Monoclonal antibody against hexachlorocyclohexane and use thereof

Abstract

The application discloses a kind of monoclonal antibody of anti-hexachlorocyclohexane and application thereof, the application is with five chlorobenzoic acid-carrier protein conjugate as immunogen immunizes mouse, after cell fusion and screening, obtain the hybridoma cell strain capable of stably secreting anti-hexachlorocyclohexane monoclonal antibody.The amino acid sequence of the variable region of the light chain and the heavy chain of the monoclonal antibody and its encoding nucleotide sequence, and each complementarity determining region sequence are completely disclosed.The monoclonal antibody has very high sensitivity to hexachlorocyclohexane, and the half inhibitory concentration (IC 50 ) is as low as 5 ng / mL.The cross-reactivity of common veterinary drugs such as gentamicin and chloramphenicol is less than 0.1%.Based on the antibody, a rapid on-site screening for hexachlorocyclohexane residues in the environment and food can be developed, which 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 hexachlorocyclohexane and its application. Background Technology

[0002] Hexachlorocyclohexane (HCH) is a broad-spectrum organochlorine insecticide that was once widely used for agricultural pest control and sanitary pest control. Hexachlorocyclohexane achieves its insecticidal effect by acting on chloride ion channels in the insect's nervous system, interfering with the normal transmission of nerve signals.

[0003] However, hexachlorocyclohexane possesses high chemical stability and lipid solubility, making it extremely difficult to degrade in the environment, with a half-life lasting for years or even decades. Hexachlorocyclohexane can accumulate in organisms through the food chain and eventually enter the human body. Toxicological studies have shown that hexachlorocyclohexane has significant neurotoxicity, hepatotoxicity, and reproductive and developmental toxicity, and the International Agency for Research on Cancer (IARC) has classified it as a Group 2B possible carcinogen. Nevertheless, in regions where hexachlorocyclohexane has historically been used extensively, its residues can still be detected in soil, water bodies, and agricultural products. Furthermore, due to the illegal use of pre-banned stockpiles of this drug in some areas, incidents of excessive hexachlorocyclohexane residues in animal-derived foods (such as pork, chicken, liver, and kidneys) still occur frequently, posing a continued threat to consumer health.

[0004] Currently, the main methods for detecting hexachlorocyclohexane and its isomers include instrumental analysis methods such as gas chromatography (GC), gas chromatography-tandem mass spectrometry (GC-MS / MS), and high-performance liquid chromatography (HPLC). Although instrumental analysis methods offer high accuracy and sensitivity, they typically require expensive equipment, professional operators, complex sample pretreatment procedures, and long detection cycles, making them unsuitable for rapid on-site screening of large batches of samples.

[0005] Immunological detection methods, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography, offer advantages such as ease of operation, rapid detection, low cost, and suitability for large-scale on-site screening. They have become important supplementary methods for detecting veterinary drug residues and persistent organic pollutants. The core reagent in 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.

[0006] In the prior art, Chinese patent CN107759689A discloses a method for preparing zearalenone monoclonal antibodies. This method uses zearalenone standards as raw materials to prepare zearalenone monoclonal antibodies, synthesizes immunogens using the carbodiimide method, and screens for hybridoma cell lines capable of stably secreting antibodies. However, this patent targets the mycotoxin zearalenone, whose hapten design, immunogen preparation method, and antibody recognition epitopes are completely different from those of hexachlorocyclohexane compounds. Therefore, the disclosed technical solution cannot be directly applied to the preparation of hexachlorocyclohexane antibodies. Furthermore, this patent does not disclose the amino acid or nucleotide sequence information of the antibody, limiting further engineering modifications.

[0007] Chinese patent CN110272876A discloses a hybridoma cell line and its construction method using a monoclonal antibody against diethyl phthalate. This invention uses diethyl phthalate as a hapten and prepares the immunoantigen and coating antigen using a mixed anhydride method and a carbodiimide method, respectively. The hybridoma cell line is obtained through mouse immunization, cell fusion, and screening. However, diethyl phthalate belongs to the phthalate ester class of endocrine disruptors, and its molecular structure (containing a benzene ring and diester groups) differs significantly from hexachlorocyclohexane (containing a chlorocyclohexane structure), resulting in completely different hapten design strategies and antibody recognition characteristics. This patent also does not disclose the antibody's sequence information.

[0008] The existing technologies for the immunoassay of hexachlorocyclohexane have the following main shortcomings: First, hexachlorocyclohexane is a small molecule compound (molecular weight approximately 290.8) and is not immunogenic in itself. A well-designed hapten coupled with a carrier protein is required to elicit an immune response, but existing technologies lack effective hapten design schemes targeting the structural characteristics of hexachlorocyclohexane. Second, currently available antibodies against hexachlorocyclohexane are mainly polyclonal antibodies derived from the serum of immunized animals. These antibodies exhibit significant batch-to-batch variability, cannot be supplied consistently and stably, and have considerable room for improvement in sensitivity and specificity. Third, there is a lack of publicly available complete sequence information for anti-hexachlorocyclohexane monoclonal antibodies in this field.

[0009] In summary, there is an urgent need in this field to develop a monoclonal antibody against hexachlorocyclohexane that is more sensitive, more specific, has a well-defined antibody sequence, and can be stably produced, 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 monitoring hexachlorocyclohexane residues in the environment and food. Summary of the Invention

[0010] To address the problems of existing hexachlorocyclohexane detection antibodies, which are mainly polyclonal antibodies, lacking sensitivity, lacking clear sequence information, and difficult to stably produce, this invention provides a monoclonal antibody against hexachlorocyclohexane and its applications. This invention uses a self-designed hexachlorocyclohexane hapten (pentachlorobenzoic acid) and a carrier protein conjugate as an immunogen. Hybridoma cell lines that stably secrete anti-hexachlorocyclohexane 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 develop detection reagents, kits, and colloidal gold immunochromatographic test strips, which can be used for rapid and sensitive detection of hexachlorocyclohexane residues in the environment and food.

[0011] In a first aspect, the present invention provides a monoclonal antibody against hexachlorocyclohexane, which is prepared by immunizing animals with a hexachlorocyclohexane hapten-carrier protein conjugate and is capable of specifically recognizing hexachlorocyclohexane.

[0012] Furthermore, the anti-hexachlorocyclohexane 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.

[0013] Specifically:

[0014] The amino acid sequence of the light chain complementarity-determining region CDR1 is shown in SEQ ID NO. 18;

[0015] The amino acid sequence of the light chain complementarity-determining region CDR2 is KVS;

[0016] The amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO. 21;

[0017] The amino acid sequence of the heavy chain complementarity-determining region CDR1 is shown in SEQ ID NO. 2;

[0018] The amino acid sequence of the heavy chain complementarity-determining region CDR2 is shown in SEQ ID NO. 4;

[0019] The amino acid sequence of the heavy chain complementarity-determining region CDR3 is shown in SEQ ID NO. 6.

[0020] 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.

[0021] 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.

[0022] Secondly, the present invention provides a method for preparing the above-mentioned monoclonal antibody against hexachlorocyclohexane, comprising the following steps:

[0023] (1) Animal immunization: Balb / c mice were immunized with hexachlorocyclohexane hapten-carrier protein conjugate as an immunogen;

[0024] (2) Cell fusion: Spleen cells from immunized mice were fused with myeloma cells SP2 / 0;

[0025] (3) Hybridoma screening: Indirect ELISA and competitive ELISA were used to screen for positive hybridoma cell lines that could secrete monoclonal antibodies against hexachlorocyclohexane;

[0026] (4) Cloning: positive hybridoma cells are cloned by limiting dilution to obtain hybridoma cell lines that stably secrete monoclonal antibodies;

[0027] (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.

[0028] Preferably, the carrier protein is bovine serum albumin (BSA) or ovalbumin (OVA).

[0029] Preferably, the hexachlorocyclohexane hapten is pentachlorobenzoic acid.

[0030] Thirdly, the present invention provides the application of the above-mentioned anti-hexachlorocyclohexane monoclonal antibody in the detection of hexachlorocyclohexane.

[0031] Specifically, a reagent for detecting hexachlorocyclohexane comprises the anti-hexachlorocyclohexane monoclonal antibody described in this invention.

[0032] Furthermore, a kit for detecting hexachlorocyclohexane comprises the anti-hexachlorocyclohexane 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.

[0033] Fourthly, the present invention provides the application of the above-mentioned anti-hexachlorocyclohexane monoclonal antibody in the preparation of detection reagents or kits for detecting hexachlorocyclohexane.

[0034] Fifthly, the present invention provides a method for detecting hexachlorocyclohexane in a sample, comprising the step of performing an immunoassay on the sample using the anti-hexachlorocyclohexane monoclonal antibody described in this invention. The method includes, but is not limited to, enzyme-linked immunosorbent assay (ELISA), immunochromatography, and immunosensor methods.

[0035] The beneficial effects of this invention are:

[0036] (1) The half-maximal inhibitory concentration (IC50) of the monoclonal antibody provided by this invention against hexachlorocyclohexane. 50 With a concentration as low as 5 ng / mL, its sensitivity is significantly better than that of polyclonal antibodies in existing technologies, and it can meet the more stringent requirements for the detection of veterinary drug residues and environmental pollutants.

[0037] (2) The antibody of the present invention has high specificity to hexachlorocyclohexane 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.

[0038] (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 hexachlorocyclohexane monoclonal antibodies in the prior art.

[0039] (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.

[0040] In summary, this invention provides a highly sensitive, specific, well-defined, and stably produced technical solution for the rapid detection of hexachlorocyclohexane residues, which is suitable for on-site testing. This is of great practical significance for ensuring environmental and food safety and implementing monitoring of persistent organic pollutants. Attached Figure Description

[0041] Figure 1 This is a synthetic route diagram for the coupling of the hexachlorocyclohexane hapten with the carrier protein of the present invention;

[0042] Figure 2 This is a diagram showing the homology comparison results of the heavy chain gene sequence of the anti-hexachlorocyclohexane monoclonal antibody of this invention;

[0043] Figure 3 This is a diagram showing the homology comparison results of the heavy chain amino acid sequence of the anti-hexachlorocyclohexane monoclonal antibody of this invention;

[0044] Figure 4This is a diagram showing the homology comparison results of the light chain gene sequence of the anti-hexachlorocyclohexane monoclonal antibody of this invention;

[0045] Figure 5 This is a diagram showing the homology comparison results of the light chain amino acid sequence of the anti-hexachlorocyclohexane monoclonal antibody of this invention;

[0046] Figure 6 This is a standard ELISA inhibition curve of the anti-hexachlorocyclohexane monoclonal antibody against hexachlorocyclohexane in this invention (the horizontal axis represents the concentration of hexachlorocyclohexane, and the vertical axis represents OD). 450 (nm value). Detailed Implementation

[0047] 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.

[0048] The sequences involved in this invention are shown in Tables 1 and 2 below.

[0049] The hexachlorocyclohexane standard used in the embodiments of this invention is a standard substance conventionally used in the art. Various isomers of hexachlorocyclohexane (including α-HCH, β-HCH, γ-HCH, δ-HCH, etc.) all share the same core molecular skeleton—the hexachlorocyclohexane ring—differenceing only in the spatial orientation of the chlorine atom on the ring. This invention uses pentachlorobenzoic acid as a hapten, and its design concept utilizes the common structural feature of a 'six-membered ring skeleton with polychlorinated atom substitution' to prepare antibodies. As shown in Examples 6-7, this antibody can effectively recognize the hexachlorocyclohexane standard, indicating that it recognizes the common core epitope of hexachlorocyclohexane compounds and can cover various isomer forms. Therefore, the method establishment and performance verification using hexachlorocyclohexane standards in the following embodiments can represent the detection capability of the antibody of this invention for hexachlorocyclohexane compounds.

[0050] Table 1: Nucleotide and amino acid sequences of IGH (heavy chain)

[0051] FR1 1 EVKLVESGGGSVKPGGALKLSCVAS 9 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTCAGTGAAGCCTGGAGGGGCCCTGAAACTCTCCTGTGTAGCCTCT CDR1 2 GFTLSSYA 10 GGATTCACTCTCAGCAGTTATGCC FR2 3 VSWVRQTPEKRLEWVAS 11 GTGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCC CDR2 4 ISSGGTA 12 ATTAGTAGTGGTGGTACCGCC FR3 5 YYSDSVKGRFTISRDNARNILYLQINSLRSEDTAMYYC 13 TACTATTCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTTCAAATTAACAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGT CDR3 6 ATIYNGYEAWFVY 14 GCAACCATCTACAATGGTTACGAGGCCTGGTTTGTTTAC FR4 7 WGQGTLVTVSA 15 TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA V(D)J-IMGT 8 EVKLVESGGGSVKPGGALKLSCVASGFTLSSYAVSWVRQTPEKRLEWVASISSGGTAYYSDSVKGRFTISRDNARNILYLQINSLRSEDTAMYYCATIYNGYEAWFVYWGQGTLVTVSA 16 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTCAGTGAAGCCTGGAGGGGCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTCTCAGCAGTTATGCCGTGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGGTGGTACCGCCTACTATTCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTTCAAATTAACAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAACCATCTACAATGGTTACGAGGCCTGGTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0052] Table 2: Nucleotide and amino acid sequences of IGK (light chain)

[0053] FR1 17 AVLLTQTPLSLPVSLGDQASISCRSS 24 GCTGTTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT CDR1 18 QSFVHSNGDTY 25 CAGAGCTTTGTACATAGTAATGGAGACACCTAT FR2 19 LEWYLQKPGRSPKLLIY 26 TTGGAGTGGTACCTGCAGAAACCAGGCCGGTCTCCTAAGCTCCTGATCTAC CDR2 — (Note 1) KVS — (Note 2) AAAGTTTCC FR3 20 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC 27 AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC CDR3 21 FQGSHVPRT 28 TTTCAAGGTTCACATGTTCCTCGGACG FR4 22 FGGGTKLEIK 29 TTCGGTGGAGGCACCAAGCTGGAAATCAAA V(D)J-IMGT 23 AVLLTQTPLSLPVSLGDQASISCRSSQSFVHSNGDTYLEWYLQKPGRSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK 30 GCTGTTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCTTTGTACATAGTAATGGAGACACCTATTTGGAGTGGTACCTGCAGAAACCAGGCCGGTCTCCTAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0054] Note 1: The amino acid sequence of the light chain complementarity-determining region CDR2 is KVS (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.

[0055] Note 2: The nucleotide sequence encoding the light chain complementarity-determining region CDR2 is AAAGTTTCC (9 nucleotides). Since it is less than 10 nucleotides, it is not assigned a SEQ ID NO according to the patent sequence naming convention.

[0056] Example 1:

[0057] Preparation of hexachlorocyclohexane complete antigen.

[0058] In this embodiment, hexachlorocyclohexane immunogenic antigen and coating antigen were prepared respectively, and the synthetic routes are as follows: Figure 1 As shown.

[0059] Hexachlorocyclohexane itself lacks active functional groups that can couple with carrier proteins, making it impossible to directly prepare a complete antigen. Through research, the inventors selected pentachlorobenzoic acid as a hapten. Pentachlorobenzoic acid has a cyclic molecular backbone with polychlorinated atom substitution, exhibiting structural similarity to hexachlorocyclohexane in spatial conformation and chlorine atom substitution patterns. Simultaneously, its carboxyl group can act as an active linker arm to couple with carrier proteins, forming an immunogenic complete antigen.

[0060] Accurately weigh 30 mg of pentachlorobenzoic acid (hapten) and dissolve it in 1.0 mL of anhydrous DMF, ensuring complete dissolution. Add 20 mg of N-hydroxysuccinimide (NHS) and 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) sequentially to the solution. Stir magnetically at room temperature in the dark for 4–6 hours to complete the activation of pentachlorobenzoic acid.

[0061] Accurately weigh 40 mg of bovine serum albumin (BSA) and dissolve it in 4.0 mL of 0.1 M borate buffer (pH 8.0). Place the solution in an ice-water bath (0–4 °C). Turn on magnetic stirring and very slowly add the "activation solution" prepared in step one to the protein solution. After the addition is complete, stir the solution 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.

[0062] 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, and the pentachlorobenzoic acid-BSA conjugation ratio was calculated to be within the optimal range.

[0063] The preparation method of the coating antigen pentachlorobenzoic acid-OVA is the same as above, except that the carrier protein is replaced with ovalbumin (OVA).

[0064] Example 2:

[0065] Animal immunization and serum titer detection.

[0066] Pentachlorobenzoic acid-BSA was used as the immunogen to immunize 6-8 week old female Balb / c mice. 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.

[0067] The detection method is as follows:

[0068] (1) Coating: Dilute the pentachlorobenzoic acid-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;

[0069] (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;

[0070] (3) Sample addition: Mouse serum was serially diluted from 1:5000. 50 μL of serum diluent and 50 μL of hexachlorocyclohexane standard (final concentration 100 ppb) or PBS blank control were added to each well. The mixture was reacted at 37°C for 1 hour and washed 3 times.

[0071] (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;

[0072] (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;

[0073] (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%).

[0074] The test results are shown in Table 3.

[0075] Table 3: Serum titer and inhibition detection in immunized mice

[0076] Note: Inhibition rate at a dilution of 1:320000 was not calculated (indicated by "—"); NC is the negative control, "—" indicates it is not applicable.

[0077] The results showed that the serum titer of the immunized mice was high and it had a significant inhibitory effect on hexachlorocyclohexane. Mice with high titer and good inhibition rate were selected for spleen cell fusion.

[0078] Example 3:

[0079] Cell fusion and hybridoma screening.

[0080] Spleens were removed from mice with the best immunogenicity in Example 2 under aseptic conditions, and spleen cell suspensions were 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.

[0081] 7-10 days after fusion, when the hybridoma cell colonies have grown to 1 / 3-1 / 2 of the bottom of the well, the supernatant is collected and positive wells are screened using an indirect ELISA method. The positive wells are then further analyzed using an indirect competitive ELISA method to determine the inhibition rate: the coating antigen is pentachlorobenzoic acid-OVA (1 μg / mL), and the competitor is 10 ppb hexachlorocyclohexane standard; the specific procedure is the same as in Example 2. Based on the OD difference and inhibition rate, hybridoma cell lines with superior performance are screened. The results are shown in Table 4.

[0082] Table 4: Titer and Inhibition Detection of Fusion Monoclonal Cells

[0083] The results showed that clone number 2C11G10 had the highest inhibition rate (88%) and a high titer, and cells from this well were selected for cloning.

[0084] Example 4:

[0085] Preparation and purification of monoclonal antibodies.

[0086] The positive hybridoma cells (2C11G10) screened in Example 3 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-hexachlorocyclohexane monoclonal antibody was obtained and named 2C11G10.

[0087] 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 2C11G10 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 1M Tris-HCl (pH 9.0). The eluent was dialyzed against PBS overnight to obtain purified anti-hexachlorocyclohexane monoclonal antibody. Antibody purity was assessed by SDS-PAGE (>95%), and antibody concentration was determined by the BCA method.

[0088] Example 5:

[0089] Total RNA extraction, reverse transcription, antibody variable region gene amplification, and sequencing.

[0090] The 2C11G10 hybridoma cells from Example 4 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.

[0091] 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.

[0092] Sequence analysis:

[0093] 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).

[0094] 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 KVS, 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.

[0095] c. Sequence alignment analysis:

[0096] 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 2 As shown, the heavy chain amino acid sequence alignment results are as follows: Figure 3 As shown, the light chain gene sequence alignment results are as follows: Figure 4 As shown, the light chain amino acid sequence alignment results are as follows: Figure 5 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.

[0097] Example 6:

[0098] Identification of the sensitivity of monoclonal antibodies.

[0099] The sensitivity of the purified monoclonal antibody from Example 4 was determined using an indirect competitive ELISA method.

[0100] Operating steps:

[0101] (1) Coating: Dilute the pentachlorobenzoic acid-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;

[0102] (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;

[0103] (3) Sample addition: Dilute the hexachlorocyclohexane standard with PBS to a series of concentrations (0, 2.5, 5, 10, 20, 40, 80 ng / mL), add 50 μL of standard solution and 50 μL of diluted anti-hexachlorocyclohexane monoclonal antibody (working concentration determined by checkerboard titration) to each well, react at 37°C for 1 hour, and wash 3 times;

[0104] (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;

[0105] (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%).

[0106] The test results are shown in Table 5.

[0107] Table 5: Results of Sensitivity Assessment of Indirect ELISA

[0108] 0 1.4163 0.00% 2.5 1.0136 28.43% 5 0.7048 50.23% 10 0.5247 62.95% 20 0.4132 70.82% 40 0.2753 80.56% 80 0.1669 88.22%

[0109] Plot the standard curve based on the data in Table 5. Figure 6 The half-maximal inhibitory concentration (IC50) of the monoclonal antibody against hexachlorocyclohexane was calculated. 50 The concentration was 5 ng / mL, indicating that the monoclonal antibody of this invention has extremely high sensitivity.

[0110] Example 7:

[0111] Monoclonal antibody specificity identification.

[0112] The specificity of the purified monoclonal antibody from Example 4 was identified using an indirect competitive ELISA method. Hexachlorocyclohexane 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 6, with the detection concentrations of each drug being: hexachlorocyclohexane 0-80 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 % = (hexachlorocyclohexane IC) 50 / IC of the drug to be tested 50 (×100%)

[0113] The test results are shown in Table 6.

[0114] Table 6: Results of Indirect ELISA Cross-Reactivity Identification

[0115] Hexachlorocyclohexane 5 100% Gentamicin >1000 <0.1% Chloramphenicol >1000 <0.1%

[0116] The results showed that the monoclonal antibody of the present invention has high specificity for hexachlorocyclohexane, 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.

[0117] 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 hexachlorocyclohexane, 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 monoclonal antibody against hexachlorocyclohexane 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 hexachlorocyclohexane 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 hexachlorocyclohexane, characterized in that, The monoclonal antibody comprising the anti-hexachlorocyclohexane as described in any one of claims 1-3.

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

6. The use of the anti-hexachlorocyclohexane monoclonal antibody as described in any one of claims 1-3 in the preparation of a detection reagent or kit for detecting hexachlorocyclohexane.

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

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