A tetracycline hapten shs, artificial antigen, antibody and preparation method and application thereof

Abstract

The application discloses a tetracycline hapten SHS, an artificial antigen, an antibody and a preparation method and application thereof. The tetracycline hapten SHS is provided firstly, and then the artificial antigen obtained by coupling the tetracycline hapten SHS with hemocyanin is used as an immunogen to immunize mice, so that a monoclonal antibody is obtained. The titer, broad spectrum and affinity of the obtained antibody are good, the minimum detection limits (LOD) of the antibody to tetracycline, terramycin and aureomycin are 3.16 ng / mL, 2.37 ng / mL and 0.42 ng / mL respectively, the antibody has the characteristics of strong broad spectrum, wide linear range and high sensitivity; the monoclonal antibody has high organic solvent tolerance, the detection process can be carried out in a solution containing a certain concentration of organic solvent, the detection sensitivity is improved and the sample pretreatment operation is simplified, and the monoclonal antibody has good application prospect and wide development space in simple, rapid, accurate and effective detection of tetracycline drug residues.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. More specifically, it relates to a tetracycline hapten SHS, an artificial antigen, an antibody, a method for preparing the same, and their applications. Background Technology

[0002] Tetracycline antibiotics (TCS) are a class of broad-spectrum antibiotics produced by actinomycetes, including tetracycline, oxytetracycline, chlortetracycline, and semi-synthetic derivatives such as doxycycline. At high concentrations, they have bactericidal effects and are widely used in edible animals as veterinary drugs for the prevention and treatment of livestock and poultry diseases or as feed additives to promote livestock and poultry growth. However, due to mismanagement, improper use, or failure to comply with withdrawal period regulations, residues can occur in livestock products. According to the National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs in Food (GB 31650-2019), the maximum residue limit for tetracycline (residue markers are oxytetracycline, chlortetracycline, and single or combined tetracycline) in aquatic products is 200 μg / kg to 1200 μg / kg. However, in the process of livestock and poultry farming, the practice of illegally increasing drug dosages or failing to comply with withdrawal period regulations in order to quickly control diseases continues to occur, directly resulting in drug residues in sold products not being degraded to below the standard limits. Therefore, a rapid, sensitive, and efficient detection method is needed to achieve rapid detection of tetracycline / oxytetracycline / chlortetracycline.

[0003] Common methods for detecting tetracycline / oxytetracycline / chlortetracycline include instrumental analysis methods such as high-performance liquid chromatography (HPLC), fluorescence spectroscopy, gas chromatography (GC), ultraviolet spectroscopy (UV GC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, instrumental analysis methods are unsuitable for large-scale sample screening due to their cumbersome sample pretreatment and measurement processes, high cost, and the need for professional personnel. Enzyme-linked immunosorbent assay (ELISA), on the other hand, offers advantages such as high sensitivity, strong specificity, low cost, simple operation, fast speed, large sample volume per test, low instrument requirements, and relatively simple sample pretreatment. It is suitable for market monitoring and on-site surveillance. Sensitivity is the lowest concentration of the target molecule in a sample that the detection method can reliably detect, i.e., the limit of detection (LOD). Immunoassays based on antigen-antibody specific binding mainly use monoclonal and polyclonal antibodies. For immunoassays, antibodies are the core raw material, and the effectiveness of antibodies largely depends on the antigen structure that induces an immune response in the corresponding animal. Furthermore, ELISA testing often requires sample pretreatment with organic solvents due to the complex matrix components such as lipids and sugars in the samples. However, the introduction of non-physiological conditions like organic solvents during pretreatment can reduce the recognition activity of antibodies intolerant to these solvents, affecting the interpretation of test results. Therefore, organic solvents must be removed using methods such as nitrogen blowing, which accounts for approximately 60%–70% of the entire analytical process and is time-consuming. Therefore, providing antibodies capable of immunoreaction in solutions containing a certain concentration of organic solvents can improve detection accuracy and further simplify sample pretreatment. Thus, providing a method for the rapid, sensitive, and accurate detection of tetracycline / oxytetracycline / chlortetracycline is of great significance, as it allows for the preparation of broad-spectrum antibodies resistant to organic solvents. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a tetracycline hapten SHS, an artificial antigen, an antibody, and their preparation methods and applications.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] This invention first provides a tetracycline hapten SHS, with the structural formula shown in formula (Ⅰ):

[0007]

[0008] The present invention also provides a method for preparing the tetracycline hapten SHS, wherein the method uses the phenolic hydroxyl group on the tetracycline as the arm extension site, and introduces the carboxylic acid arm by a substitution reaction between the phenolic hydroxyl group and 6-bromohexanoic acid.

[0009] The tetracycline hapten SHS of this invention is obtained by using the phenolic hydroxyl group on tetracycline as an arm extension site, and introducing a carboxylic acid-containing arm through a substitution reaction between the phenolic hydroxyl group and 6-bromohexanoic acid. Since the benzene ring in tetracycline has a rigid structure, introducing an arm through the hydroxyl group on the benzene ring can expose the characteristic structures of all tetracycline drugs, thus enabling broader detection of tetracycline drugs. Furthermore, the carbon chain length of the introduced linker arm is suitable, and the steric hindrance of the carrier protein does not affect the exposure of the hapten's characteristic structure, while the immune system's recognition of the hapten's characteristic structure remains unaffected. Therefore, this hapten structure can effectively prepare broad-spectrum antibodies with high affinity and strong specificity.

[0010] Furthermore, the preparation method includes the following steps:

[0011] S1. Dissolve tetracycline and triethylamine in an organic solvent, stir, and then slowly add 6-bromohexanoic acid dissolved in the organic solvent. Stir, heat, and react.

[0012] S2. Extract the organic phase after the reaction is complete;

[0013] S3. After mixing the extracted organic phases, separate and purify them. The resulting white oily substance is the tetracycline artificial antigen SHS.

[0014] Preferably, the organic solvent in step S1 is DMF.

[0015] Preferably, the molar ratio of tetracycline, triethylamine, and 6-bromohexanoic acid in step S1 is 1:2:2.

[0016] Preferably, the heating in step S1 is heating to 80°C.

[0017] Preferably, the reaction in step S1 is carried out for 24 hours.

[0018] Preferably, the extraction in step S2 is performed using saturated brine and ethyl acetate.

[0019] Preferably, the separation and purification in step S3 is carried out by vacuum distillation.

[0020] The present invention also provides the use of the tetracycline hapten SHS in the preparation of tetracycline artificial antigens or antibodies.

[0021] Furthermore, the preparation of the tetracycline artificial antigen involves coupling the carboxyl terminus of the tetracycline hapten SHS with a carrier protein to obtain the artificial antigen.

[0022] This invention provides a tetracycline artificial antigen, obtained by coupling the aforementioned tetracycline hapten SHS with a carrier protein, and its structural formula is shown in formula (II):

[0023] Furthermore, the carrier protein is ovalbumin (OVA) or keyhole limpet hemocyanin (KLH).

[0024] This invention provides the application of the aforementioned tetracycline artificial antigen in the preparation of tetracycline antibodies.

[0025] Furthermore, the antibody is a monoclonal antibody, a polyclonal antibody, or a genetically engineered antibody.

[0026] Preferably, the antibody is a monoclonal antibody.

[0027] This invention provides a monoclonal antibody against tetracycline antibiotics, which is prepared by using the aforementioned tetracycline artificial antigen as an immunogen.

[0028] Furthermore, the tetracycline antibiotic is one or more of tetracycline, oxytetracycline, and chlortetracycline.

[0029] Furthermore, the tetracycline artificial antigen is obtained by conjugating the tetracycline hapten SHS with hemocyanin.

[0030] Furthermore, the method for preparing the anti-tetracycline monoclonal antibody is as follows: using the above-mentioned tetracycline artificial antigen SHS-KLH as an immunogen to immunize mice, fusing mouse spleen cells with SP2 / 0 myeloma cells to obtain hybridoma cells, inoculating the hybridoma cells into female Balb / c mice for culture, obtaining ascites containing a high concentration of monoclonal antibody, and purifying the ascites to prepare the anti-tetracycline monoclonal antibody.

[0031] The present invention also provides a monoclonal antibody against tetracycline antibiotics (2C8A), the amino acid sequence of which is shown in SEQ ID No.1 and the amino acid sequence of which is shown in SEQ ID No.2.

[0032] Specifically, the monoclonal antibody 2C8A includes four framework regions (FR1, FR2, FR3, FR4) and three complementarity-determining regions (CDR1, CDR2, CDR3) in the light chain variable region, and the four framework regions and three complementarity-determining regions in the heavy chain variable region are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0033] The present invention also provides a gene encoding the monoclonal antibody, the nucleotide sequence of which is shown in SEQ ID No. 3 and the nucleotide sequence of which is shown in SEQ ID No. 4.

[0034] Since the present invention has provided the amino acid sequence of the monoclonal antibody 2C8A and the gene sequence encoding the monoclonal antibody, those skilled in the art can obtain the monoclonal antibody described in this application using known recombinant DNA techniques. Therefore, any recombinant vectors or recombinant cells that can be used to prepare the monoclonal antibody described in this invention should also be within the scope of protection of this invention.

[0035] This invention provides the application of any of the above-described monoclonal antibodies in the detection of tetracycline antibiotics.

[0036] Furthermore, the tetracycline antibiotic is one or more of tetracycline, oxytetracycline, and chlortetracycline.

[0037] The present invention also provides a method for detecting tetracycline antibiotics, using the above-mentioned tetracycline artificial antigen as a coating antigen and any of the above-mentioned anti-tetracycline antibiotic monoclonal antibodies as detection antibodies; the carrier protein on the coating antigen is chicken ovalbumin.

[0038] Furthermore, the detection is performed using an indirect competitive ELISA method.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention first provides a tetracycline hapten SHS, which has a high degree of overlap with the cytoskeleton of the analyte tetracycline, effectively improving its immunogenicity. Furthermore, an artificial antigen obtained by conjugating this tetracycline hapten SHS with hemocyanin is used as an immunogen to immunize mice. Hybridoma cells are obtained through cell fusion, and ascites is prepared by in vivo inoculation of hybridoma cells. The purified ascites is then a monoclonal antibody. The obtained antibody exhibits good titer, broad spectrum, affinity, and organic tolerance. Its half-maximal inhibitory concentration (IC50) against tetracycline, oxytetracycline, and chlortetracycline is also high. 50 The concentrations were 17.7, 17.1, and 18.65 ng / mL, respectively, with linear ranges (IC50, 17.1, 18.65 ng / mL). 20 ~IC 80 The concentrations of the hapten provided by this invention are 5.96–52.5 ng / mL, 4.91–59.4 ng / mL, and 1.7–203.8 ng / mL, respectively, with limits of detection (LODs) of 3.16, 2.37, and 0.42 ng / mL, respectively. The resulting antibodies exhibit very high tolerance to organic solvents, tolerating organic solvents at concentrations of approximately 60%. Furthermore, at appropriate concentrations, organic solvents (methanol, ethanol, and n-hexane) can enhance the activity of the antibodies. Therefore, the antibodies prepared using the hapten provided by this invention possess broad-spectrum activity, a wide linear range, high sensitivity, and strong tolerance to organic solvents. They have promising application prospects and broad development potential in achieving simple, rapid, accurate, and effective detection of tetracycline drug residues. Attached Figure Description

[0041] Figure 1 This is a mass spectrometry diagram for identifying the tetracycline hapten SHS.

[0042] Figure 2 Ultraviolet scan images of carrier proteins (KLH, OVA), SHS, and artificial antigens SHS-KLH and SHS-OVA.

[0043] Figure 3 This is a schematic diagram of the amino acid sequence and domain division of the variable region of the light chain of the anti-tetracycline monoclonal antibody 2C8A.

[0044] Figure 4 This is a schematic diagram of the amino acid sequence and domain division of the variable region of the heavy chain of the anti-tetracycline monoclonal antibody 2C8A.

[0045] Figure 5 This is a standard curve for an indirect competitive ELISA based on the anti-tetracycline monoclonal antibody 2C8A.

[0046] Figure 6 Figure showing the organic solvent tolerance results of tetracycline monoclonal antibody 2C8A. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] Example 1: Preparation of tetracycline hapten SHS

[0050] A method for preparing tetracycline hapten SHS includes the following steps:

[0051] 1. Dissolve 1 mmol tetracycline (444.43 mg) and 2 mmol (202 mg) triethylamine in 5 mL DMF and stir for 10 min. Then slowly add 2 mmol (390 mg) 6-bromohexanoic acid dissolved in 2 mL DMF. During stirring, heat to 80 °C and react for 24 hours.

[0052] 2. After the reaction is complete, extract with saturated brine and ethyl acetate;

[0053] 3. After mixing the three organic phases, perform vacuum distillation to obtain a white oily substance, which is the tetracycline hapten.

[0054] The prepared tetracycline hapten has the structural formula shown in formula (I):

[0055]

[0056] The mass spectrum of the tetracycline hapten SHS is as follows: Figure 1 As shown in the mass spectrum, 559.3 is the positive ion molecular peak of the hapten SHS, and its calculated relative molecular mass is 558, which is consistent with the actual relative molecular mass, indicating that the tetracycline hapten SHS was successfully prepared.

[0057] Example 2: Preparation of Tetracycline Artificial Antigen

[0058] A method for preparing a tetracycline artificial antigen includes the following steps:

[0059] The tetracycline hapten SHS prepared in Example 1 was conjugated with ovalbumin (OVA) and keyhole lipophilic hemocyanin (KLH) via an active ester method. The tetracycline hapten SHS was dissolved in DMF, and 1.5 eq NHS and EDC were added to the solution. The mixture was reacted overnight at 4°C, and this solution was designated as solution A. Solution A was added dropwise to a PBS buffer solution containing the carrier protein, and the reaction was continued at 4°C for 8 hours. The reaction solution was dialyzed at 4°C for 3 days, with the dialysate changed twice daily for a total of 6 dialyzes. The solution collected from the dialysate bag yielded the tetracycline artificial antigens (tetracycline complete antigen SHS-KLH and tetracycline complete antigen SHS-OVA). The structural formula of the tetracycline artificial antigens is shown in formula (II).

[0060] The carrier proteins are ovalbumin (OVA) and keyhole limpet hemocyanin (KLH).

[0061] The carrier proteins (OVA, KLH), SHS, and artificial antigens SHS-OVA and SHS-KLH were identified by ultraviolet scanning (200–400 nm), and the results are as follows: Figure 2 As shown in the figure, the results indicate that the characteristic absorption peaks of the prepared artificial antigen show a significant blue shift compared to the carrier proteins (KLH, OVA) and the hapten SHS, indicating that the artificial antigen was successfully prepared.

[0062] Example 3: Identification of Tetracycline Artificial Antigen

[0063] (1) Animal Immunization

[0064] Healthy 6-week-old Balb / c female mice were used as experimental animals. The complete antigen SHS-KLH was used as the immunogen, and the mice were subcutaneously injected into the neck, back, and peritoneum. Each immunization dose was 0.5 mL (containing 0.5 mg of immunogen). For the initial immunization, 0.5 mL of complete Freund's adjuvant was emulsified with the antigen. Four weeks later, a booster immunization was performed using 0.5 mL of incomplete Freund's adjuvant emulsified with the antigen. Subsequent immunizations were performed every two weeks. During this period, a small amount of blood was collected from the tail vein for antibody quality assessment. After antibody stabilization, mice with the best performance were selected for cell fusion. Three days before cell fusion, mice were given a second booster immunization via intraperitoneal injection of 0.5 mg of immunogen.

[0065] (2) Evaluation of antiserum efficacy

[0066] Using the tetracycline complete antigen SHS-OVA prepared in Example 2 as the coating antigen, and the collected mouse serum as the detection antibody, the antiserum titer and inhibition rate of the mouse serum were determined by indirect competitive ELISA. The titer and inhibition rate of each antiserum were comprehensively considered for evaluation. The specific operating steps are as follows:

[0067] 1) Plate coating: Dilute tetracycline complete antigen SHS-OVA to 1000 ng / mL with 0.05 M carbonate buffer (pH = 9.6), add 100 μL / well, and coat overnight at 4°C; discard the coating solution, wash twice with PBST, add 120 μL of blocking solution (5% skim milk) to each well, and block at 37°C for 3 h; discard the blocking solution, dry at 37°C for 60 min, and store in a sealed bag at 4°C for later use to obtain the coated ELISA plate.

[0068] 2) Serum titer and inhibition assay: For the enzyme-labeled plate prepared in step 1), add 50 μL of PBS and 50 μL of serum diluted sequentially (1K, 2K, 4K, 8K, 16K, 32K, 64K) to each well in the titer column; add 50 μL of diluted 1000 ng / mL drug (1 tetracycline, 2 chlortetracycline, 3 oxytetracycline) and 50 μL of serum diluted sequentially (1K, 2K, 4K, 8K, 16K, 32K, 64K) to each well in the inhibition column. Perform two parallel assays. Incubate at 37℃ for 40 min, wash five times with PBST, blot dry the liquid in the wells, add 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP), incubate at 37℃ for 30 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL TMB substrate solution, and develop color at 37℃ in the dark for 10 min; add 50 μL stop solution (2M H2SO4) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0069] (3) Experimental Results

[0070] The inhibition results of the antiserum obtained from immunizing Balb / c female mice are shown in Table 1. It can be seen that all immunized mice produced an effective immune response. The obtained antiserum showed inhibitory effects on the target analytes tetracycline, oxytetracycline, and chlortetracycline at a SHS-OVA coating concentration of 1000 ng / mL and a drug concentration of 1000 ng / mL, with the most significant inhibitory effect observed in mouse number 1. This indicates that the hapten prepared in this invention can be used for subsequent preparation of monoclonal antibodies and the establishment of immunoassay methods.

[0071] Table 1. Antiserum titer and inhibition rate

[0072]

[0073] Example 4: Preparation of Tetracycline Monoclonal Antibody

[0074] (1) Cell fusion

[0075] Spleen cells from immunized mouse No. 1 were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Then, preheated fusion agent (PEG4000) was slowly added over 45 seconds to fuse the cells. The cells were then suspended in HAT medium and a suitable amount of feeder cells were added. The mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 5 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0076] (2) Screening for positive hybridomas

[0077] After cell fusion, when the cells reached 1 / 4 of the culture well area, hybridoma cells were screened using a stepwise screening method. Initial selection was performed using an indirect ELISA method. The ELISA plate was coated with a coating antigen (pre-titrated using a checkerboard method to determine the optimal coating concentration and positive serum dilution), and the culture supernatant of the test wells was added. After incubation and washing, goat anti-mouse IgG-HRP was added, followed by TMB substrate solution for colorimetric reaction. Positive wells were then screened using an indirect competitive ELISA method. The cell supernatant was mixed with equal volumes of 1000 ng / mL tetracycline, oxytetracycline, and chlortetracycline, incubated at 37°C for 30 min, and then added to the coated ELISA plate. PBS was used as a control instead of the drugs, and the remaining steps were the same. If the OD of the cells was blocked by the drugs... 450nm If the value drops to below 50% of the control well, it is considered positive. Wells that are positive after 2 or 3 tests are immediately subcloned using limiting dilution. After 4 rounds of limiting dilution, the monoclonal cell line 2C8A is obtained.

[0078] (3) Large-scale culture of hybridoma cells

[0079] Hybridoma cells obtained after 2-3 subclonings were cultured extensively, and the supernatant was collected for titer determination by indirect ELISA and then frozen. Balb / c mice aged 8-10 weeks were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse, and 7-10 days later, 1-2 × 10⁶ hybridoma cells were injected intraperitoneally. 6 / mouse, 7-10 days later, ascites fluid was extracted from mice, centrifuged to obtain the supernatant, the potency was determined, and the fluid was frozen for later use.

[0080] Example 5: Sequencing and determination of the amino acid sequence of the gene encoding the anti-tetracycline monoclonal antibody 2C8A.

[0081] 1. Total RNA extraction

[0082] Total RNA was extracted using the Trizol reagent method from Guangzhou Jiebes Biotechnology Co., Ltd.

[0083] The specific steps are as follows:

[0084] Take approximately 1×10 6 Centrifuge cells at 3000 rpm for 5 min, collect cells into centrifuge tubes, and carefully discard all culture medium supernatant. Gently tap the bottom of the centrifuge tube to loosen the cell pellet, immediately add 2 mL of lysis buffer (TRNsol), disperse the pellet, and collect it into a 2 mL centrifuge tube. Add 0.2 mL of chloroform to each 1 mL of the lysis buffer. Cap the centrifuge tube, gently shake up and down for 15 s, incubate on ice for 5 min, and centrifuge at 12000 rpm for 10 min at room temperature. Transfer the upper aqueous phase to a new centrifuge tube, slowly add 0.7 volumes of anhydrous ethanol, and mix well; transfer the resulting solution and pellet together into a GBC adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; add 500 μL of Wash Buffer I to the GBC adsorption column, centrifuge for 1 min, and discard the waste liquid; add 600 μL of Wash Buffer II to the GBC adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid. Centrifuge at 12000 rpm for 1 min, discard the waste liquid, and allow the column to air dry under a clean bench with the lid open to remove any residual washing solution. Transfer the GBC adsorption column to a new centrifuge tube, add 30–100 μL of LNase-free ddH2O, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to obtain the RNA solution.

[0085] 2. cDNA Synthesis

[0086] Using RNA as a template, cDNA first-strand synthesis was performed according to the instructions of the Takara First-Strand Reverse Transcription Kit. Specifically, 3 μg of RNA was used as a template, and 1 μL of Oligo(dT) 18 primer was added to a 1.5 mL nuclease-free centrifuge tube. The mixture was then incubated at 65°C for 5 min, followed by cooling on ice for 2 min. After the reaction, 4 μL of 5×Reaction Buffer, 1 μL of RNase inhibitor, 2 μL of 10 mM dNTP Mix, and 1 μL of M-MuLV reverse transcriptase (200 U / μL) were added to the resulting 20 μL mixture. The mixture was incubated at 42°C for 60 min, followed by incubation at 70°C for 5 min to inactivate the reverse transcriptase. The reaction mixture was then stored at -80°C for later use.

[0087] 3. Amplification of antibody variable region genes

[0088] (1) PCR cloning of V H V L Gene

[0089] Using cDNA synthesized by reverse transcription as a template, genes in the variable regions of the antibody heavy and light chains were cloned using universal primers. The universal primers are shown in Table 2.

[0090] Table 2. Universal primers for amplifying the variable regions of the heavy and light chains of single-chain antibodies.

[0091]

[0092]

[0093] Using the first strand of cDNA as a template, the variable region of the light chain was amplified using the LB18 / LF1 primer set. The reaction system and specific parameters are shown in Table 3. After PCR, the bands were identified by gel electrophoresis at 120V for 25min. The bands in the range of 300bp to 500bp were recovered by gel cutting using the kit.

[0094] Table 3. PCR amplification system and reaction conditions for the light chain variable region

[0095]

[0096] ② Heavy chain variable region gene cloning

[0097] Using the first strand of cDNA as a template, the variable region of the heavy chain was amplified using the HB6 / HB2 primer set. The reaction system and specific parameters are shown in Table 4. After PCR, the bands were identified by gel electrophoresis at 120V for 25min. The bands in the range of 300bp to 500bp were recovered by gel excision using a kit.

[0098] Table 4. PCR amplification system and reaction conditions for the heavy chain variable region

[0099]

[0100] (2) DNA gel recovery kit for recovering amplified V H V L Gene

[0101] The OMEGAgel extraction kit was used to extract PCR gel products. The specific steps are as follows: Cut off the target band with a clean blade and weigh it. Add an equal volume of Binding Buffer (XP2). Incubate at 55–60°C for 10 minutes until the gel is completely melted, shaking occasionally every 2–3 minutes. Transfer the melted gel to a HiBind DNA column and centrifuge at 10,000 rpm for 1 minute. Discard the liquid in the collection tube, add 300 μL of Binding Buffer (XP2), and centrifuge at 10,000 rpm for 1 minute. Discard the liquid in the collection tube, add 700 μL of SPW Wash Buffer (add ethanol to SPW Wash Buffer before first use), and centrifuge at 10,000 rpm for 1 minute. Repeat the process of adding SPW Wash Buffer. Wash with buffer; discard the liquid in the collection tube, centrifuge at 10000 rpm for 2 min; transfer the adsorption membrane to a 1.5 mL centrifuge tube, add 15 μL of sterile water, centrifuge at 10000 rpm for 2 min, collect the liquid in the tube, measure the DNA concentration using a nanodrop micro-UV spectrophotometer, and store at -20℃. The recovered V... H V L Genes were ligated into an 18-T Vector Cloning vector (TakaRa).

[0102] (3) Transformation

[0103] Competent DH5α cells were placed on ice and allowed to thaw for 5 minutes. 5 μL of the target vector was added, gently mixed, and incubated on ice for 25 minutes. The cells were then heat-shocked in a 42°C water bath for 45 seconds, immediately returned to ice, and incubated for 2 minutes. 400 μL of preheated LB broth (37°C) was added, and the cells were incubated at 37°C and 250 rpm for 1 hour. 100 μL of the culture was then evenly spread onto LB-A plates and incubated upside down at 37°C overnight. The following day, multiple single colonies were randomly selected from the plates for colony PCR and DNA sequencing identification and analysis.

[0104] 4. Antibody heavy chain and light chain gene sequence analysis

[0105] After adjusting the sequencing results using DNAman software, the complete forward sequence was obtained and entered into IMGT in FASTA format. https: / / www.imgt.org / IMGT_vquest / analysis The variable region gene sequence of the murine antibody was analyzed. The amino acid sequences of the heavy chain variable region (HF4-HB17) and the light chain variable region (LF1-LB18) were obtained through sorting and analysis.

[0106] 5. Experimental Results

[0107] The amino acid sequences of the light chain variable region and heavy chain variable region of the anti-tetracycline monoclonal antibody 2C8A prepared according to Example 4 are as follows: Figure 3 and Figure 4 As shown.

[0108] The amino acid sequence of the variable region of the 2C8A light chain of the anti-tetracycline monoclonal antibody is shown in SEQ ID No. 1:

[0109] DIVMTQSPAIMSASPGEKVTMTCSASSSVNYMHWYQQKSGASLKRWIYDTSNLASGVPARFSSSGSGTSYSLTISTMEAEDAATYYCQQWTSYPPTFGGGTKLEIK

[0110] The amino acid sequence of the variable region of the 2C8A heavy chain of the anti-tetracycline monoclonal antibody is shown in SEQ ID No. 2:

[0111] QVQLQQSGPELVEPGASMKISCKASGSSFTGYTMNWMKQSRGQNLEWIGLISPYNGVTRYNQKFKGKATITVDRSSSTAYMEVLSLTSEDSAVYFCARSYRYDVMENYFDYWGPGTTLTVSS

[0112] The nucleotide sequence encoding the light chain variable region of the anti-tetracycline monoclonal antibody 2C8A is shown in SEQ ID No. 3:

[0113] GACATTGTGATGACCCAGTCTCCTGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTTAATTACATGCACTGGTATCAGCAGAAGTCAGGCGCCTCTCTCAAAAGATGGATTTATGACACATCCAATCTG GCTTCTGGAGTCCCTGCTCGCTTCAGTAGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCACCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTTATCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAA

[0114] The nucleotide sequence encoding the heavy chain variable region of the anti-tetracycline monoclonal antibody 2C8A is shown in SEQ ID No. 4:

[0115] CAGGTTCAACTGCAGCAGTCTGGACCTGAGCTGGTGGAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTCCTCATTCACTGGCTACACCATGAACTGGATGAAGCAGAGCCGTGGACAGAACCTTGAGTGGATTGGACTTATTAGTCCTTACAATGGTGTTACTAGATACAAC CAGAAGTTCAAGGGCAAGGCCACAATAACTGTAGACAGGTCATCCAGCACCGCCTACATGGAGGTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGATCTTATAGGTACGACGTAATGGAGAACTACTTTGACTACTGGGGCCCGGGCACCACTCTCACAGTCTCCTCG

[0116] Example 6: Establishment of a standard curve for indirect competitive ELISA based on monoclonal antibodies

[0117] 1. Wrapping and sealing

[0118] Dilute the SHS-OVA coating agent to 500 ng / mL with coating buffer and coat overnight at 37°C. The next day, wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), add 2% skim milk powder, 120 μL per well, block at 37°C for 3 h, discard the blocking solution, dry at 37°C for 60 min, and store in a sealed bag at 4°C for later use.

[0119] 2. Establishment of the standard curve

[0120] (1) Experimental methods

[0121] Add 50 μL of 3.5 μg / mL anti-tetracycline monoclonal antibody and a series of 50 μL tetracycline standards of different concentrations to each well of the packaged ELISA plate. Incubate at 37°C for 40 min. Wash five times with PBST, blot dry the liquid in the wells, add 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP), incubate at 37°C for 40 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL TMB substrate solution, and develop color at 37°C in the dark for 10 min. Add 50 μL of stop solution (2M H2SO4) to terminate the reaction. Read the absorbance at 450 nm using an ELISA reader. Plot the tetracycline / oxytetracycline / chlortetracycline standard concentrations against the x-axis, and B / B0 (OD value of the wells containing tetracycline / oxytetracycline / chlortetracycline) against the y-axis. 450nm OD of pores without tetracycline / oxytetracycline / chlortetracycline 450nm Using y as the ordinate, establish an indirect competition standard curve.

[0122] (2) Experimental Results

[0123] The standard curve of indirect competitive ELISA based on monoclonal antibodies is shown below. Figure 5 As shown, the standard curve exhibits an S-shape, indicating good linear correlation. It also shows good half-maximal inhibition (IC50) for tetracycline, oxytetracycline, and chlortetracycline. 50 The concentrations were 17.7, 17.1, and 18.65 ng / mL, respectively, with linear ranges (IC50, 17.1, 18.65 ng / mL). 20 ~IC 80 The detection limits were 5.96–52.5 ng / mL, 4.91–59.4 ng / mL, and 1.7–203.8 ng / mL, respectively, with limits of detection (LOD) of 3.16, 2.37, and 0.42 ng / mL, respectively. The detection sensitivity was high and the linear range was wide.

[0124] Example 7: Organic Solvent Tolerance Test of Tetracycline Monoclonal Antibody 2C8A

[0125] (1) Experimental methods

[0126] Tetracycline monoclonal antibody 2C8A was diluted to the same working concentration with solutions containing 0%, 10%, 20%, 30%, 40%, 50%, and 60% methanol, ethanol, acetonitrile, and n-hexane, respectively, to determine the antibody-antigen binding ability. The binding ability of antibody diluted with 0% organic solvent was taken as 100%, and the tolerance of tetracycline monoclonal antibody 2C8A to methanol, ethanol, acetonitrile, and n-hexane was evaluated. The specific method is as follows:

[0127] Add 50 μL of diluted tetracycline monoclonal antibody 2C8A and 50 μL of PBS to the pre-packaged ELISA plate, incubate at 37°C for 40 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of LMB substrate solution, and develop the color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0128] (2) Experimental Results

[0129] The activity curves of tetracycline monoclonal antibody 2C8A with different ratios of organic solvent / PBS as diluents are shown in the figure. Figure 6 As shown in the figure, tetracycline monoclonal antibody 2C8A exhibits enhanced antibody activity when the concentration of methanol in the organic solvent is below 50%; ethanol in the organic solvent also enhances antibody activity when the concentration is below 30%, with antibody activities reaching 80% and 44% at 50% and 60% concentrations, respectively; acetonitrile in the organic solvent enhances antibody activity at 30% and 40% concentrations, respectively; and n-hexane in the organic solvent enhances antibody activity at all concentrations. These results indicate that tetracycline monoclonal antibody 2C8A has very high tolerance to organic solvents, and that methanol, ethanol, and n-hexane in the organic solvents enhance antibody activity at appropriate concentrations.

[0130] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A tetracycline hapten SHS, characterized in that, The structural formula is shown in equation (Ⅰ): 。 2. The method for preparing the tetracycline hapten SHS according to claim 1, characterized in that, It is obtained by introducing a carboxylic acid arm through a substitution reaction between the phenolic hydroxyl group on tetracycline and 6-bromohexanoic acid, using the phenolic hydroxyl group as the arm extension site.

3. The use of the tetracycline hapten SHS according to claim 1 in the preparation of tetracycline artificial antigens or antibodies.

4. A tetracycline artificial antigen, characterized in that, The tetracycline hapten SHS described in claim 1 is conjugated with a carrier protein, and its structural formula is shown in formula (II): .

5. The tetracycline artificial antigen according to claim 4, characterized in that, The carrier protein is chicken oocyte albumin or hemocyanin.

6. The use of the tetracycline artificial antigen according to claim 4 or 5 in the preparation of anti-tetracycline antibodies.

7. The application according to claim 6, characterized in that, The antibody is a monoclonal antibody, a polyclonal antibody, or a genetically engineered antibody.

8. A monoclonal antibody against tetracycline antibiotics, characterized in that, It is prepared by using the tetracycline artificial antigen described in claim 4 or 5 as an immunogen, and the amino acid sequence of its light chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID No.

2.

9. The use of the monoclonal antibody according to claim 8 in the detection of tetracycline antibiotics for non-disease diagnostic and therapeutic purposes.

10. A method for detecting tetracycline antibiotics for non-disease diagnosis and treatment purposes, characterized in that, The detection was performed using the tetracycline artificial antigen of chicken ovalbumin as the carrier protein described in claim 5 as the coating antigen, and the monoclonal antibody described in claim 8 as the detection antibody.

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