A monoclonal antibody of doramectin, a detection test strip and application thereof

Abstract

The application discloses a doramectin monoclonal antibody, a detection test strip and application thereof, and relates to the field of animal health. 50 The heavy chain and light chain variable regions of the monoclonal antibody have specific complementarity determining region sequences, and the half inhibition concentration IC of the monoclonal antibody to doramectin is as low as 3-5 ng / mL, which shows excellent sensitivity; the cross reaction rates of the monoclonal antibody to abamectin, ivermectin and acetyl abamectin are 75%, 75% and 60% respectively, and the monoclonal antibody has no cross reaction with common antibiotics such as tetracycline and gentamicin. Based on the antibody, the application also develops a colloidal gold immunochromatography test strip, which is simple in operation, rapid in detection, visual in result and has a detection limit of 5 ng / mL, and is suitable for on-site rapid screening of doramectin and abamectin drug residues in animal-derived food, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a doramectin monoclonal antibody, a test strip, and their applications. Background Technology

[0002] Doramectin, a 16-membered macrolide antiparasitic drug, is an important member of the avermectin family. It is produced through fermentation of a new strain of recombinant Streptomyces avermitilis and possesses advantages such as a broad antiparasitic spectrum, long-lasting efficacy, and no allergic reactions. It is widely used in animal husbandry to treat internal and external parasites in cattle, sheep, pigs, and other animals. However, doramectin is slowly metabolized in animals and has high fat solubility, easily leading to residues in animal-derived foods (such as meat and dairy products). Long-term consumption of food containing doramectin residues in humans may have potential toxic effects on their nervous system and development. Therefore, many countries and regions, including China, have established strict maximum residue limits (MRLs) for doramectin in animal-derived foods.

[0003] Currently, the main methods for detecting doramectin residues are high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While these instrumental analytical methods are accurate and sensitive, they typically require expensive equipment, specialized operators, and complex sample pretreatment procedures, resulting in high detection costs and long processing times, making them unsuitable for rapid on-site screening of large-scale samples. In contrast, immunological detection methods, especially enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies and colloidal gold immunochromatographic strip methods, offer advantages such as speed, simplicity, low cost, and suitability for on-site operation, and have become an important technological direction in the field of residue detection.

[0004] There are some existing studies and reports on immunoassay techniques for avermectin-based drugs. For example, Northeast Agricultural University disclosed an anti-avermectin monoclonal antibody and its hybridoma cell line 2F2 (CGMCC No. 1413) in patent CN1733910A published in 2005. This antibody has a half-maximal inhibitory concentration (IC50) against avermectin. 50 The concentration of the antibody was 101 ng / mL, and the cross-reactivity with ivermectin and doramectin was less than 0.01%, indicating that the antibody only specifically recognizes ivermectin and has extremely low affinity for doramectin, making it unsuitable for sensitive detection of doramectin.

[0005] In recent years, Jiangnan University has disclosed a hybridoma cell line (CGMCC No. 45477) that secretes avermectin-like monoclonal antibodies in patent CN118480513A. This antibody exhibits high sensitivity (IC50) to avermectin, ivermectin, emamectin benzoate, and ephemeratin. 50 The concentrations were 0.95, 1.72, 0.46, and 0.62 ng / mL, respectively, but its IC50 against doramectin was... 50 With a concentration as high as 17.71 ng / mL, its sensitivity is significantly lower than other avermectin drugs, making it difficult to meet the requirements for accurate detection of doramectin residues. Furthermore, this patent does not disclose the specific amino acid or nucleotide sequence of the antibody, making it impossible for those skilled in the art to easily obtain antibodies with the same performance based on the disclosed information, and also hindering subsequent engineering modifications and application development.

[0006] In summary, the existing technology still has the following shortcomings: On the one hand, there is a lack of a method with high sensitivity (IC50) to doramectin. 50 On the one hand, monoclonal antibodies that can reach the ng / mL level or even lower are needed; on the other hand, among the currently published avermectin antibodies, those that can simultaneously recognize doramectin and have excellent sensitivity to it have not been reported, and antibody sequence information is generally lacking, which limits their development and application in rapid detection products. Therefore, developing a doramectin monoclonal antibody and test strip with high sensitivity, good specificity, well-defined sequence, and suitable for rapid on-site detection is of great practical significance and application value for ensuring the safety of animal-derived food and strengthening the monitoring of doramectin residues. Summary of the Invention

[0007] To address the aforementioned issues, this invention discloses a doramectin monoclonal antibody, a test strip, and their applications. This invention provides a novel monoclonal antibody sequence and further develops a colloidal gold test strip that can be used for rapid on-site detection.

[0008] In a first aspect, a doramectin monoclonal antibody is prepared by immunizing animals with a doramectin-BSA immunogen, which can specifically recognize doramectin and has good cross-recognition ability with avermectin, ivermectin and acetylavermectin.

[0009] Furthermore, the doramectin monoclonal antibody includes a light chain variable region and a heavy chain variable region. The light chain variable region includes three complementarity-determining regions (CDRs): light chain CDR1, light chain CDR2, and light chain CDR3. The heavy chain variable region includes three complementarity-determining regions (CDRs): heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3.

[0010] Specifically: The amino acid sequence of the light chain complementarity-determining region CDR1 is shown in SEQ ID NO. 18 (QNIVHSDGITY). The amino acid sequence of the light chain complementarity-determining region CDR2 is KVS; The amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO. 21 (FQGSHVPYT). The amino acid sequence of the heavy chain complementarity-determining region CDR1 is shown in SEQ ID NO. 2 (GYTFSNHW). The amino acid sequence of the heavy chain complementarity-determining region CDR2 is shown in SEQ ID NO. 4 (IYPGSGHI). The amino acid sequence of the heavy chain complementarity-determining region CDR3 is shown in SEQ ID NO. 6 (VFGNSYNYAMDY).

[0011] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 23; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8.

[0012] SEQ ID NO. 23 (light chain variable region amino acid): DVLMTQIPLSLPVSLGDQASISCRSSQNIVHSDGITYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK.

[0013] SEQ ID NO. 8 (heavy chain variable region amino acid): QVQLQQSGAELVRPGTSVKMSCKAAGYTFSNHWIGWVKQRPGHGLEWVGDIYPGSGHIDYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAIYYCVFGNSYNYAMDYWGQGTSVTVSS.

[0014] More preferably, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 30; and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 16.

[0015] SEQ ID NO. 30 (Light chain variable region gene): GATGTTTTGATGACCCAAATTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGTAGATCTAGTCAGAACATTGTACATAGTGATGGAATCACTTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCTTGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA.

[0016] SEQ ID NO. 16 (heavy chain variable region gene): CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGATGTCCTGCAAGGCTGCTGGATACACCTTCAGTAATCACTGGATAGGTTGGGTAAAACAGAGGCCTGGACATGGCCTTGAGTGGGTTGGAGATATTTATCCTGGAAGTGGTCATATTGATTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCCATCTATTACTGTGTTTTTGGTAACTCATATAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0017] Secondly, the present invention provides a method for preparing the above-mentioned doramectin monoclonal antibody, including steps such as animal immunization, cell fusion and screening, cloning and ascites preparation.

[0018] Thirdly, the present invention provides applications of the doramectin monoclonal antibody, including its use in the preparation of doramectin detection reagents or test strips. Specifically, a colloidal gold immunochromatographic test strip for detecting doramectin comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane (NC membrane), an absorbent pad, and a backing plate. The gold-labeled pad is coated with the colloidal gold-labeled doramectin monoclonal antibody of the present invention; the NC membrane has a detection line (T line) and a control line (C line), the T line is coated with a doramectin-carrier protein conjugate, and the C line is coated with goat anti-mouse IgG secondary antibody.

[0019] The beneficial effects of this invention are: The doramectin monoclonal antibody provided by this invention has a half-maximal inhibitory concentration (IC50) against doramectin. 50 The antibody exhibits excellent sensitivity with a concentration of 3-5 ng / mL. It shows a 75% cross-reactivity with avermectin, a 75% cross-reactivity with ivermectin, and a 60% cross-reactivity with acetylavermectin, enabling simultaneous recognition of multiple avermectin-like drugs and broadening its application range. Furthermore, the antibody shows no cross-reactivity with other common antibiotics such as tetracycline and gentamicin (cross-reactivity rate <0.1%), ensuring the accuracy of the detection results. This invention fully discloses the amino acid sequences and encoding nucleotide sequences of the antibody's light chain variable region, heavy chain variable region, and complementarity-determining region (CDR), providing clear sequence resources for antibody engineering and detection reagent development in related fields. The colloidal gold immunochromatographic test strip developed based on this antibody is easy to operate, provides rapid detection (5-10 minutes), and delivers visible results. With a detection limit of up to 5 ng / mL, it is suitable for rapid on-site screening and large-scale initial screening of doramectin and avermectin residues in animal-derived foods. In summary, this invention provides a highly sensitive, specific, and sequence-defined technical solution for the rapid detection of doramectin residues, which is suitable for on-site detection and has good application prospects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of the doramectin-BSA artificial antigen used in this invention; Figure 2 The indirect competitive ELISA standard curves (OD450nm as a function of concentration) of doramectin, avermectin, ivermectin, and acetylavermectin provided for this invention. Figure 3 A diagram showing the homology alignment results of the monoclonal antibody heavy chain variable region gene sequence provided by this invention; Figure 4 This is a diagram showing the homology comparison results of the amino acid sequence of the heavy chain variable region of the monoclonal antibody provided by this invention. Figure 5A diagram showing the homology comparison results of the gene sequence of the variable region of the light chain of the monoclonal antibody provided by this invention; Figure 6 A diagram showing the homology comparison results of the amino acid sequence of the variable region of the light chain of the monoclonal antibody provided by this invention. Figure 7 The image shows the specificity test results of the doramectin monoclonal antibody colloidal gold test strip provided by this invention. Detailed Implementation

[0021] The present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to these embodiments. Unless otherwise specified, in the following embodiments, all units are weight units; all raw materials are commercially available raw materials in the art; and all methods are conventional methods in the art.

[0022] Table 1: Nucleotide and amino acid sequences of IGH (heavy chain)

[0023] Table 2: Nucleotide and amino acid sequences of IGK (light chain)

[0024] Example 1 Preparation of doramectin-BSA artificial antigen.

[0025] The doramectin-BSA artificial antigen of the present invention is prepared by the carbodiimide / N-hydroxysuccinimide method (EDC / NHS method), and its structure is as follows: Figure 1 As shown.

[0026] (1) Weigh 10 mg of doramectin and dissolve it in 1 mL of 0.1 M 2-morpholinoethanesulfonic acid (MES) buffer (pH 4.7). Then add 5 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 3 mg of NHS (N-hydroxysuccinimide) in sequence. Stir at room temperature for 30 minutes to activate the solution and obtain an activated doramectin solution.

[0027] (2) Weigh 20 mg of bovine serum albumin (BSA) and dissolve it in 5 mL of 0.01 M phosphate buffer (PBS, pH 7.4) to obtain BSA solution.

[0028] (3) The activated doramectin solution obtained in step (1) is slowly added dropwise to the BSA solution in step (2) under magnetic stirring, and the reaction is stirred overnight (12-16 hours) at 4°C.

[0029] (4) Transfer the reaction solution to a dialysis bag (molecular weight cutoff 14 kDa), place it in 0.01 M PBS (pH 7.4), and dialyze at 4°C for 72 hours, changing the solution every 8 hours. After dialysis, centrifuge the solution in the dialysis bag (5000 rpm, 10 minutes) and collect the supernatant, which is the purified doramectin-BSA artificial antigen.

[0030] (5) The protein concentration and coupling ratio of the conjugate were determined by ultraviolet spectrophotometry, and the conjugate was aliquoted and stored at -20℃ for later use.

[0031] In the above method, the carrier protein can also be replaced with ovalbumin (OVA) to prepare doramectin-OVA coated antigen. The prepared artificial antigen can be verified by ultraviolet scanning, SDS-PAGE electrophoresis, or animal immunization effects.

[0032] Example 2 Preparation of doramectin monoclonal antibody.

[0033] 1. Animal Immunization: Doramectin-BSA immunogen was thoroughly emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites on the back of 6-8 week old female BALB / c mice. The immunization dose per mouse was 50-100 μg. Three weeks later, a second immunization was performed using an equal volume of immunogen emulsified with Freund's incomplete adjuvant. Three weeks after that, a third immunization was performed (without adjuvant, directly injected intraperitoneally). Seven to ten days after the third immunization, tail blood was collected, and serum titer and inhibition rate were detected by indirect ELISA. The results are shown in Table 3. Mice with high titers and good inhibition rates (Mice No. 1, whose serum was still positive after a 160,000-fold dilution and whose inhibition rate against doramectin 100 ppb was greater than 50%) were selected for spleen cell fusion.

[0034] Table 3: Results of serum titer and inhibition rate in mice after triple immunization

[0035] 2. Cell fusion and screening: a. Preparation: Take the spleen from the mouse with the best immune titer (mouse #1) and prepare a spleen cell suspension. At the same time, prepare SP2 / 0 myeloma cells in good growth condition.

[0036] b. Fusion: Spleen cells and myeloma cells are mixed in a ratio of 5:1 to 10:1 and fused under the action of PEG (polyethylene glycol).

[0037] c. Screening: The fused cells were cultured in HAT selective medium. When the hybridoma cell colonies reached 1 / 3-1 / 2 of the bottom of the well, the supernatant was collected and positive wells were screened using an indirect ELISA method. The positive wells were further screened using an indirect competitive ELISA method (coating antigen: doramectin-BSA, competitor: 100 ppb doramectin standard) to determine the inhibition rate. Based on the OD difference and inhibition rate, hybridoma cell lines with superior performance were screened.

[0038] 3. Cloning: The selected positive hybridoma cells (e.g., 2E12D4) are cloned using a limiting dilution method. After 2-3 cloning cycles, until all cloned cell wells show 100% positivity and stable inhibition rate, a monoclonal cell line that can stably secrete monoclonal antibodies is obtained.

[0039] 4. Ascites preparation and purification: 10-12 week old BALB / c mice were sensitized by intraperitoneal injection of liquid paraffin. 7-14 days later, the mice were injected intraperitoneally with 1×10⁻⁶ mol / L of paraffin. 6 -2×10 6 Hybridoma cells were collected. Ascites fluid was collected from mice 7-10 days later. After centrifugation to remove lipids and cell debris, the ascites fluid was purified using a Protein G affinity chromatography column to obtain high-purity doramectin monoclonal antibody.

[0040] Example 3 Cloning and sequence analysis of antibody variable region genes.

[0041] Total RNA extraction and reverse transcription: Hybridoma cells were collected, and total RNA was extracted using Trizol reagent. Using the total RNA as a template, cDNA was synthesized via reverse transcription using Oligo(dT) primers.

[0042] PCR amplification and sequencing: Using universal primers for the variable regions of mouse immunoglobulins, the antibody heavy chain variable region (VH) and light chain variable region (VL) genes were amplified, respectively. The PCR products were recovered and purified, ligated into a T vector, transformed into *E. coli*, and positive clones were screened for sequencing.

[0043] Sequence analysis: a. The obtained nucleotide sequences are shown in SEQ ID NO.30 (light chain variable region gene) and SEQ ID NO.16 (heavy chain variable region gene). The deduced amino acid sequences are shown in SEQ ID NO.23 (light chain variable region) and SEQ ID NO.8 (heavy chain variable region).

[0044] b. Analysis using the IMGT / V-QUEST database: The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.18 (QNIVHSDGITY), CDR2 is KVS, and CDR3 is shown in SEQ ID NO.21 (FQGSHVPYT). The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.2 (GYTFSNHW), CDR2 is shown in SEQ ID NO.4 (IYPGSGHI), and CDR3 is shown in SEQ ID NO.6 (VFGNSYNYAMDY).

[0045] c. Homology comparison analysis Figures 3-6 ): Heavy chain gene sequence ( Figure 3 It showed the highest homology with the variable region of mouse immunoglobulin heavy chain; Heavy chain amino acid sequence ( Figure 4 It showed the highest homology with the variable region of mouse immunoglobulin heavy chain; Light chain gene sequence ( Figure 5 It showed the highest homology with the variable region of the mouse immunoglobulin light chain; Light chain amino acid sequence ( Figure 6 It has the highest homology with the variable region of mouse immunoglobulin light chain.

[0046] The comparison results show that the antibody sequence provided by this invention is highly novel.

[0047] Example 4 Sensitivity and specificity identification of doramectin monoclonal antibody.

[0048] Sensitivity and cross-reactivity were determined using an indirect competitive ELISA method.

[0049] Procedure: Coat the ELISA plate with doramectin-BSA and incubate overnight at 4°C; after washing and blocking, add different concentrations of doramectin, avermectin, ivermectin, acetylavermectin, tetracycline, gentamicin, and other standards, as well as diluted doramectin monoclonal antibody; after incubation at 37°C, add HRP-labeled goat anti-mouse secondary antibody; finally, add TMB substrate for color development and measure the OD450nm value.

[0050] Sensitivity results: For doramectin standards, IC50... 50 The concentration is 3-5 ng / ml. Specific data are shown in Table 4, and the standard curve is shown below. Figure 2 .

[0051] Table 4: Sensitivity Assessment Results (Indirect ELISA)

[0052] Cross-reactivity results: see Table 5.

[0053] Table 5: Results of Cross-Reactivity Identification

[0054] The above results demonstrate that the monoclonal antibody of this invention exhibits extremely high sensitivity (IC50) to doramectin. 50 =3 ng / ml), with good cross-recognition ability with avermectin, ivermectin and acetylavermectin, and no cross-reaction with unrelated drugs such as tetracycline and gentamicin, with good specificity.

[0055] Example 5 Preparation and application of doramectin colloidal gold test strips.

[0056] 1. Preparation of colloidal gold: Take 100 ml of 0.01% chloroauric acid aqueous solution, heat to boiling, quickly add 1.5 ml of 1% trisodium citrate aqueous solution, continue boiling for 15 minutes, and after cooling, obtain a colloidal gold solution with a particle size of about 40 nm.

[0057] 2. Preparation of gold-labeled antibody: Adjust the pH of the colloidal gold solution to 8.2 with 0.1 M K₂CO₃. Under magnetic stirring, add the optimal concentration of doramectin monoclonal antibody dropwise (8-12 μg antibody per mL of colloidal gold). Stir at room temperature for 30 minutes, add 10% BSA to a final concentration of 1%, and block for 15 minutes. Centrifuge at 12000 rpm for 30 minutes, discard the supernatant, and resuspend the precipitate in gold-labeled antibody reconstitution solution (20 mM borate buffer containing 2% BSA, 0.05% PEG20000, and 0.1% NaN₃) to 1 / 10 of the original volume. Store at 4°C.

[0058] 3. Test strip assembly: a. Gold-labeled pad: Spray the above gold-labeled antibody solution evenly onto the glass fiber pad, and freeze-dry or dry at 37°C for 2 hours.

[0059] b. NC membrane: Doramectin-BSA (T line, concentration 1.0 mg / mL) and goat anti-mouse IgG (C line, concentration 1.0 mg / mL) were coated onto the NC membrane using a coating instrument at a rate of 1.0 μL / cm and dried at 37°C for 12 hours.

[0060] c. Assembly: Attach the sample pad, gold label pad, NC membrane, and absorbent pad sequentially to the PVC backing board. Overlap each component by 1-2 mm to ensure smooth chromatography. Cut the strips into 3.05 mm wide strips using a strip cutter, pack them into plastic cartridges, add desiccant, and seal for storage.

[0061] 4. Test strip usage and result interpretation: Take 100 μL of the sample solution to be tested (such as milk or tissue extract) and add it to the sample well of the test strip. Incubate at room temperature for 5-10 minutes.

[0062] Positive: C line shows color, T line does not show color (or the color is lighter than the control line). This indicates that the doramectin residue in the sample is higher than the detection limit.

[0063] Negative: Both the C and T lines show color, with the T line being darker than or equal to the control line. This indicates that there is no doramectin residue in the sample or the residue level is below the detection limit.

[0064] Invalid: If the C line does not develop color, the test strip is invalid regardless of whether the T line develops color.

[0065] Example 6 Performance testing of test strips 1. Sensitivity Test: Doramectin standard solutions of different concentrations (0, 2, 5, 10, 20, 50 ng / mL) were prepared using standard diluent, and the test strips prepared in Example 5 were used for detection. The results showed that the visual detection limit of the test strip for doramectin was 5 ng / mL.

[0066] 2. Specificity test: Doramectin (10 ng / mL), avermectin (10 ng / mL), ivermectin (10 ng / mL), acetylavermectin (10 ng / mL), gentamicin (1000 ng / mL), and chloramphenicol (1000 ng / mL) standard solutions were tested respectively.

[0067] The results are as follows Figure 7 As shown: the T line color of the test strips for doramectin, avermectin, ivermectin, and acetylavermectin is significantly weakened, indicating a positive test; while the T line color of the test strips for gentamicin and chloramphenicol is clear, indicating a negative test.

[0068] The results confirm that the test strip prepared in this invention has good recognition ability for doramectin and avermectin drugs, and has no cross-reaction with unrelated drugs such as gentamicin and chloramphenicol, and has good specificity.

[0069] 3. Stability Test: The test strip was placed in a 37℃ constant temperature incubator for accelerated preservation and tested on days 0, 3, 6, 9, and 12. The results showed that the test strip could be stably stored at 37℃ for at least 6 months, indicating that it has good thermal stability.

[0070] In summary, this invention provides a highly sensitive, highly specific, and sequence-defined doramectin monoclonal antibody, and based on this antibody, a colloidal gold immunochromatographic test strip for rapid on-site detection has been successfully developed, suitable for rapid screening of doramectin and avermectin residues in animal-derived foods.

[0071] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A doramectin monoclonal antibody, characterized in that, The antibody includes a light chain variable region and a heavy chain variable region. The light chain variable region includes three complementarity-determining regions: light chain complementarity-determining region CDR1, light chain complementarity-determining region CDR2, and light chain complementarity-determining region CDR3. The heavy chain variable region includes three complementarity-determining regions: heavy chain complementarity-determining region CDR1, heavy chain complementarity-determining region CDR2, and heavy chain complementarity-determining region CDR3. The amino acid sequence of the light chain complementarity-determining region CDR1 is shown in SEQ ID NO. 18, the amino acid sequence of the light chain complementarity-determining region CDR2 is KVS, and the amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO.

21. The amino acid sequence of the heavy chain complementarity-determining region CDR1 is shown in SEQ ID NO. 2, the amino acid sequence of the heavy chain complementarity-determining region CDR2 is shown in SEQ ID NO. 4, and the amino acid sequence of the heavy chain complementarity-determining region CDR3 is shown in SEQ ID NO.

6.

2. The doramectin monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 23, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

8.

3. The doramectin monoclonal antibody according to claim 2, characterized in that: The nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 30, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.

16.

4. A doramectin test strip, characterized in that, The sample pad includes a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad. The gold-labeled pad is coated with a colloidal gold-labeled doramectin monoclonal antibody as described in any one of claims 1-3. The nitrocellulose membrane has a detection line and a control line. The detection line is coated with a doramectin-carrier protein conjugate, and the control line is coated with goat anti-mouse IgG antibody.

5. The doramectin test strip according to claim 4, characterized in that: The doramectin-carrier protein conjugate is doramectin-BSA or doramectin-OVA.

6. The method for preparing the doramectin test strip as described in claim 4 or 5, characterized in that, Includes the following steps: (1) Preparation of gold-labeled pad: Colloidal gold solution was prepared by the trisodium citrate reduction method, the pH was adjusted to 8.0-8.5, and doramectin monoclonal antibody as described in any one of claims 1-3 was added for labeling. After blocking, centrifugation and resuspension, the gold-labeled antibody solution was sprayed onto the glass fiber pad and dried for later use. (2) Coating of NC membrane: Doramectin-carrier protein conjugate and goat anti-mouse IgG antibody were coated on the detection line and control line of nitrocellulose membrane respectively, and dried for later use; (3) Assembly: The sample pad, gold label pad, NC film and absorbent pad are attached to the backing plate in sequence, overlapping each other, and then cut to the required width.

7. The use of the doramectin monoclonal antibody according to any one of claims 1-3 in the preparation of a kit or biosensor for detecting doramectin residues.

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