O45 escherichia coli detection antibody and application thereof

Abstract

This invention relates to the field of biomedicine, specifically to an antibody for detecting Escherichia coli O45 and its application method. The antibody is a monoclonal antibody or its antigen-binding fragment, comprising the heavy chain CDR sequence shown in SEQ ID NO:1-3 and the light chain CDR sequence shown in SEQ ID NO:4-6. This invention also provides the nucleic acid molecule encoding the antibody, the expression vector, the host cell, and the preparation method thereof. Specificity identification results show that the antibody specifically binds to Escherichia coli O45 and shows no cross-reactivity with other Escherichia coli serotypes such as O157, O26, O103, O111, O121, and O145, as well as closely related serotypes of O55, and also shows no cross-reactivity with common foodborne pathogens. The indirect ELISA detection method based on this antibody has a detection limit of 1×10⁻⁶ for Escherichia coli O45. 2 The detection rate is CFU / mL, and the entire testing process can be completed within 24 hours. The monoclonal antibody provided by this invention can be used to prepare detection kits, test strips, and other products, and is suitable for rapid screening of O45 Escherichia coli in imported and exported food.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to an antibody for detecting Escherichia coli O45 and its application in immunological detection. Background Technology

[0002] Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic foodborne bacterium that produces one or more Shiga toxins. Based on serotype differences, it can be divided into two main categories: O157 EHEC and non-O157 EHEC. In the past, food safety control has largely focused on O157 EHEC. However, in recent years, reports of outbreaks caused by non-O157 EHEC, especially the internationally recognized "Top Six" non-O157 serotypes (O26, O45, O103, O111, O121, and O145), have increased significantly, making them a key target for global public health and food safety management.

[0003] Escherichia coli O45 is an important Shiga toxin-producing Escherichia coli serotype. Its O antigen is encoded by the rfb gene cluster, and the O antigen polymerase encoded by the specific wzy gene can be used for serotype identification. Strains of O45 can carry the stx1 and / or stx2 Shiga toxin genes and the eae adhesion gene, possessing the pathogenic potential to cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. Of particular note is the O45:H2 subtype, which carries the intestinal exfoliation site (LEE) pathogenic island, exhibiting a virulence spectrum highly similar to the highly pathogenic O103:H2 strain; while the O45:H15 subtype is associated with typical enteropathogenic Escherichia coli (EPEC) and can cause foodborne diarrhea outbreaks. Furthermore, serologically, there is some antigenic cross-reactivity between serotypes O45 and O55, making accurate differentiation difficult with traditional serological agglutination assays, significantly increasing the difficulty of screening and identifying this strain.

[0004] As a major food importer and exporter, my country imports large quantities of high-risk animal-based foods such as beef and raw milk every year. These foods are susceptible to contamination by Shiga toxin-producing Escherichia coli (E. coli) other than O157, such as O45. The risk of imported exogenous pathogens continues to rise, seriously threatening domestic food safety and public health. To strictly control pathogen contamination and improve the food safety testing system, my country has successively issued a number of national and industry standards in recent years. GB 4789.49-2024, "National Food Safety Standard - Microbiological Examination of Food - Examination of Shiga Toxin-Producing Escherichia coli," which officially came into effect in 2024, explicitly includes the O45 serotype in the statutory testing scope, designating the standard strain number as CMCC(B)43242 / GDMCC1.3808. At the same time, the General Administration of Customs issued industry standards such as SN / T 5439.5-2022 to regulate the testing requirements for Shiga toxin-producing Escherichia coli in imported and exported foods. However, imported and exported food at ports is characterized by a large number of batches and a large number of samples to be tested. Moreover, trade clearance has strict requirements for testing timeliness. Traditional microbial culture and identification methods, due to their long testing cycle and cumbersome testing procedures, cannot fully meet the testing needs of rapid clearance at ports. There is an urgent need to develop rapid, specific, sensitive testing technologies that can be adapted to screening large numbers of samples.

[0005] Currently, the mainstream detection methods for O45 serotype are polymerase chain reaction (PCR) and immunomagnetic bead separation (IMS). While PCR boasts high detection sensitivity, it relies on sophisticated instruments and requires qualified personnel, resulting in high costs and poor portability, making it unsuitable for rapid on-site screening at ports of entry. Immunomagnetic bead separation, on the other hand, uses specific antibody-coated magnetic beads to enrich and capture target strains, offering advantages such as high sensitivity, high throughput, and ease of operation, making it an ideal solution for rapid screening of large batches of samples at ports of entry. However, there are significant shortcomings in the core biomaterials for O45 strain detection, with a severe shortage of commercially available antibodies, severely hindering the practical application of immunomagnetic bead separation technology and various immunological detection methods in O45 strain detection.

[0006] Therefore, developing highly specific and high-affinity monoclonal antibodies against O45 Escherichia coli and establishing a rapid immunological detection system suitable for port applications can optimize the detection process for non-O157 Escherichia coli and have practical application value in ensuring the biosafety of imported and exported food. Summary of the Invention

[0007] The purpose of this invention is to provide an antibody for detecting Escherichia coli O45 and its application.

[0008] Specifically, the purpose of this invention is to provide a highly specific and high-affinity monoclonal antibody against Escherichia coli O45.

[0009] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to Escherichia coli O45, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3; and the light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6.

[0010] In a preferred embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0011] In a preferred embodiment, the heavy chain variable region comprises an amino acid sequence with greater than 80% identity to the sequence of SEQ ID NO:7; the light chain variable region comprises an amino acid sequence with greater than 80% identity to the sequence of SEQ ID NO:8.

[0012] In a preferred embodiment, the monoclonal antibody is of type IgG1 with a κ light chain and is named 5D1.

[0013] In a second aspect, the present invention provides a nucleic acid molecule comprising a polynucleotide sequence encoding the aforementioned monoclonal antibody or its antigen-binding fragment.

[0014] In a third aspect, the present invention provides a carrier containing the aforementioned nucleic acid molecules.

[0015] In a preferred embodiment, the vector includes any one or more of plasmids, viral vectors, and transposons; the viral vector is selected from lentiviruses, adenoviruses, and AAV viruses.

[0016] In a fourth aspect, the present invention provides a host cell containing the aforementioned vector or having the aforementioned nucleic acid molecule integrated into its genome; the host cell is a eukaryotic cell capable of expressing antibodies, preferably a CHO cell.

[0017] In a fifth aspect, the present invention provides a method for preparing the aforementioned monoclonal antibody, comprising the following steps: (a) Culture the aforementioned host cells to obtain a culture containing monoclonal antibodies; (b) Isolation and recovery of monoclonal antibodies from cultures; (c) Optionally, the resulting antibody may be purified or modified.

[0018] In a sixth aspect, the present invention provides a detection kit comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof that specifically binds to Escherichia coli O45.

[0019] In a preferred embodiment, the detection kit is selected from immunohistochemical detection kits, immunoblotting detection kits, enzyme-linked immunosorbent assay kits, immunofluorescence detection kits, or flow cytometry detection kits.

[0020] In another preferred embodiment, the kit is an ELISA detection kit, suitable for rapid screening of Escherichia coli O45 in food samples.

[0021] In a seventh aspect, the present invention provides a method for detecting Escherichia coli O45 in food samples for non-disease diagnostic purposes, comprising the following steps: (a) The food sample to be tested is brought into contact with the monoclonal antibody or its antigen-binding fragment described in this invention; (b) Detect the binding of antibodies to target antigens in the sample to determine whether the sample contains Escherichia coli O45.

[0022] In an eighth aspect, the present invention provides the use of the aforementioned monoclonal antibody in the preparation of O45 Escherichia coli detection reagents, test strips, detection kits, or immunomagnetic beads.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The monoclonal antibody 5D1 provided by the present invention has high specificity against Escherichia coli O45 and no obvious cross-reaction with other pathogenic Escherichia coli serotypes and common foodborne pathogens. It can effectively avoid interference from closely related serotypes such as O55 and has high identification accuracy. (2) The ELISA detection method based on this antibody is simple and fast to operate, and the entire detection process can be completed within 24 hours, making it suitable for rapid on-site screening at customs ports; (3) The detection kit of the present invention has high sensitivity, and the detection limit for Escherichia coli O45 can reach 1×10⁻⁶. 2 CFU / mL, meeting the requirements for food safety testing. Attached Figure Description

[0024] Figure 1 This is an indirect ELISA potency assay curve.

[0025] Figure 2 This is the sensitivity analysis standard curve for detecting O45 Escherichia coli using the indirect ELISA method. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0027] Example 1: Antigen Preparation

[0028] The standard strain of Escherichia coli O45 (CMCC(B)43242) was inoculated into LB liquid medium and cultured at 37°C with shaking at 180 rpm for 6-8 hours until the logarithmic growth phase. The bacterial cells were collected, washed three times with sterile PBS (centrifuged at 8000 rpm for 5 minutes), and the supernatant was discarded. The bacterial cells were resuspended in 0.5% formaldehyde solution and inactivated at room temperature for 24 hours. After inactivation, the cells were washed three times with PBS to remove residual formaldehyde. The bacterial cell concentration was adjusted to 1×10⁻⁶ with PBS. 9 CFU / mL, used as an immunogenic antigen, stored at 4°C for later use.

[0029] Example 2: Preparation of Monoclonal Antibodies 2.1 Animal Immunization Healthy female BALB / c mice aged 6-8 weeks were selected and immunized according to the following program: For the initial immunization, inactivated O45 cells were mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified. Each mouse was subcutaneously injected with 200 μL of antigen (containing 1 × 10⁻⁶ cells). 8 CFU inactivated bacteria); To boost immunization, administer the second and third immunizations at two-week intervals. Use Freund's incomplete adjuvant instead of Freund's complete adjuvant, emulsify it with the bacterial cells in an equal volume, and inject it at the same dose as the first immunization. Seven days after the last immunization, blood was collected by enucleation of the eyeball, serum was separated and stored at -20°C. At the same time, the spleen was harvested for cell fusion.

[0030] 2.2 Valence determination Serum titers of immunized mice were determined using an indirect ELISA method. (1) Coating: Dilute the inactivated O45 cells with carbonate buffer (pH 9.6) to a final concentration of 1×10⁻⁶. 6 Add 100 μL of CFU / mL to each well and incubate overnight at 4°C. (2) Sealing: Shake off the liquid, add 200 μL of 5% skim milk powder / PBST to each well, and seal at 37°C for 1 hour; (3) Primary antibody reaction: Wash 3 times with PBST, serially dilute the immunized mouse serum from 1:1000 to 2-fold, add 100 μL to each well, and incubate at 37°C for 1 hour; (4) Secondary antibody reaction: Wash 3 times with PBST, add 100 μL of HRP-labeled goat anti-mouse IgG (1:5000 dilution) to each well, and incubate at 37°C for 1 hour; (5) Color development: Wash 5 times with PBST, add 100 μL of TMB color development solution to each well, and develop color for 15 minutes in the dark; (6) Termination: Add 50 μL of 2 M H2SO4 to each well to terminate the reaction; (7) Measurement: Read OD using an ELISA reader 450 The positive value was defined as P / N ≥ 2.1, and the highest positive dilution was used as the serum titer. Mice with the highest titer (≥ 1:64000) were selected for cell fusion.

[0031] 2.3 Cell Fusion (1) Take the mice with the highest titer, remove the eyeballs and bleed them to death, aseptically remove the spleen, and prepare spleen cell suspension using incomplete RPMI-1640 medium; (2) Take SP2 / 0 myeloma cells in logarithmic growth phase, mix them with spleen cells at a ratio of 1:10, centrifuge at 800 rpm for 8 minutes, and discard the supernatant; (3) In a 37°C water bath, slowly add 50% PEG 1450 (0.8 mL) dropwise while gently shaking for 1 minute; (4) Slowly add incomplete RPMI-1640 medium to terminate the fusion, and centrifuge at 800 rpm for 8 minutes; (5) Resuspend the cells in HAT selective medium (containing 10% FBS, 1×HT, 1×HAT, 100 U / mL penicillin, and 100 μg / mL streptomycin), seed them in 96-well cell culture plates, and culture at 37°C and 5% CO2. (6) After culturing for 7-10 days, positive clones are screened by indirect ELISA.

[0032] 2.4 Hybridoma Screening and Subcloning Positive clones were screened using an indirect ELISA method: bacterial cells were inactivated with O45 (1×10⁻⁶). 7 CFU / mL) was used to coat ELISA plates. Serum from immunized mice served as a positive control, while serum from unimmunized mice served as a negative control. OD was selected. 450 High-value clones were selected. Positive clones were subcloned three times using limiting dilution until monoclonalization was achieved, resulting in hybridoma cell lines that stably secreted anti-O45 monoclonal antibodies.

[0033] 2.5 Antibody purification and titer determination Collect hybridoma cell culture supernatant (density approximately 1×10⁻⁶). 6Monoclonal antibodies were purified using a Protein G affinity chromatography column (GE Healthcare), with a concentration of cells / mL. The purity of the purified product was verified by SDS-PAGE electrophoresis (a single band was visible). The titer of the purified antibody was determined by indirect ELISA, achieving a titer of 1:128000 (cells / mL). Figure 1 ).

[0034] 2.6 Antibody Subclass Identification and Sequencing The antibody subclass was determined using a mouse monoclonal antibody subclass identification kit (Sigma), and the results showed that the antibody was of type IgG1 and the light chain was κ chain.

[0035] Total RNA was extracted from hybridoma cells using the RNAiso Plus kit (TaKaRa), and RNA concentration and purity were measured. cDNA was synthesized via reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser. PCR amplification was performed using universal primers for the variable region of a mouse antibody (Shanghai Sangon Biotech Co., Ltd.). The purified product was then sent to BGI Genomics Co., Ltd. for Sanger sequencing. Sequencing results were assembled and analyzed using DNAStar software, and CDR sequences were annotated using the Kabat system.

[0036] The amino acid sequences of VH and VL are as follows (where the CDR sequence is marked with bold underline): HCDR1: SDYFT (SEQ ID NO:1) HCDR2:AISYRGPNYYAPVLKS (SEQ ID NO:2) HCDR3: GKDSFDY (SEQ ID NO:3) LCDR1:GAKSVIYGALN (SEQ ID NO:4) LCDR2: GAWVLAD (SEQ ID NO:5) LCDR3: QQHLTNPFT (SEQ ID NO:6) VH: EVQLQESGAGLAKPSQTLSLTCSVTGYSITSDYFTWIRIFPGNKLEHMGAISYRGPNYYAPVLKSRISITRDTSKNQFYLQLNSVTTEDTATYFCAGGKDSFDYWGQGTTLTVSS (SEQ ID NO:7) VL: DIQMTQSPASLSGSVGETVTITCGAKSVIYGALNWYQRKQEKSPQLLIYGAWVLADGMSSRFSGSRSGRQYSLKISSLHPDDVATYYCQQHLTNPFTFGGGTKLEIK (SEQ ID NO: 8) Example 3: Specificity identification of monoclonal antibodies 3.1 Test strains The test strains include: Standard strains of Escherichia coli with different serotypes: O157:H7 (CMCC(B)44939), O26 (CMCC(B)43221), O103 (CMCC(B)43234), O111 (CMCC(B)43211), O121 (CMCC(B)43228), O145 (CMCC(B)43243), O45 (CMCC(B)43242), O55 (ATCC 12014); Common foodborne pathogens: Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19115, Vibrio parahaemolyticus ATCC 17802, Shigella flexneri CMCC(B)51572.

[0037] 3.2 Indirect ELISA test Inactivated bacterial cells of each strain (1×10⁻⁶) 7 Coat ELISA plates with 100 μL of purified antibody 5D1 (1 μg / mL) per well and incubate overnight at 4°C. After blocking, add purified antibody 5D1 (1 μg / mL) and incubate at 37°C for 1 hour. After washing, add HRP-labeled goat anti-mouse IgG (1:5000) and incubate at 37°C for 1 hour. Develop color with TMB, stop with 2M H2SO4, and measure OD. 450 Value. Experimental data show that the antibody 5D1 of this invention only produced a strong positive reaction against strain O45, while the absorbance of other strains was close to blank, indicating no cross-reactivity; among them, the OD of strain O55 was... 450 The value was 0.080, indicating no antigen cross-contamination. The specificity identification results are shown in Table 1.

[0038] Table 1 Test Results Example 4: Monoclonal Antibody Sensitivity Analysis 4.1 Plotting the Standard Curve The O45 Escherichia coli (CMCC(B)43242) culture was serially diluted 10-fold with PBS to prepare 1×10⁻⁶ solutions. 2 1×10 31×10 4 1×10 5 1×10 6 CFU / mL series of bacterial suspensions. 100 μL of each concentration of bacterial suspension was used to coat ELISA plates. A blank control (PBS) and a negative control (O157:H7 CMCC(B)44939, 1×10⁻⁶) were also included. 6 (CFU / mL). Three replicates were used per concentration, and detection was performed using an indirect ELISA procedure to measure OD. 450 value.

[0039] Plot the logarithm of O45 bacterial concentration (CFU / mL) on the x-axis, OD 450 The values ​​are plotted on the ordinate to create a standard curve, and linear regression analysis is performed using GraphPad Prism 8.0 software.

[0040] 4.2 Determination of detection limit According to the standard curve, use the blank control OD 450 The positive cut-off value was calculated by adding three times the standard deviation to the value. The lowest concentration below this threshold was defined as the limit of detection (LOD). The sensitivity analysis results of the indirect ELISA method for detecting Escherichia coli O45 are shown in Table 2.

[0041] Table 2. Sensitivity analysis of indirect ELISA for detecting Escherichia coli O45 With log 10 (Concentration) is the x-axis, OD 450 Linear regression analysis was performed using the ordinate, and the standard curve equation is: y = 0.5471 × log10(x) - 0.8236, with a correlation coefficient R0. 2 = 0.9920. Blank control OD 450 The value is 0.058 ± 0.008, and the cut-off value calculated using Blank + 3SD is 0.082. 1 × 10 2 OD of CFU / mL 450 The value was 0.271, higher than the cut-off value, and was therefore considered positive. Thus, the detection limit of this method is 1 × 10⁻⁶. 2 CFU / mL.

[0042] Example 5: O45 Escherichia coli Detection Kit Based on monoclonal antibody 5D1, an indirect ELISA detection kit for Escherichia coli O45 is provided, comprising the following components: (1) Microplate: 96-well polystyrene microplate; (2) Coating buffer: carbonate buffer (pH 9.6); (3) Primary antibody: O45 Escherichia coli monoclonal antibody 5D1 (1 mg / mL); (4) Enzyme-labeled secondary antibody: HRP-labeled goat anti-mouse IgG, working dilution 1:5000; (5) Colorimetric solution: TMB substrate solution (A solution: B solution = 1:1 mixture); (6) Termination solution: 2MH2SO4; (7) Washing solution: PBS containing 0.05% Tween-20 (PBST); Blocking solution: 5% skim milk powder / PBST; (8) Positive control: O45:H2 inactivated bacterial cells (1×10⁻⁶) 6 CFU / mL); Negative control: O157:H7 inactivated bacteria (1×10⁻⁶ CFU / mL); 6 (CFU / mL).

[0043] Testing steps: ① Sample preparation: Take 25g of the food sample to be tested, add 225mL of BPW enrichment broth, and incubate at 37℃ for 6-8 hours; ② Take 1 mL of enrichment broth, centrifuge at 12000 rpm for 2 minutes, discard the supernatant, and resuspend the precipitate in 100 μL PBS; ③ Add an equal volume of 2× sample lysis buffer (containing 0.2% SDS and 0.5% Triton X-100), boil in a water bath for 5 minutes, and cool to obtain the sample to be tested; ④ Coating: Take the sample to be tested, positive control, and negative control, adjust the bacterial concentration with coating buffer, add 100 μL to each well of the microplate, and coat at 37°C for 2 hours; ⑤ Shake off the liquid, add 200 μL of blocking solution to each well, block at 37°C for 1 hour, wash 3 times with PBST, and pat dry; ⑥ Add 100 μL of anti-O45 monoclonal antibody 5D1 (1 μg / mL) to each well, incubate at 37°C for 1 hour, wash 3 times with PBST, and pat dry; ⑦ Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) to each well, incubate at 37°C for 1 hour, wash 5 times with PBST, and pat dry; ⑧ Add 100 μL of TMB colorimetric solution to each well and develop the color at 37°C in the dark for 15 minutes; ⑨ Add 50 μL of stop solution to each well and read the OD value using a microplate reader. 450 value; ⑩ Result determination: Sample OD 450 A value ≥0.082 (cut-off value) is considered positive; OD 450 A value <0.082 is considered negative.

[0044] The entire testing process (including enrichment culture) can be completed within 24 hours, making it suitable for rapid inspection and quarantine of imported and exported food by customs.

[0045] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to Escherichia coli O45, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1, CDR2, and CDR3, respectively. The light chain variable region comprises CDR1, CDR2, and CDR3, respectively.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:7, or the heavy chain variable region contains an amino acid sequence having more than 80% identity with SEQ ID NO:7; the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:8, or the light chain variable region contains an amino acid sequence having more than 80% identity with SEQ ID NO:

8.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a polynucleotide sequence encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2.

4. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 3 or the vector of claim 4.

6. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that, The method involves culturing the host cells as described in claim 5 to generate the antibody.

7. The method according to claim 6, characterized in that, It further includes the recovery of the monoclonal antibody or its antigen-binding fragment from the host cell or cell culture.

8. A test kit, characterized in that, The test kit comprises any one of the monoclonal antibodies or antigen-binding fragments thereof as claimed in claims 1-2.

9. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2 in the preparation of O45 Escherichia coli detection reagents, test strips, detection kits or immunomagnetic beads.

10. A method for detecting Escherichia coli O45 in food samples for non-disease diagnostic purposes, comprising the following steps: (a) The food sample to be tested is brought into contact with the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2; (b) Detect the binding of antibodies to target antigens in the sample to determine whether the sample contains Escherichia coli O45.

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