light chains. The heavy chain includes a first VH domain or a second VH domain and a heavy chain constant region. The light chain includes a first VL domain or a second VL domain corresponding to the heavy chain and a light chain constant region. Those skilled in the art can select the specific sequence composition of the heavy chain constant region and the light chain constant region according to actual needs. For example, the heavy chain constant region can be selected from any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, and the light chain constant region can be selected from the κ chain or the λ chain. Furthermore, those skilled in the art can adjust the sequences or modifications of the selected heavy chain constant region and the light chain constant region using conventional methods according to actual needs.

[0053] It should be noted that the modules, scFV molecules, Fv molecules, Fab molecules, and complete antibody molecules provided above can be obtained through artificial synthesis. Complete antibody molecules can also be obtained by constructing hybridoma cells and expressing and secreting them.

[0054] In a seventh aspect, the present invention provides a first monoclonal antibody having a first VH domain and a first VL domain as described in the foregoing embodiments.

[0055] Eighthly, the present invention provides a second monoclonal antibody having the second VH domain and the second VL domain as described in the foregoing embodiments.

[0056] In a ninth aspect, the present invention provides a kit for detecting OXA-48 enzyme, the kit containing the OXA-48 enzyme-binding molecule described in the foregoing embodiments or the monoclonal antibody described in the foregoing embodiments.

[0057] In some optional embodiments, the kit includes an immunochromatographic assay card with a labeled first or second monoclonal antibody against OXA-48 enzyme embedded in the sample conjugation pad and a second or first monoclonal antibody against OXA-48 enzyme coated on the test line (T). If the test sample is positive, the OXA-48 enzyme binds to the labeled first or second monoclonal antibody to form a complex. Under chromatography, the complex moves forward along the paper strip and is captured by the pre-coated second or first monoclonal antibody against OXA-48 enzyme when it passes the test 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 test 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, the first monoclonal antibody is embedded in the sample conjugation pad, and the second monoclonal antibody is coated on the test line.

[0058] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0059] Example 1: Preparation of Antigen

[0060] This invention obtained the conserved sequences of the OXA-48 enzymes (OXA-48-162-163, OXA-48-181, OXA-48-204, OXA-48-232, OXA-48-247, OXA-48-244-245) through NCBI (National Center for Biotechnology Information). Using standard molecular biology techniques such as enzyme digestion and ligation, the expression plasmid pET-28a(+)-PM was constructed, and 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 using standard methods, and the plasmid was extracted for PCR identification to confirm the presence of the target gene. The extracted expression plasmid pET-28a(+)-PMAA was transformed into E. coli BL21(DE3) competent cells, plated on selective medium, and single colonies resistant to 100 μg / mL ampicillin were screened. The culture was then incubated overnight. 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 0.5–0.6). IPTG (1 mmol / L) was added, and the culture was induced at 16°C for 3 h. The fermentation broth was purified by nickel column chromatography, and high-purity protein was obtained through prokaryotic gene expression.

[0061] Example 2: Animal Immunization

[0062] 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 OXA-48 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. A booster immunization is given two weeks later, followed by booster immunizations every week for a total of six immunizations. Starting from the third immunization, collect 200–500 μL of blood from the marginal ear vein of the rabbits one week after immunization to determine titer and affinity. After the final immunization, harvest the spleen for cell fusion to prepare hybridoma cells.

[0063] Example 3: Preparation and screening of hybridoma cells

[0064] 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 in the 96-well plates were diluted with cell culture medium to 1–3 cells / mL and added to the plates. The plates were then incubated 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.

[0065] Example 4: Isolation of antibody variable region gene from hybridoma cells using RT-PCR

[0066] After homogenizing hybridoma cells, RNA was extracted using 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 New Zealand rabbit-specific primers, with hybridoma cell cDNA as a template, to amplify the variable regions of the antibody's heavy and light chains. A 50 μL system contained 5 μL cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM specific primers. PCR amplification was performed under the following conditions: pre-denaturation at 94℃ for 5 min; 35 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 50 s; 72℃ for 7 min. The obtained PCR products were identified by 1% agarose gel electrophoresis, the target fragments were recovered, and sequenced. The sequencing results were compared with the IMGT database (http: / / www.imgt.org / IMGT_vquest / vquest), yielding two sets of antibody variable region gene fragments. The first monoclonal antibody possesses the first VH domain (amino acid sequence as shown in SEQ ID). The first monoclonal antibody has a first VL domain (amino acid sequence shown in SEQ ID No. 13) and a second VH domain (amino acid sequence shown in SEQ ID No. 15) and a second VL domain (amino acid sequence shown in SEQ ID No. 16).

[0067] Example 5: Construction, expression, and purification of monoclonal antibodies

[0068] Homologous recombination primers were used to add homologous recombination arms and signal peptide nucleic acid fragments to both ends of the heavy chain variable region genes and the light chain variable region genes of the two groups of antibodies, respectively. The amino acid sequence of the signal peptide corresponding to the heavy chain variable region is shown in SEQ ID No. 17 (MDWTWRFLFVVAAATGVQS), and the amino acid sequence of the signal peptide corresponding to the light chain variable region is shown in SEQ ID No. 18 (MDMRVPAQLLGLLLLWLSGARC). The resulting nucleotide fragments encoding the heavy chain variable region (containing the signal peptide) of the first monoclonal antibody are shown in SEQ ID No. 19, the light chain variable region (containing the signal peptide) of the first monoclonal antibody are shown in SEQ ID No. 20, the heavy chain variable region (containing the signal peptide) of the second monoclonal antibody are shown in SEQ ID No. 21, and the light chain variable region (containing the signal peptide) of the second monoclonal antibody are shown in SEQ ID No. 22.

[0069] Then, the expression plasmid containing the constant regions of rabbit antibody heavy and light chain IgG1 was linearized using a dual-enzyme approach to generate homologous recombination arms. The variable region gene fragment with added homologous recombination arms and the linearized plasmid were then linked together by homologous recombination to form a complete expression vector. The recombination product was then transformed into TOP10 E. coli competent cells to amplify the plasmid.

[0070] The two groups of 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 incubated 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 supernatant of the expressed antibody was purified using the AKTA protein purification system and the affinity purification method (Protein A) to obtain the monoclonal antibody against IMP enzyme. The specific steps were as follows: (1) The supernatant of the expressed antibody was centrifuged at 2500×g at room temperature for 10 min to remove the precipitate; (2) The affinity purification column containing Protein A was thoroughly washed with 10 times the volume of binding buffer; (3) The expression supernatant was passed through the purification column at a flow rate of 5 mL / min; (4) The purification column was thoroughly washed with 20 times the volume of binding buffer; (5) 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.

[0071] Example 6: Molecular weight determination

[0072] 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, the lanes from left to right are the Marker, the first monoclonal antibody, and the second monoclonal antibody.

[0073] Example 7: Potency determination

[0074] The affinity activity (titer) of monoclonal antibody for OXA-48 enzyme was detected by ELISA. The main steps are as follows: (1) Dilute OXA-48 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 concentrate the purified solution. The antibody was serially diluted from 1:1000 to 1:2560000 and added to each well, 100 μL per well, and incubated at 37°C for 1 h; (4) the antibody diluent was discarded, the antibody was washed 6 times with PBST, goat anti-rabbit IgG-HRP was diluted with 1:5000 blocking buffer, 100 μL per well was added and incubated at 37°C for 1 h; (5) the secondary antibody diluent was discarded, the antibody was washed 6 times with PBST, TMB was added, 100 μL / well, and the antibody 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 two groups of monoclonal antibodies screened (where 1# and 2# correspond to the first and second monoclonal antibodies, respectively) have a strong binding ability to OXA-48 enzyme, and the titer against OXA-48 enzyme antigen reaches 1:1280000 (OD value > 0.5).

[0075] Example 8: Comparison with existing monoclonal antibodies

[0076] The affinity (titer) for OXA-48 enzyme was detected using ELISA with the monoclonal antibody prepared in Example 5 of this invention, the natural antibody (rabbit serum), and the published murine anti-OXA-48 enzyme monoclonal antibody (purchased from Zhuhai Bomei Biotechnology Co., Ltd.), respectively. The specific steps are as described in Example 7. The results are shown in Table 1, indicating that the monoclonal antibody provided by this invention has increased affinity and enhanced biological activity compared with the prior art.

[0077] Table 1. Comparison of titers of the monoclonal antibody obtained in this invention with existing monoclonal antibodies.

[0078]

[0079] Example 9: Cross-reaction

[0080] Microplates were coated with KPC, NDM, VIM, IMP, and OXA-48 enzymes, respectively, with a coating amount of 50 ng per well. The two groups of monoclonal antibodies prepared in Example 5 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 taken. Results are as follows: Figure 3 and 4 As shown, Figure 3 This is the result of cross-reactivity of the first monoclonal antibody. Figure 4 The results of the cross-reaction of the second monoclonal antibody are shown in Figures 3 and 4. As can be seen, the two groups of monoclonal antibodies provided by this invention do not cross-react with other types of carbapenemases and have high specificity.

[0081] Example 10: Antibody Pairing Verification

[0082] The first monoclonal antibody prepared in Example 5 was used as the capture antibody, and the second monoclonal antibody was used as the labeling antibody (HRP enzyme labeling). The capture antibody was also HRP enzyme labeled as a control group. The capture antibody was coated onto an antigen plate. First, serially diluted antigen was added, incubated, and then unbound antigen was washed away. Next, the labeled antibody was added, incubated, and then unbound labeled antibody was washed away. Finally, chromogenic solution was added for color development. If color development was successful, 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 was unsuccessful, it indicates that the labeled antibody cannot bind to the antigen and is thus eluted, and the capture antibody and labeled antibody are not a paired antibody pair. The results shown below demonstrate that these two antibodies have the best ability to pair and bind to the antigen.

[0083]

[0084] 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 OXA-48 enzyme binding molecule characterized in that, comprises a first VH domain and a first VL domain, the first VH domain comprises a first CDR-H1 with an amino acid sequence of SEQ ID No. 1, a first CDR-H2 with an amino acid sequence of SEQ ID No. 2 and a first CDR-H3 with an amino acid sequence of SEQ ID No. 3; the first VL domain comprises a first CDR-L1 with an amino acid sequence of SEQ ID No. 7, a first CDR-L2 with an amino acid sequence of SEQ ID No. 8 and a first CDR-L3 with an amino acid sequence of SEQ ID No.

9. the amino acid sequence of the first VH domain is SEQ ID No.

13.

2. The OXA-48 enzyme binding molecule according to claim 1, characterized in that, the amino acid sequence of the first VL domain is SEQ ID No.

15.

3. The OXA-48 enzyme binding molecule according to claim 1, characterized in that, the OXA-48 enzyme binding molecule is selected from Fv, Fab or intact antibody that specifically binds to OXA-48 enzyme; 4. The OXA-48 enzyme binding molecule according to any one of claims 1 to 3, characterized in that, the intact antibody is a monoclonal antibody. the monoclonal antibody has the first VH domain of claim 2 and the first VL domain of claim 3.

5. The OXA-48 enzyme binding molecule according to claim 4, characterized in that, 6. Use of the OXA-48 enzyme binding molecule of any one of claims 1-5 in the preparation of OXA-48 enzyme antigen detection products, or in the in vitro detection of OXA-48 enzyme antigen for purposes other than disease diagnosis or treatment. the detection method comprises double antibody sandwich indirect ELISA method or immunochromatography method. the label in the immunochromatography method is selected from colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres or quantum dots.

7. Use according to claim 6, characterized in that, the kit contains the OXA-48 enzyme binding molecule of any one of claims 1-5.

8. A kit for the detection of OXA-48 enzymes, characterized in that, the kit comprises an immunochromatographic detection card, and the immunochromatographic detection card comprises the OXA-48 enzyme binding molecule of any one of claims 1-5 and a label.

9. The kit of claim 8, wherein the label is selected from colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres or quantum dots.

10. The kit of claim 9, wherein the label is colloidal gold.

11. The kit of claim 10, wherein ​

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