Nano antibody for detecting enrofloxacin and application thereof

Abstract

The invention discloses an anti-enrofloxacin nano antibody and application thereof, and belongs to the technical field of nano antibodies. The invention aims to provide a nano antibody which is small in molecular mass, high in stability, strong in penetrating power, low in production cost and capable of being specifically combined with enrofloxacin. The invention provides a nano-antibody for detecting enrofloxacin. The amino acid sequence of a CDR1 of the nano-antibody is shown as SEQ ID NO.6, the amino acid sequence of a CDR2 of the nano-antibody is shown as SEQ ID NO.7, and the amino acid sequence of a CDR3 of the nano-antibody is shown as SEQ ID NO.8. The invention further provides a preparation method of the nano-antibody for detecting enrofloxacin. The indirect competitive ELISA method established by the invention has relatively high sensitivity, precision, specificity and correctness, provides a new technical support for detection of enrofloxacin residues in animal-derived food, is simple, rapid, sensitive and low in cost, and can effectively detect the enrofloxacin residues in the animal-derived food.

Description

Technical Field

[0001] This invention belongs to the field of nanobodies, specifically relating to a nanobody for detecting enrofloxacin and its application. Background Technology

[0002] Enrofloxacin (ENR) is a third-generation quinolone antibiotic with the molecular formula C2. 19 H 22 FN3O3, with a molecular weight of 359.40 Da, is a pale yellow crystalline powder. It is slightly soluble in methanol, has low solubility in water, and is readily soluble in sodium hydroxide or hydrochloric acid solutions. Its target is bacterial DNA gyrase and topoisomerase IV, exerting its effect by interfering with DNA replication, recombination, and gene expression. It inhibits bacteria at low concentrations and kills bacteria at high concentrations. Long-term or excessive use of ENR may lead to excessive drug residues in animal-derived foods, increasing bacterial resistance, causing allergic reactions in humans, and posing risks such as liver and kidney toxicity, thus posing a potential threat to human health. Establishing efficient and sensitive detection methods to monitor ENR residue levels in animal-derived foods is of great significance for ensuring food safety.

[0003] Currently, the main detection technologies used for ENR residues include chromatography, microbial inhibition methods, electrochemical methods, and immunoassay. Chromatography offers advantages such as strong separation capabilities, high sensitivity, and fast analysis speed, but it suffers from drawbacks such as cumbersome and time-consuming sample pretreatment, and high instrument and maintenance costs. Microbial inhibition methods are characterized by low sensitivity, poor specificity, and long processing times. Electrochemical methods may encounter interference from other electroactive substances in complex samples (such as blood, urine, or food). Among immunoassay methods, enzyme-linked immunosorbent assay (ELISA) is the most commonly used. This method is suitable for component analysis in complex matrices such as animal tissues, and it offers advantages such as ease of operation and low cost, making it ideal for rapid screening in grassroots settings. Current immunological analysis methods for ENR are mostly based on polyclonal and monoclonal antibodies. Nanobodies, with their small molecular weight, can recognize antigenic epitopes that are difficult for conventional antibodies to recognize, and have significant advantages such as high stability, low production cost, and strong affinity, which can significantly improve the accuracy, sensitivity, and reliability of immunological detection. Summary of the Invention

[0004] The purpose of this invention is to provide a nanobody for detecting enrofloxacin that has a small molecular weight, high stability, strong antigen-binding activity, and can specifically recognize antigenic epitopes that are difficult for traditional antibodies to recognize.

[0005] The present invention provides a nanobody for detecting enrofloxacin. The amino acid sequence of CDR1 of the variable region of the nanobody is shown in SEQ ID NO.6, the amino acid sequence of CDR2 of the variable region of the nanobody is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 of the variable region of the nanobody is shown in SEQ ID NO.8.

[0006] Further specifying, the nanobody also includes four framework regions, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.5.

[0007] The present invention provides a nucleotide sequence encoding the above-mentioned nanobody.

[0008] This invention provides a nanobody for detecting enrofloxacin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] The present invention provides a recombinant vector containing the above-mentioned nucleotide sequence.

[0010] The present invention provides a recombinant microbial cell containing the above-mentioned nucleotide sequence.

[0011] The present invention provides a kit for detecting enrofloxacin, the kit containing the above-mentioned nanobody.

[0012] This invention provides the application of the above-mentioned nanobody, the above-mentioned nucleotide sequence, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cell in the preparation of a kit for detecting enrofloxacin.

[0013] Further, the testing will be limited to enrofloxacin residues in the food, environmental, or medical fields.

[0014] This invention provides a method for detecting enrofloxacin residues, which utilizes the aforementioned nanobody for indirect competitive ELISA.

[0015] Beneficial effects: This invention compares the thermal stability of the prepared nanobody with that of commercially available ENR monoclonal antibody. The nanobody Nb-E1 retained 75% antigen-binding activity after treatment at 90℃ for 5 min and maintained 60% activity after treatment at 85℃ for 1 h, while the monoclonal antibody was completely inactivated after treatment at 70℃ for 5 min. It can be seen that the thermal stability of the nanobody is better than that of the monoclonal antibody.

[0016] This invention compares the organic solvent tolerance of the prepared nanobody with that of commercially available ENR monoclonal antibody. The nanobody Nb-E1 showed significantly better tolerance to methanol, acetonitrile, dichloromethane, and dimethyl sulfoxide than the monoclonal antibody.

[0017] An indirect competitive ELISA method for detecting ENR based on nanobody Nb-E1 was established. 50 The effective value was 13.54 ng / mL, the linear detection range was 4.49–100.12 ng / mL, the limit of detection was 3.19 ng / mL, and the limit of quantitation was 4.28 ng / mL. This method exhibits high sensitivity, precision, accuracy, and specificity.

[0018] Compared to monoclonal antibodies with a molecular weight of approximately 150 kDa, nanobodies have a molecular weight of only 15 kDa. They recognize epitopes that are difficult for traditional antibodies to reach, have strong specific binding ability, extremely high stability, and can be efficiently expressed in prokaryotic expression systems, greatly shortening the production cycle and reducing production costs. Attached Figure Description

[0019] Figure 1 This is a diagram of the molecular structure of enrofloxacin. Figure 2 Synthesis route diagrams for ENR-BSA and ENR-OVA; Figure 3 The images show the UV scans of ENR-BSA and ENR-OVA; A is the UV spectral scan of ENR-BSA, and B is the UV spectral scan of ENR-OVA. Figure 4 Gel electrophoresis identification images of ENR-BSA (A) and ENR-OVA (B); M is protein marker, 1 is BSA, 2 is ENR-BSA, 3 is OVA, and 4 is ENR-OVA; Figure 5 Image of Phage ELISA results; Figure 6 The image shows the results of bacterial culture PCR identification; M represents DNA Ladder 2000, 1 represents ER2738 negative control, and 2 represents E1. Figure 7 This is a schematic diagram of double enzyme digestion of the prokaryotic expression vector pET28a-E1; M is DNA Ladder 5000, 1 is pET28a, 2 is pET28a-E1, and 3 is pET28a-E1 after enzyme digestion. Figure 8 SDS-PAGE (A) and Western blotting results (B) for Nb-E1; M is the protein marker, and 1 is the purified Nb-E1; Figure 9 The image shows the results of antigen-binding activity analysis of the nanobody Nb-E1. Figure 10 The graph shows the comparison of the thermal stability of nanobody Nb-E1 and monoclonal antibody; A represents the incubation of Nb-E1 and monoclonal antibody at 4℃~90℃ for 5 min, and B represents the incubation of Nb-E1 and monoclonal antibody at 85℃ for 0~60 min. Figure 11 The figure shows the comparison results of organic solvent tolerance between the nanobody Nb-E1 and the monoclonal antibody; A is methanol, B is acetonitrile, C is dichloromethane, and D is dimethyl sulfoxide. Figure 12 This is a standard curve for enrofloxacin ic-ELISA based on Nb-E1. Detailed Implementation

[0020] Example 1. Complete antigen synthesis and identification of enrofloxacin The chemical formula of enrofloxacin described in the following embodiments of the present invention is as follows: Figure 1 As shown.

[0021] 1. Complete antigen synthesis of enrofloxacin This invention studies the preparation of complete antigens ENR-BSA and ENR-OVA using the carbodiimide method. The synthetic route is as follows: Figure 2 Accurately weigh 20.3 mg of ENR, 9.31 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 5.19 mg of N-hydroxysuccinimide (NHS), and dissolve them in 1 mL of N,N-dimethylformamide (DMF). After stirring magnetically at room temperature in the dark for 2 h, centrifuge at 2000 rpm for 10 min, discard the precipitate, and collect the supernatant as solution A. Weigh 5 mg of bovine serum albumin (BSA) and dissolve it in 1 mL of pre-chilled PBS to obtain solution B. Slowly add solution A dropwise to solution B, and stir at 4°C in the dark for 4 h. After the reaction is complete, dialyze the mixture against the light using PBS at 4°C for 3 days. After dialysis, collect the solution in the dialysis bag, centrifuge at 2000 rpm for 10 min, and collect the supernatant, which is the complete ENR-BSA antigen, and store it at -20°C for later use.

[0022] The same method was used to couple ENR with chicken ovalbumin (OVA) to generate ENR-OVA.

[0023] 2. Ultraviolet scanning identification The successful synthesis of complete antigens ENR-BSA and ENR-OVA was determined using ultraviolet absorption spectroscopy. Figure 3As shown in Figure A, ENR exhibits significant characteristic absorption peaks at 271 nm and 334 nm, while BSA's characteristic peak appears at 278 nm. After coupling, ENR-BSA shows characteristic absorption peaks near the original peaks at 276 nm and 332 nm, and these peaks are shifted to some extent compared to the uncoupled peak positions of ENR and BSA. Figure 3 As shown in Figure B, the characteristic absorption peak of OVA is at 280 nm, while the characteristic absorption peaks of the coupled ENR-OVA appear at 279 and 332 nm, exhibiting a similar peak position adjustment and new peak formation phenomenon. Based on the above results, it can be preliminarily determined that the coupling of ENR-BSA and ENR-OVA was successful.

[0024] 3. SDS-PAGE identification The migration positions of BSA, ENR-BSA, OVA, and ENR-OVA bands were observed by SDS-PAGE electrophoresis. Figure 4 As shown in A, the band of BSA is located around 66 kDa. The molecular weight of ENR-BSA is increased, so the migration rate is slower during electrophoresis and the band position shifts slightly upward. Figure 4 As shown in Figure B, the band of OVA is located at approximately 45 kDa, and the molecular weight of ENR-OVA has also increased slightly. These results demonstrate that both ENR-BSA and ENR-OVA have been successfully coupled.

[0025] Example 2. Panning and Identification of Enrofloxacin Nanobodies 1. Screening of enrofloxacin nanobodies A 300 µL alpaca natural nanobody library was inoculated into 100 mL of 2×YTG(AT) medium and cultured at 37°C and 200 rpm until OD500. 600 After reaching a multiplicity of infection (MOI) of 0.6, helper phage M13KO7 was added at a MOI of 20:1. The mixture was incubated at 37°C for 30 min with shaking for 30 min, centrifuged at 4°C and 3300 rpm, and the precipitate was resuspended in 100 mL of 2×YT (ATK) medium and incubated overnight at 30°C with shaking. Simultaneously, 2 mL of 100 µg / mL ENR-BSA and BSA solutions were added to immunotubes and incubated overnight at 4°C. The next day, the bacterial culture was purified by centrifugation at 4°C and 5000 rpm, precipitated with PEG / NaCl, and resuspended in PBS. The supernatant titer was measured to determine the phage input amount. After blocking and washing the BSA immunotubes, phage supernatant was added to remove interference. The treated phage was then added to the ENR-BSA immunotubes and bound at 37°C. After washing with PBST and PBS, the phage was eluted and neutralized with 0.1 mol / L triethylamine. The eluent was then mixed with E. coli. E. coliER2738 bacterial suspension (Beijing Baokewei Food Safety, HB13005) was incubated at 37℃ for 30 min. Part of the bacterial suspension was diluted and plated to measure the output phage quantity. The remaining bacterial suspension was centrifuged, resuspended, and plated on petri dishes. Colonies were scraped to collect the bacterial suspension. 300 µL was used for the second round of sieving, and the remainder was added to 15% glycerol and stored at -80℃.

[0026] The alpaca natural nanobody library underwent four rounds of adsorption-elution-amplification screening. The input and output amounts of phages in each round were calculated, and the results are shown in Table 1.

[0027] Table 1. Enrichment of phages from four rounds of screening.

[0028] Recovery rate = Output phage volume (PFU) / Input phage volume (PFU) 2. Phage ELISA identification Ninety-six colonies were randomly selected from the fourth round of screening plates to prepare phage supernatant. The binding activity of the recombinant phage to ENR was detected using the phage ELISA method. The results are shown in [Figure number missing]. Figure 5 The clone with the highest P / N value was selected and named E1.

[0029] 3. PCR identification and sequencing analysis The VHH gene of strain E1 was amplified. The gel electrophoresis results are as follows: Figure 6 As shown, compared with the negative control ER2738 bacterial culture, a band of approximately 400 bp appeared, which is consistent with the size of the target fragment, indicating that the strain has successfully inserted the VHH gene.

[0030] Example 3. Preparation of enrofloxacin nanobodies 1. Construction of prokaryotic expression recombinant plasmids Based on the sequence obtained from sequencing strain E1, the pET28a-E1 prokaryotic expression recombinant plasmid was constructed. The plasmid pET28a-E1 was then... Bam HI and Xho I double digestion, results as follows Figure 7 As shown, a fragment of about 400 bp appeared after enzyme digestion, indicating that the prokaryotic expression recombinant plasmid was successfully constructed.

[0031] 2. Expression and purification of nanobodies Plasmid pET28a-E1 was transformed into BL21(DE3). Colonies with correct sequencing results were expanded and cultured. After induction at 16°C for 18 h with 0.4 mmol / L IPTG, bacterial protein was extracted, and antibodies were purified by nickel column chromatography. The purified protein was analyzed by SDS-PAGE electrophoresis and Western blotting. Results are shown below. Figure 8 A distinct band appeared near 15 kDa, and no obvious impurities were observed, indicating good protein purification. The purified nanobody was named Nb-E1.

[0032] The amino acid sequence of the nanobody Nb-E1 corresponds to SEQ ID NO.1, which contains complete structural domains: four FR backbone regions and three CDR variable regions. The amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.5; the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.8.

[0033] The amino acid sequence of the nanobody Nb-E1 (SEQ ID NO.1): QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCINYSDRTPNHADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAATVDPCTFYDGSYFRNSWGQGTQVTVSS; The amino acid sequence of FR1 (SEQ ID NO.2): QVQLQESGGGLVQPGGSLRLSCAAS; The amino acid sequence of FR2 (SEQ ID NO.3): IGWFRQAPGKEREGVSC; The amino acid sequence of FR3 (SEQ ID NO.4): NHADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYC; The amino acid sequence of FR4 (SEQ ID NO.5): WGQGTQVTVSS; The amino acid sequence of CDR1 (SEQ ID NO.6): GFTLDYYA; The amino acid sequence of CDR2 (SEQ ID NO.7): INYSDRTP; The amino acid sequence of CDR3 (SEQ ID NO.8): AATVDPCTFYDGSYFRNS; Nanobody Nb-E1 gene sequence, SEQ ID NO.9: caggtgcagctgcaggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctgggttcactttggattattatgccattggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattaattacagtgatcgtacaccaaaccatgcagactccgtgaagggccgattcaccatttccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacaggcgtttattactgtgcagcaacggtcgacccctgtacattttatgatggtagttactttcgaaactcctggggccaggggacccaggtcaccgtctcctcac; FR1 gene sequence, SEQ ID NO.10: caggtgcagctgcaggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctct; FR2 gene sequence, SEQ ID NO.11: attggctggttccgccaggccccagggaaggagcgtgagggggtctcatgt; FR3 gene sequence, SEQ ID NO.12: aaccatgcagactccgtgaagggccgattcaccatttccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacaggcgtttattactgt; FR4 gene sequence, SEQ ID NO.13: tggggccaggggacccaggtcaccgtctcctcac; CDR1 gene sequence, SEQ ID NO.14: gggttcactttggattattatgcc; CDR2 gene sequence, SEQ ID NO.15: attaattacagtgatcgtacacca; CDR3 gene sequence, SEQ ID NO.16: gcagcaacggtcgacccctgtacattttatgatggtagttactttcgaaactcc.

[0034] Example 4. Biological characteristics of nanobody Nb-E1 1. Antigen binding activity analysis The purified Nb-E1 nanobody was diluted to different concentrations (0.5 µg / mL, 2 µg / mL, 4 µg / mL, 20 µg / mL), and the binding activity of the nanobody to the target antigen was identified using an indirect ELISA method. Figure 9 The results show that, with the antigen ENR-OVA concentration remaining constant, OD increases with increasing Nb-E1 concentration. 450 The value also increased accordingly. These results indicate that the nanobody Nb-E1 has good antigen-binding activity.

[0035] 2. Thermal stability analysis The nanobody Nb-E1 and the commercially available ENR monoclonal antibody (Shenzhen Antibio, AT01ENORAb) were treated at different temperatures (4℃, 37℃, 50℃, 70℃, 80℃, 90℃) for 5 min each. The binding activity of the antibodies to the antigen was detected by indirect ELISA. Another group of nanobodies and monoclonal antibodies were treated at 85℃ for different times (0 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min), and the binding activity of the antibodies to the antigen was also detected by indirect ELISA to evaluate the thermal stability of the nanobody Nb-E1.

[0036] Depend on Figure 10 As shown in Figure A, the activity of the nanobody Nb-E1 remained relatively stable with increasing temperature, retaining over 75% of its activity even after heating at 90℃ for 5 minutes. In contrast, the activity of monoclonal antibodies decreased significantly with increasing temperature, essentially losing their ability to bind to antigens at 70℃. Figure 10 As shown in Figure B, monoclonal antibodies essentially lose their antigen-binding activity after being heated at 85°C for 5 minutes; while the nanobody Nb-E1 retains nearly 60% of its antigen-binding activity after being heated at 85°C for 1 hour, indicating that the nanobody Nb-E1 has high thermal stability.

[0037] 3. Organic solvent tolerance analysis Methanol, acetonitrile, dichloromethane, and dimethyl sulfoxide were selected as solvents and prepared into solutions with concentrations of 20%, 40%, 60%, and 80%, respectively. Using these solvents of different concentrations, the nanobody Nb-E1 and ENR monoclonal antibodies were diluted to working concentrations, and the binding activity between the antibodies and antigens was detected using an indirect ELISA method. Results are shown below. Figure 11 Under 40% methanol, the Nb-E1 nanobody retained approximately 60% of its activity, while the activity of the monoclonal antibody decreased to approximately 38%. Under 60% acetonitrile, the binding activity of the Nb-E1 nanobody remained at 45%, while the monoclonal antibody essentially lost its binding activity. The Nb-E1 nanobody exhibited good tolerance to dichloromethane, retaining approximately 90% of its activity at a 40% dichloromethane concentration, while the activity of the monoclonal antibody under the same conditions decreased to less than 50%. The activities of both the Nb-E1 nanobody and the monoclonal antibody decreased with increasing dimethyl sulfoxide concentration, but the activity of the nanobody remained consistently higher than that of the monoclonal antibody. These results indicate that the Nb-E1 nanobody exhibits better organic solvent tolerance compared to the monoclonal antibody.

[0038] Example 5. Establishment of an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method for enrofloxacin based on nanobodies. (1) Coating: 1 µg / mL ENR-OVA was used as the coating antigen. 100 µL / well was added to the microplate and incubated overnight at 4°C.

[0039] (2) Blocking: Discard the coating solution, wash 3 times with PBS, and pat dry. Add 300 µL of 3% skim emulsion to each well and block at 37°C for 2 h.

[0040] (3) Competitive binding: Discard the blocking solution, wash 3 times each with PBST and PBS, and pat dry. After homogenizing animal liver or muscle samples, extract 50 µL of the supernatant and add it to the well, then add 50 µL of 4 μg / mL Nb-E1, and bind at 37℃ for 1.5 h.

[0041] (4) Incubation of secondary antibody: Pour out the liquid in the plate, wash 3 times each with PBST and PBS, and pat dry. Add 100 µL of HRP-anti-His (Wuhan Sanying Biotechnology, HRP-66005) antibody diluted 1:20000 to each well, and incubate at 37℃ for 1 h.

[0042] (5) Color development: Discard the enzyme-labeled secondary antibody, wash 3 times each with PBST and PBS, and pat dry. Add 100 µL of TMB color development solution to each well and react at 37°C in the dark for 20 min.

[0043] (6) Termination: Add 50 µL of 2 mol / L H2SO4 to each well to stop the color development, and then detect the OD using a microplate reader. 450 Read value.

[0044] (7) Establishment of standard curve: 50 µL of ENR standard solutions diluted to different concentrations (0.2 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 1000 ng / mL) were measured. The results were fitted using a Logistic function to establish a standard curve for the enrofloxacin ic-ELISA method based on the Nb-E1 nanobody. The IC50 was then calculated. 50 The value was 13.54 ng / mL, and the linear detection range (IC50) was [missing value]. 20 ~IC 80 The concentrations ranged from 4.49 to 100.12 ng / mL, and the standard curve equation was Y = -20.70ln(X) + 110.0, R0. 2 =0.9922. The obtained standard curve is as follows: Figure 12 As shown.

[0045] (8) Result determination: Substitute the absorbance of the measured sample into the standard curve to obtain the actual concentration.

[0046] Example 6. Evaluation of the enrofloxacin ic-ELISA method based on nanobody Nb-E1 1. Sensitivity Analysis OD of 10 additive-free drug samples was measured 450 Calculate the mean and standard deviation of these 10 values. Calculate the value of (mean - 2 × standard deviation) and substitute it into the standard curve to obtain a concentration of 3.19 ng / mL, which is the limit of detection for this method. Calculate the value of (mean - 3 × standard deviation) and substitute it into the standard curve to obtain a concentration of 4.28 ng / mL, which is the limit of quantitation for this method.

[0047] 2. Precision analysis The established ic-ELISA method was used to perform 10 independent tests on the same test sample (5 ng / mL ENR standard solution), and the absorbance values ​​were read. The 10 sets of data were statistically analyzed, and the mean and standard deviation were calculated. The intra-batch coefficient of variation was found to be 6.1%. Ten independent determinations were performed on the same test sample (5 ng / mL ENR standard solution) by different operators, and the inter-batch coefficient of variation was found to be 8.8%.

[0048] Coefficient of variation = Standard deviation / Measured mean × 100% 3. Specificity analysis Seven quinolone antibiotics (excluding ENR) and three other commonly used antibacterial drugs were selected and tested using the ic-ELISA method. Standard curves were plotted, and IC50 was calculated. 50 The cross-reactivity rate of each drug relative to the ENR is calculated to evaluate the specificity of this method. Cross-reactivity rate (%) = IC50 50 (ENR) / IC 50 (Other drugs) ×100%. The cross-reactivity rates of the established ic-ELISA method with ciprofloxacin, dalofaxin, ofloxacin, norfloxacin, sarafloxacin, enoxacin, and lomefloxacin were 11.38%, 6.75%, 7.21%, 4.62%, 8.30%, 4.76%, and 6.86%, respectively, and no cross-reactivity was detected with the three non-quinolone drugs (see Table 2), indicating that the enrofloxacin ic-ELISA method based on the Nb-E1 nanobody has good specificity.

[0049] Table 2. Determination of Cross-Reactivity Rate

[0050] Note: ND (not detected) 4. Recovery rate determination ENR standards (10 μg / kg, 50 μg / kg, and 100 μg / kg) were added to SPF pork, pork liver, SPF chicken, and chicken liver. The tissue supernatant was extracted and analyzed by ic-ELISA. The recovery rates were calculated. The results are shown in Table 3. The recovery rates in pork samples ranged from 82.55% to 108.57%, in pork liver samples from 90.45% to 107.22%, in chicken samples from 83.52% to 109.02%, and in chicken liver samples from 90.07% to 106.79%. The national standard GB / T 33411-2016 stipulates that the recovery rate of enzyme-linked immunosorbent assay (ELISA) kits should be within the range of 60% to 120%. Therefore, the recovery rate of the established ic-ELISA method meets the requirements.

[0051] Table 3 Recovery rate determination (n=6)

Claims

1. A nanobody for detecting enrofloxacin, characterized in that, The amino acid sequence of CDR1 of the variable region of the nanobody is shown in SEQ ID NO.6, the amino acid sequence of CDR2 of the variable region of the nanobody is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 of the variable region of the nanobody is shown in SEQ ID NO.

8.

2. The nanobody according to claim 1, characterized in that, The nanobody further includes four framework regions: the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.

5.

3. The nucleotide sequence encoding the nanobody of claim 1.

4. A nanobody for detecting enrofloxacin, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleotide sequence as described in claim 3.

6. A recombinant microbial cell, characterized in that, The recombinant microbial cells contain the nucleotide sequence described in claim 3.

7. A reagent kit for detecting enrofloxacin, characterized in that, The kit contains the nanobody as described in claim 1, 2 or 4.

8. The use of the nanobody of claim 1, 2 or 4, the nucleotide sequence of claim 3, the recombinant vector of claim 5 or the recombinant microbial cell of claim 6 in the preparation of a kit for detecting enrofloxacin.

9. The application according to claim 8, characterized in that, Detection of enrofloxacin residues in the food, environmental, or medical fields.

10. A method for detecting enrofloxacin residues, characterized in that, A method for indirect competitive ELISA using the nanobody described in claim 1, 2 or 4.

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