Monoclonal antibody against quinofenone and application thereof

Abstract

The application discloses a kind of monoclonal antibody of anti quinofenone and application thereof, belong to immunodetection technical field.The application is quinofenone carboxyl hapten-carrier protein conjugate as immunogen immunizes mouse, by cell fusion and screening, obtain the hybridoma cell strain capable of stable secretion anti quinofenone monoclonal antibody.The amino acid sequence of the light chain and heavy chain variable region of the monoclonal antibody and its coding nucleotide sequence, and each complementarity determining region sequence are completely disclosed in the application.The monoclonal antibody has very high sensitivity to quinofenone, half inhibitory concentration (IC 50 ) It is as low as 0.2 ng / mL, and the cross reactivity of common veterinary drugs such as gentamicin and chloramphenicol is less than 0.1%;It can be used for on-site rapid screening of quinofenone residues in animal-derived food, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of immunochemistry and veterinary drug residue detection technology, specifically relating to a monoclonal antibody against quinolone and its application. Background Technology

[0002] Quinacetone (molecular formula C) 18 H 14 N2O3 belongs to the quinoxaline-1,4-dioxide class of veterinary drugs. It has an inhibitory effect on a variety of intestinal pathogens (especially Gram-negative bacteria), can significantly reduce the incidence of diarrhea in livestock and poultry, promote animal growth and improve feed conversion rate.

[0003] However, with in-depth research, the safety of quinolones and their metabolites has increasingly attracted attention. Quinolones can be metabolized into various products in animals, the main metabolites being desoxyquinacetone and 3-methylquinoxaloline-2-carboxylic acid (MQCA). Toxicological studies have shown that quinoxaloline-1,4-dioxide compounds have potential mutagenicity and genotoxicity, and their mechanism of action is closely related to the metabolic reduction process of the N→O group on the parent nucleus in vivo. Due to insufficient understanding of the physicochemical properties and pharmacological and toxicological characteristics of drugs among livestock producers, there are instances of irrational use and even abuse in the breeding process, leading to frequent occurrences of excessive residues of quinolones and their metabolites in animal-derived foods (such as pork, chicken, liver, and kidneys). These residues can enter the human body through the food chain, posing a potential threat to consumer health.

[0004] Currently, methods for detecting quinolones and their metabolites mainly include instrumental analytical methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). For example, Chinese patent CN103342683A discloses a quinolone hapten and its preparation method, which can be used to prepare antibodies against quinolones and their metabolites, but it mainly relies on traditional chromatography-mass spectrometry for detection. Although instrumental analytical methods have high accuracy and sensitivity, they usually require expensive equipment, professional operators, complex sample pretreatment procedures, and long detection cycles, making it difficult to meet the needs of rapid on-site screening of large batches of samples.

[0005] Immunological detection methods, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography, have advantages such as simple operation, rapid detection, low cost, and suitability for large-scale on-site screening, and have become an important supplementary means for veterinary drug residue detection. The core reagent of immunoassay methods is the specific antibody that recognizes the target analyte; the sensitivity and specificity of the antibody directly determine the performance of the detection method. Chinese patent CN104034888A discloses an ELISA kit for detecting quinolone, which uses a conjugate of the quinolone metabolite MQCA and a carrier protein as an immunogen to prepare a quinolone-specific polyclonal antibody. However, the polyclonal antibody used in this kit is derived from the serum of immunized animals, resulting in large batch-to-batch variability, inability to maintain a stable supply, and low half-inhibitory concentration (IC50). 50 The concentration is only about 5 ng / mL, indicating significant room for improvement in both sensitivity and specificity. Furthermore, none of the aforementioned existing technologies disclose the amino acid or nucleotide sequence information of the antibody, limiting further engineering modifications and optimization of detection methods.

[0006] Monoclonal antibodies have attracted increasing attention in the field of immunoassay due to their advantages such as single source, high specificity, high affinity, and the ability to be stably produced in unlimited quantities. Currently, there are few reports on monoclonal antibodies against quinolone, especially lacking complete publicly available antibody sequence information. On the other hand, another technical challenge in quinolone detection is that quinolone is metabolized into multiple products in animals; an ideal detection antibody should be able to simultaneously recognize the quinolone parent drug and its major metabolites to improve detection coverage and accuracy.

[0007] In summary, there is an urgent need in this field to develop an anti-quinolone monoclonal antibody with higher sensitivity, stronger specificity, a well-defined antibody sequence, and stable production capability, and to develop an immunological detection product based on this antibody suitable for rapid on-site detection, so as to meet the actual needs of quinolone residue monitoring in animal-derived foods. Summary of the Invention

[0008] To address the problems of existing quinolone detection antibodies, which are mainly polyclonal antibodies with insufficient sensitivity, lack of clear sequence information, and difficulty in stable production, this invention provides a monoclonal antibody against quinolone and its applications. This invention uses a self-designed quinolone carboxyl hapten (QCT-COOH) and a carrier protein conjugate as an immunogen. Hybridoma cell lines that stably secrete anti-quinolone monoclonal antibodies are obtained through hybridoma technology screening. The variable region sequence and complementarity-determining region (CDR) sequence of this antibody are fully disclosed. Based on this antibody, those skilled in the art can further develop detection reagents, kits, and colloidal gold immunochromatographic test strips for rapid and sensitive detection of quinolone residues in animal-derived foods.

[0009] In a first aspect, the present invention provides a monoclonal antibody against quinolone, which is prepared by immunizing animals with a quinolone carboxyl hapten-carrier protein conjugate and is capable of specifically recognizing quinolone and its major metabolites.

[0010] Furthermore, the anti-quinolone monoclonal 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.

[0011] Specifically:

[0012] The amino acid sequence of the light chain complementarity-determining region CDR1 is shown in SEQ ID NO. 18;

[0013] The amino acid sequence of the light chain complementarity-determining region CDR2 is EAS;

[0014] The amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO. 21;

[0015] The amino acid sequence of the heavy chain complementarity-determining region CDR1 is shown in SEQ ID NO. 2;

[0016] The amino acid sequence of the heavy chain complementarity-determining region CDR2 is shown in SEQ ID NO. 4;

[0017] The amino acid sequence of the heavy chain complementarity-determining region CDR3 is shown in SEQ ID NO. 6.

[0018] 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.

[0019] 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.

[0020] Secondly, the present invention provides a method for preparing the above-mentioned anti-quinolone monoclonal antibody, comprising the following steps:

[0021] (1) Animal immunization: Balb / c mice were immunized with quinolone carboxyl hapten-carrier protein conjugate as an immunogen;

[0022] (2) Cell fusion: Spleen cells from immunized mice were fused with myeloma cells SP2 / 0;

[0023] (3) Hybridoma screening: Indirect ELISA and competitive ELISA were used to screen for positive hybridoma cell lines that could secrete anti-quinolone monoclonal antibodies;

[0024] (4) Cloning: positive hybridoma cells are cloned by limiting dilution to obtain hybridoma cell lines that stably secrete monoclonal antibodies;

[0025] (5) Ascites preparation and purification: Hybridoma cells were injected into the peritoneal cavity of mice to prepare ascites, and monoclonal antibodies were obtained by affinity chromatography purification.

[0026] Preferably, the carrier protein is bovine serum albumin (BSA) or ovalbumin (OVA).

[0027] Preferably, the quinone carboxyl hapten is 2-(3-(4-carboxyphenyl)acryloyl)-3-methylquinoxaline-1,4-dioxide (QCT-COOH).

[0028] Thirdly, the present invention provides the application of the above-mentioned anti-quinolone monoclonal antibody in the detection of quinolone and its metabolites.

[0029] Specifically, a reagent for detecting quinolone comprises the antiquinolone monoclonal antibody described in this invention.

[0030] Furthermore, a kit for detecting quinolone includes the anti-quinolone monoclonal antibody described in this invention. The kit may be an enzyme-linked immunosorbent assay (ELISA) kit, a chemiluminescent immunoassay kit, or a fluorescent immunoassay kit, etc.

[0031] Fourthly, the present invention provides the application of the above-mentioned anti-quinolone monoclonal antibody in the preparation of detection reagents or kits for detecting quinolone and its metabolites.

[0032] Fifthly, the present invention provides a method for detecting quinolone in a sample, comprising the step of performing an immunoassay on the sample using the anti-quinolone monoclonal antibody described in this invention. The method includes, but is not limited to, enzyme-linked immunosorbent assay (ELISA), immunochromatography, and immunosensor methods.

[0033] The beneficial effects of this invention are:

[0034] (1) The half-maximal inhibitory concentration (IC50) of the monoclonal antibody provided by this invention against quinolone 50 With a detection rate as low as 0.2 ng / mL, its sensitivity is significantly better than that of polyclonal antibodies in existing technologies (approximately 5 ng / mL), representing an improvement of about 25 times, and can meet more stringent requirements for veterinary drug residue detection.

[0035] (2) The antibody of the present invention has high specificity for quinolone and cross-reactivity with common veterinary drugs such as gentamicin and chloramphenicol is less than 0.1%, which can effectively avoid false positive results and ensure detection accuracy.

[0036] (3) This invention fully discloses the amino acid sequence and encoding nucleotide sequence of the light chain variable region, heavy chain variable region and complementarity-determining region (CDR) of the antibody, providing clear sequence resources for those skilled in the art to carry out antibody engineering, humanization, affinity maturation and detection method optimization, and filling the gap in the lack of sequence information of quinolone monoclonal antibodies in the prior art.

[0037] (4) The monoclonal antibody of the present invention is derived from hybridoma cells and can be produced stably in unlimited quantities, overcoming the defects of large batch-to-batch differences and unstable supply of polyclonal antibodies, and has good prospects for industrial application.

[0038] In summary, this invention provides a highly sensitive, specific, well-defined, stably produced, and on-site detection technical solution for the rapid detection of quinolone residues. This is of great practical significance for ensuring the safety of animal-derived food and implementing veterinary drug residue monitoring. Attached Figure Description

[0039] Figure 1 This is a synthetic route diagram for the quinone hapten QCT-COOH of the present invention;

[0040] Figure 2 This is a synthetic route diagram for the quinone complete antigen (QCT-COOH-carrier protein conjugate) of the present invention;

[0041] Figure 3 This is a diagram showing the homology comparison results of the heavy chain variable region gene sequence of the anti-quinolone monoclonal antibody of this invention;

[0042] Figure 4 This is a diagram showing the homology comparison results of the amino acid sequence of the heavy chain variable region of the anti-quinolone monoclonal antibody of this invention;

[0043] Figure 5 This is a diagram showing the homology comparison results of the light chain variable region gene sequence of the anti-quinolone monoclonal antibody of this invention;

[0044] Figure 6 This is a diagram showing the homology comparison results of the amino acid sequence of the light chain variable region of the anti-quinolone monoclonal antibody of this invention;

[0045] Figure 7 This is a standard ELISA inhibition curve of the anti-quinolone monoclonal antibody of the present invention against quinolone (the horizontal axis is the concentration of quinolone, and the vertical axis is the OD450nm value). Detailed Implementation

[0046] 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.

[0047] The sequences involved in this invention are shown in Tables 1 and 2 below.

[0048] Table 1: Nucleotide and amino acid sequences of IGH (heavy chain)

[0049] FR1 1 QVQLQQPGAEVVKPGASVKLSCKAS 9 CAGGTCCAACTCCAGCAGCCTGGGGCTGAAGTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCT CDR1 2 GYTFTSYW 10 GGCTACACCTTCACCAGCTACTGG FR2 3 MHWVKLRPGQGFEWIGE 11 ATGCACTGGGTGAAGCTGAGGCCTGGACAAGGCTTTGAGTGGATTGGAGAG CDR2 4 INPSNGGT 12 ATTAATCCTAGCAATGGTGGTACT FR3 5 NYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYC 13 AACTACAATGAGAAGTTCAAGAGAAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGT CDR3 6 TPSYYAPFAY 14 ACGCCCTCGTACTACGCCCCGTTTGCTTAC FR4 7 WGQGTLVTVSA 15 TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA V(D)J-IMGT 8 QVQLQQPGAEVVKPGASVKLSCKASGYTFTSYWMHWVKLRPGQGFEWIGEINPSNGGTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTPSYYAPFAYWGQGTLVTVSA 16 CAGGTCCAACTCCAGCAGCCTGGGGCTGAAGTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCTGAGGCCTGGACAAGGCTTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGAAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACGCCCTCGTACTACGCCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0050] Table 2: Nucleotide and amino acid sequences of IGK (light chain)

[0051] FR1 17 DIQMNQSPSSLSASLGDTITITCHAS 24 GACATCCAGATGAATCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGT CDR1 18 QNINVW 25 CAGAACATTAATGTTTGG FR2 19 LSWYQEKPGNFPKLLIY 26 TTAAGCTGGTACCAGGAGAAACCAGGAAATTTTCCTAAACTATTGATCTAT CDR2 — (Note 1) EAS — (Note 2) GAGGCTTCC FR3 20 NLHTGVPSRFSGSGSGTGFTLTISSLRPEDIATYYC 27 AACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTGACCATCAGCAGCCTGCGGCCTGAAGACATTGCCACTTACTACTGT CDR3 21 QQGQSLPYT 28 CAACAGGGTCAAAGTCTTCCGTACACG FR4 22 FGGGTKLEVR 29 TTCGGAGGGGGGACCAAGCTGGAAGTAAGA V(D)J-IMGT 23 DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQEKPGNFPKLLIYEASNLHTGVPSRFSGSGSGTGFTLTISSLRPEDIATYYCQQGQSLPYTFGGGTKLEVR 30 GACATCCAGATGAATCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTAAGCTGGTACCAGGAGAAACCAGGAAATTTTCCTAAACTATTGATCTATGAGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTGACCATCAGCAGCCTGCGGCCTGAAGACATTGCCACTTACTACTGTCAACAGGGTCAAAGTCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAGTAAGA

[0052] Note 1: The amino acid sequence of the light chain complementarity-determining region CDR2 is EAS (3 amino acids). Since it is less than 4 amino acids, it is not assigned a SEQ ID NO according to the patent sequence naming convention.

[0053] Note 2: The nucleotide sequence encoding the light chain complementarity-determining region CDR2 is GAGGCTTCC (9 nucleotides). Since it is less than 10 nucleotides, it is not assigned a SEQ ID NO according to the patent sequence naming convention.

[0054] Example 1:

[0055] Synthesis of the quinone hapten QCT-COOH.

[0056] The synthetic route for the quinone hapten QCT-COOH (2-(3-(4-carboxyphenyl)acryloyl)-3-methylquinoxaline-1,4-dioxide) is as follows: Figure 1 As shown, the specific synthesis steps are as follows:

[0057] In a clean, dry, single-necked round-bottom flask, add 2.18 g (10 mmol) of 2-acetyl-3-methylquinoxaline-1,4-dioxide (acetylmethylquinoxaline), followed by 1.50 g (10 mmol) of p-formylbenzoic acid, then inject 30-40 mL of anhydrous methanol, turn on the magnetic stirrer, and add 1-2 mL of diethylamine very slowly with a dropper. Reflux the reaction at 65-70°C for 4-8 hours.

[0058] After the reaction is complete, stop heating and cool the flask to room temperature. Slowly add a few drops of glacial acetic acid to adjust the pH of the solution to approximately 5-6. Place the mixture in a refrigerator at 4°C and allow it to crystallize for 12 hours. Filter under reduced pressure using a Buchner funnel and collect the filter cake. Wash the filter cake 2-3 times with a small amount of cold methanol to remove unreacted raw materials and byproducts. Finally, wash once with a small amount of methyl tert-butyl ether. Place the purified yellow solid in a vacuum drying oven and dry under reduced pressure at 40°C in the dark for 12 hours to obtain the quinone hapten QCT-COOH.

[0059] The obtained hapten was identified by mass spectrometry and nuclear magnetic resonance, and its structure was correct with a purity of 96.5% and a yield of 67%.

[0060] Example 2:

[0061] Preparation of quinone complete antigen.

[0062] In this embodiment, quinolone immunogen (QCT-COOH-BSA) and coating antigen (QCT-COOH-OVA) were prepared respectively, and the synthetic routes are as follows: Figure 2 As shown.

[0063] Accurately weigh 10 mg of quinone carboxylated hapten (QCT-COOH) and dissolve it in 1 mL of anhydrous DMF, ensuring complete dissolution. Add 15 mg of EDC·HCl and 10 mg of NHS sequentially. Incubate at room temperature with magnetic stirring in the dark for 4–6 hours to complete the activation of QCT-COOH.

[0064] Accurately weigh 20 mg of BSA (or OVA) and dissolve it in 3 mL of 0.01 M PBS (pH 7.4) buffer. Stir slowly at 4°C until dissolved. Add the activated QCT-COOH solution very slowly to the protein solution. After the addition is complete, stir slowly at 4°C in the dark for 12 hours. Transfer the reaction solution to a prepared dialysis bag and dialyze with 0.01 M PBS (pH 7.4) at 4°C for 2 days, changing the dialysate 3-4 times daily.

[0065] After dialysis, the solution in the dialysis bag was collected. The solution was centrifuged at 10,000 r / min for 15 minutes at 4°C, and the supernatant was collected. The purified complete antigen (supernatant) was aliquoted and stored at -20°C for later use. The protein concentration and conjugation ratio of the conjugates were determined by ultraviolet spectrophotometry. The conjugation ratio of QCT-COOH-BSA was 12:1, and the conjugation ratio of QCT-COOH-OVA was 8:1.

[0066] Example 3:

[0067] Animal immunization and serum titer detection.

[0068] Using QCT-COOH-BSA as the immunogen, 6-8 week old female Balb / c mice were immunized. The immunogen was thoroughly emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites on the back of each mouse, with an immunization dose of 50-100 μg. Three weeks later, a second immunization was performed using an equal volume of the 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 titers and inhibition rates were detected using an indirect ELISA method.

[0069] The detection method is as follows:

[0070] (1) Coating: Dilute the QCT-COOH-OVA coating antigen to an appropriate concentration with carbonate buffer (pH 9.6), add 100 μL / well to the microplate, and incubate overnight at 4°C;

[0071] (2) Washing and blocking: Discard the liquid in the wells and wash three times with PBST. Add 200 μL of blocking buffer (PBS containing 1% BSA) to each well and block at 37°C for 1 hour;

[0072] (3) Sample addition: The mouse serum was serially diluted from 1:5000. 50 μL of serum diluent and 50 μL of quinolone standard (final concentration 50 ppb) or PBS blank control were added to each well. The mixture was reacted at 37°C for 1 hour and washed 3 times.

[0073] (4) Add secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) to each well, react at 37℃ for 1 hour, and wash 3 times;

[0074] (5) Color development and termination: Add 100 μL of TMB substrate color development solution to each well, develop color at 37℃ in the dark for 15 minutes, and add 50 μL of 0.5 M H2SO4 to terminate the reaction;

[0075] (6) Measurement: The OD value of each well at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibition rate was calculated (inhibition rate % = (1 - OD value of standard well / OD value of blank control well) × 100%).

[0076] The test results are shown in Table 3.

[0077] Table 3: Serum titer and inhibition detection in immunized mice.

[0078] 1:5000 1.7898 1.0896 39% 1:10000 1.6257 0.9726 40% 1:20000 1.2783 0.6836 47% 1:40000 0.9310 0.4473 52% 1:80000 0.7066 0.2467 65% 1:160000 0.4903 0.2162 56% 1:320000 0.3290 0.1690 — NC 0.0807 — —

[0079] Note: Inhibition rate at a dilution of 1:320000 was not calculated (indicated by "—"); NC is the negative control, and its OD value has been adjusted for background, "—" indicates that it is not applicable.

[0080] The results showed that the serum titer of the immunized mice was high and that it had a significant inhibitory effect on quinolone. The mice with high titer and good inhibition rate (#1) were selected for spleen cell fusion.

[0081] Example 4:

[0082] Cell fusion and hybridoma screening.

[0083] The spleen of the mouse with the best immunogenicity in Example 3 (#1) was removed under aseptic conditions, and a spleen cell suspension was prepared. Simultaneously, SP2 / 0 myeloma cells in good growth condition were prepared. Spleen cells and myeloma cells were mixed at a ratio of 5:1 to 10:1 and fused under PEG (polyethylene glycol, molecular weight 1500) mediation. The fused cells were resuspended in HAT selective medium, aliquoted into 96-well cell culture plates, and cultured in a 37°C, 5% CO2 incubator.

[0084] Seven to ten days after fusion, when the hybridoma cell colonies had grown to 1 / 3 to 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 subjected to inhibition rate determination using an indirect competitive ELISA method: the coating antigen was QCT-COOH-OVA (1 μg / mL), and the competitor was 1 ppb quinolone standard; the specific procedure was the same as in Example 3. Based on the OD difference and inhibition rate, hybridoma cell lines with superior performance were screened. The results are shown in Table 4.

[0085] Table 4: Titer and inhibition detection of fused monoclonal cells.

[0086] 2G4E11 1.7224 0.8558 50% 3G4G1 1.5703 0.6641 58% 7H12F7 1.7224 0.4273 75% 9A2C1 1.3523 0.4559 66% 9H11B7 1.4388 0.6101 58% 10C12A7 1.2728 0.5039 60%

[0087] The results showed that clone number 7H12F7 had the highest inhibition rate (75%) and a high titer, and cells from this well were selected for cloning.

[0088] Example 5

[0089] Preparation and purification of monoclonal antibodies.

[0090] The positive hybridoma cells (7H12F7) selected in Example 4 were cloned using a limiting dilution method. After counting the cells, the suspension was diluted with complete culture medium to a concentration of 5-10 cells per milliliter, and seeded at 100 μL / well in 96-well plates. After 7-10 days of culture, the supernatant from each well was collected for ELISA detection. The wells with the highest positive results and inhibition rates were selected for the next round of cloning. After 2-3 rounds of cloning, until all cloned cell wells showed 100% positive results and stable inhibition rates, a monoclonal cell line that stably secreted anti-quinolone monoclonal antibodies was obtained and named 7H12F7.

[0091] 10-12 week old Balb / c mice were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin. 7-14 days later, the mice were injected intraperitoneally with 1×10⁻⁶ mol / L of liquid paraffin. 6 -2×10 6 7H12F7 hybridoma cells were collected. Ascites fluid was collected from mice 7-10 days later. After centrifugation at 3000 rpm for 10 minutes to remove lipids and cell debris, the ascites fluid was purified using a Protein G affinity chromatography column. The specific procedure was as follows: the ascites fluid supernatant was diluted with binding buffer (20 mM sodium phosphate, pH 7.0) and loaded onto a Protein G affinity column. The column was washed with binding buffer until the baseline stabilized. The antibody was eluted with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected and immediately neutralized to pH 7.4 with 1 M Tris-HCl (pH 9.0). The eluent was dialyzed against PBS overnight to obtain purified anti-quinolone monoclonal antibody. Antibody purity was assessed by SDS-PAGE (>95%), and antibody concentration was determined by the BCA method.

[0092] Example 6:

[0093] Total RNA extraction, reverse transcription, antibody variable region gene amplification, and sequencing.

[0094] 7H12F7 hybridoma cells from Example 5 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.

[0095] Using universal primers for the variable regions of mouse immunoglobulins, the heavy chain variable region (VH) and light chain variable region (VL) genes of antibodies were amplified by PCR. The PCR products were recovered and purified, ligated into a T-vector, transformed into *E. coli*, and positive clones were screened for sequencing.

[0096] Sequence analysis:

[0097] a. The obtained nucleotide sequences are shown in SEQ ID NO. 30 (light chain) and SEQ ID NO. 16 (heavy chain). The deduced amino acid sequences are shown in SEQ ID NO. 23 (light chain) and SEQ ID NO. 8 (heavy chain).

[0098] b. Based on the Kabat numbering system, the complementarity determination regions (CDRs) are divided as follows: for light chains, CDR1 is SEQ ID NO. 18, CDR2 is EAS, and CDR3 is SEQ ID NO. 21; for heavy chains, CDR1 is SEQ ID NO. 2, CDR2 is SEQ ID NO. 4, and CDR3 is SEQ ID NO. 6.

[0099] c. Sequence alignment analysis:

[0100] The antibody gene sequence of this invention was compared with mouse immunoglobulin sequences in public databases such as NCBI. The heavy chain gene sequence alignment results are as follows: Figure 3 As shown, the heavy chain amino acid sequence alignment results are as follows: Figure 4 As shown, the light chain gene sequence alignment results are as follows: Figure 5 As shown, the light chain amino acid sequence alignment results are as follows: Figure 6 As shown. The comparison results show that the heavy chain and light chain variable region sequences of the antibody of the present invention are different from the mouse immunoglobulin sequences published in the database, and its CDR region sequence is unique, indicating that the antibody sequence provided by the present invention is novel.

[0101] Example 7:

[0102] Identification of the sensitivity of monoclonal antibodies.

[0103] The sensitivity of the purified monoclonal antibody from Example 5 was determined using an indirect competitive ELISA method.

[0104] Operating steps:

[0105] (1) Coating: Dilute the QCT-COOH-OVA coating antigen to 1 μg / mL with carbonate buffer (pH 9.6), add 100 μL / well to the microplate, and coat overnight at 4°C;

[0106] (2) Washing and blocking: Discard the liquid in the wells, wash 3 times with PBST, add 200 μL of blocking solution (PBS containing 1% BSA) to each well, and block at 37°C for 1 hour;

[0107] (3) Sample addition: Dilute the quinolone standard with PBS to a series of concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 ng / mL), add 50 μL of standard solution and 50 μL of diluted antiquinolone monoclonal antibody (working concentration determined by checkerboard titration to be 0.125 μg / mL) to each well, react at 37℃ for 1 hour, and wash 3 times;

[0108] (4) Add secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) to each well, react at 37℃ for 1 hour, and wash 3 times;

[0109] (5) Color development and measurement: Add 100 μL of TMB substrate color development solution to each well, incubate at 37℃ in the dark for 15 minutes, then add 50 μL of 0.5 M H2SO4 to stop the reaction, and measure the OD using a microplate reader. 450 nm value, calculate inhibition rate (inhibition rate % = (1 - OD value of standard well / OD value of blank control well) × 100%).

[0110] The test results are shown in Table 5.

[0111] Table 5: Results of Sensitivity Assessment of Indirect ELISA

[0112] 0 1.4163 0.00% 0.1 1.0136 28.43% 0.2 0.7048 50.23% 0.4 0.5247 62.95% 0.8 0.4132 70.82% 1.6 0.2753 80.56% 3.2 0.1669 88.22%

[0113] Plot the standard curve based on the data in Table 5. Figure 7 The half-maximal inhibitory concentration (IC50) of the monoclonal antibody against quinolone was calculated. 50 The concentration was 0.2 ng / mL, indicating that the monoclonal antibody of the present invention has extremely high sensitivity.

[0114] Example 8:

[0115] Monoclonal antibody specificity identification.

[0116] The specificity of the purified monoclonal antibody from Example 5 was identified using an indirect competitive ELISA method. Quinolone was used as a standard, and gentamicin and chloramphenicol were selected as interfering agents for cross-reactivity testing. The specific procedure was the same as in Example 7, with the detection concentrations of each drug being: quinolone 0-3.2 ng / mL, gentamicin 0.1-1000 ng / mL, and chloramphenicol 0.1-1000 ng / mL. The half-maximal inhibitory concentration (IC50) of each drug was determined. 50 ), calculate the cross-reactivity rate (cross-reactivity rate % = (quinolone IC50) 50 / IC of the drug to be tested 50 (×100%)

[0117] The test results are shown in Table 6.

[0118] Table 6: Results of Indirect ELISA Cross-Reactivity Identification

[0119] Quinocetone 0.2 100% Gentamicin >1000 <0.1% Chloramphenicol >1000 <0.1%

[0120] The results showed that the monoclonal antibody of the present invention has high specificity for quinolone, and the cross-reactivity rate with gentamicin and chloramphenicol is less than 0.1%, indicating that the antibody can effectively avoid interference from other common veterinary drugs and ensure the accuracy of the test results.

[0121] 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 monoclonal antibody against quinolone, 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 EAS, 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 monoclonal antibody against quinolone 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 monoclonal antibody against quinolone 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 reagent for detecting quinones, characterized in that, A monoclonal antibody comprising the antiquinolone as described in any one of claims 1-3.

5. A kit for detecting quinolone, characterized in that, A monoclonal antibody comprising the antiquinolone as described in any one of claims 1-3.

6. The use of the antiquinolone monoclonal antibody as described in any one of claims 1-3 in the preparation of a detection reagent or kit for detecting quinolone and its metabolites.

7. A method for detecting quinolone in a sample, characterized in that, The method includes the step of performing an immunoassay on a sample using the antiquinolone monoclonal antibody as described in any one of claims 1-3.

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