Hybridoma cell line 14G1 and monoclonal antibodies produced therefrom and their applications

Abstract

The present invention provides a hybridoma cell line 14G1 with a deposit number of CCTCC NO: C2021201, which can secrete and produce anti-chlorfenapyr monoclonal antibodies, and the titer of the monoclonal antibodies measured by enzyme-linked immunosorbent assay is 6.4×10 4 The 50% inhibitory concentration IC of chlorfenapyr was determined by conventional competitive ELISA. 50 It is 0.0253ng / mL, has no cross reaction with diuron, chlorotoluron, thidiazuron, linuron and clofentezine, has high sensitivity and high specificity, and can be used for immunoassay determination of chlorfenapyr content, enrichment or purification of chlorfenapyr.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and specifically relates to a hybridoma cell line 14G1 and a monoclonal antibody produced by the hybridoma cell line 14G1 and an application thereof. Background Art

[0002] Chloropyrim (1-(2-chloropyridin-4-yl)-3-phenylurea, CPPU) is a synthetic phenylurea-derived cytokinin widely used as a plant growth regulator, mainly for increasing fruit size. Chloropyrim has been registered in many countries, and its widespread use in agricultural planting can significantly increase the yield of fruits and promote consumption in the agricultural economy. However, studies have found that long-term exposure to chlorpyrifos may cause protein metabolism disorders, mild emphysema and weight loss. In order to ensure food safety and life health, many countries in the world have established limit standards for the maximum residue of chlorpyrifos in various fruits and vegetables. my country's national food safety standard GB 2763-2021 "Maximum Residue Limits of Pesticides in Food" stipulates that the residue of chlorpyrifos in cucumbers, watermelons and melons is 0.1 mg / kg, and the residue in oranges, loquats, grapes and kiwis is 0.05 mg / kg. In view of the decentralized planting pattern of small farmers in my country and the widespread use of chlorfenapyr, strengthening the detection of chlorfenapyr in food and developing a highly sensitive and specific chlorfenapyr detection method are of great significance to ensuring the safety of food consumption in my country.

[0003] The existing methods for detecting chlorfenuron residues mainly include precision instrumental analysis and immunoassay. Instrumental analysis methods for detecting chlorfenuron include high performance liquid chromatography, gas chromatography and liquid chromatography-mass spectrometry, etc., which have high precision and good accuracy, but the instruments are expensive, the sample pretreatment steps are cumbersome, and it is difficult to achieve rapid on-site screening of a large number of samples. The immunoassay based on the specific binding reaction of antigen and antibody has the advantages of being sensitive, convenient, low cost and suitable for on-site batch determination, which can overcome the shortcomings of instrumental analysis and has important application prospects in the rapid screening of chlorfenuron residues. Immunoassay usually uses the biological, physical or chemical amplification of markers on antigens and antibodies to quantitatively or qualitatively analyze trace residues. Among them, antibodies are the core detection reagents of immune reactions. To study and establish immunological detection technology for chlorfenuron, it is necessary to first prepare highly sensitive and highly specific anti-chlorfenuron antibodies. Summary of the invention

[0004] The purpose of the present invention is to provide a hybridoma cell line 14G1 capable of secreting highly sensitive and highly specific anti-chlorfenapyr antibodies and the monoclonal antibodies produced by it and their applications in view of the above-mentioned problems existing in the prior art.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A hybridoma cell line 14G1, whose deposit number is CCTCC NO: C2021201.

[0007] The monoclonal antibody is secreted and produced by the hybridoma cell line.

[0008] The nucleic acid sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3;

[0009] The nucleic acid sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 4.

[0010] The amino acid sequences of the heavy chain CDR1-3 of the monoclonal antibody are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively;

[0011] The amino acid sequences of the light chain CDR1-3 of the monoclonal antibody are shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0012] The heavy chain of the monoclonal antibody is IgG1, and the light chain is a Kappa chain.

[0013] The application of the anti-forchlorfenuron monoclonal antibody in the determination of forchlorfenuron content.

[0014] The anti-chlorfenuron monoclonal antibody is used in the enrichment or purification of chlorfenuron. The anti-chlorfenuron monoclonal antibody is coupled with agarose gel and silica gel microspheres to prepare an immunoaffinity chromatography column, and the immunoaffinity chromatography column is used to enrich or purify chlorfenuron.

[0015] The anti-chlorfenapyr monoclonal antibody is modified by random or site-directed mutagenesis technology to obtain an antibody that can specifically bind to chlorfenapyr.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The hybridoma cell line 14G1 provided by the present invention can secrete and produce anti-chlorfenapyr monoclonal antibodies, and the titer of the monoclonal antibodies measured by enzyme-linked immunosorbent assay is 6.4×10 4 The 50% inhibitory concentration IC of chlorfenapyr was determined by conventional competitive ELISA. 50The concentration of the antibody is 0.0253 ng / mL, and it has no cross reaction with diuron, chlorotoluron, thidiazuron, linuron and clofentezine, and can be applied to immunoassay determination of chlorfenuron content, enrichment or purification of chlorfenuron, and can be used to obtain antibodies that can specifically bind to chlorfenuron through random or site-directed mutagenesis technology. Therefore, the invention not only has high sensitivity and high specificity, but also has wide application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The present invention is a competitive inhibition standard curve of the anti-forchlorfenuron monoclonal antibody on forchlorfenuron determined by enzyme-linked immunosorbent assay.

[0019] Figure 2 The present invention is a competitive inhibition standard curve of the anti-chlorfenuron monoclonal antibody on chlorfenuron determined by magnetic bead enzyme-linked immunosorbent assay. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0021] Description of biological deposit:

[0022] Classification and nomenclature: Hybridoma cell line 14G1, depository: China Center for Type Culture Collection, deposit number: CCTCC NO: C2021201, deposit date: December 27, 2021, deposit address: Wuhan University, Wuhan, China.

[0023] The amino acid sequences of the heavy chain CDR1-3 and the light chain CDR1-3 of the anti-chlorfenapyr monoclonal antibody of the present invention are determined by the rules of the International Immunogenetics Database IMGT.

[0024] Embodiment 1:

[0025] Hybridoma cell line 14G1, the screening method thereof is as follows:

[0026] (1) Animal immunization

[0027] CPPU-OVA purchased from Shandong Ludu Biotechnology Co., Ltd. was used as the complete antigen of chlorfenapyr. After the first immunization, CPPU-OVA was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points in the neck and abdomen of 6-week-old BALB / c mice. The second immunization was carried out 3 weeks later, and Freund's incomplete adjuvant was used to emulsify an equal volume of CPPU-OVA and inject it into the mouse peritoneum and subcutaneously at multiple points. The third immunization was 2 weeks apart from the second immunization, and the immunization method was the same. The fourth immunization was carried out 2 weeks after the third immunization, and the immunization method was the same as the second immunization. The 4 immunization doses were the same, all 80 μg per mouse. The serum after the third immunization was used to measure the mouse serum titer and inhibition rate by indirect ELISA. After the fourth immunization, mice corresponding to the serum with relatively high titer and sensitivity were selected for the last sprint immunization, and the immunization dose was 40 μg.

[0028] (2) Cell fusion

[0029] Three days after the sprint immunization, 50% PEG (molecular weight 1450) was used as a fusion agent and cell fusion was performed as follows:

[0030] After the mice were killed by cervical dislocation, they were immediately immersed in 75% medical alcohol for 5 min. Mouse spleen cells were isolated by aseptic operation, mixed with mouse SP2 / 0 myeloma cells at a ratio of 4:1, and then fused under the mediation of 50% PEG. Take 1mL of 50% PEG and slowly add it to the mixed cells within 1min, let it stand for 1min, take 1mL of RPMI-1640 culture medium (purchased from Gibco) and add it to the mixed cells within 1min, then take 2mL of RPMI-1640 culture medium and add it to the mixed cells within 1min, then take 5mL of RPMI-1640 culture medium and add it to the mixed cells within 1min, finally quickly add 20mL of RPMI-1640 culture medium to the mixed cells, centrifuge at 1000rpm for 5min, discard the supernatant, and resuspend in RPMI-1640 screening culture medium containing 15% (volume percentage) fetal bovine serum, 5% (volume percentage) growth factor, 1% (volume percentage) penicillin-streptomycin solution and 2% (weight percentage) 50×HAT (purchased from Sigma-Aldrich). The resuspended cells were added dropwise to a 96-well cell culture plate covered with a feeder layer, 100 μL / well, and cultured at 37°C and 5% CO2.

[0031] (3) Screening and cloning of cell lines

[0032] The supernatant of the cell wells was tested on the 10th day after fusion. The complete antigen CPPU-BSA (purchased from Beijing Biolong Immunotechnology Co., Ltd.) was used as the screening coating antigen. The positive cell wells were first screened by indirect ELISA, and then the chlorfenapyr standard was used as a competitor. The inhibitory effect of positive cells was determined by indirect competitive ELISA. The strong positive cell wells with good inhibition of the chlorfenapyr standard were selected and cloned by limiting dilution. The indirect competitive ELISA was still used for detection 10 days after cloning. The above method was used for 3 consecutive clonings to finally obtain a stable monoclonal cell line.

[0033] Embodiment 2:

[0034] The anti-chlorfenapyr monoclonal antibody secreted by hybridoma cell line 14G1 is prepared by:

[0035] The hybridoma cell line 14G1 obtained by screening in Example 1 was injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected. The antibody was purified by the caprylic acid-ammonium sulfate method. The specific operation was as follows:

[0036] The collected ascites was centrifuged at 5000 rpm for 10 min, the supernatant was aspirated, mixed with 3 times the volume of acetate buffer, and octanoic acid was slowly added under stirring (the volume of octanoic acid required for each milliliter of ascites was 40 μL). After mixing at room temperature for 30 min, it was allowed to stand at 4°C for 2 h, and then centrifuged at 4°C and 10000 rpm for 10 min. The supernatant was filtered with double-layer filter paper, and 1 / 10 volume of the filtrate was added with phosphate buffer (0.1 mol / L, pH=7.4), and the pH of the mixture was adjusted to 7.4 with 2 mol / L sodium hydroxide solution, and precooled at 4°C. Ammonium sulfate was slowly added while stirring to a final concentration of 0.277 g / mL of ammonium sulfate, and the mixture was allowed to stand at 4°C for 3 h, followed by centrifugation at 4°C and 10,000 rpm for 20 min, the supernatant was discarded, and the resulting precipitate was resuspended with 0.01 mol / L phosphate buffer of 1 / 10 of the original ascites volume, dialyzed for desalination, and finally the fully dialyzed monoclonal antibody solution was stored at -20°C or freeze-dried and stored at -80°C, wherein the acetate buffer was obtained by adding 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL of water, and the phosphate buffer was obtained by adding 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to 100 mL of water.

[0037] Subtype identification: The mouse monoclonal antibody subtype identification kit of Proteintech Company was used to identify that the heavy chain of the anti-chlorfenapyr monoclonal antibody prepared in this example was IgG1, and the light chain was Kappa chain.

[0038] Titer identification: Using the complete antigen CPPU-BSA as the coating antigen, the titer of the monoclonal antibody was measured by conventional non-competitive ELISA method to reach 6.4×10 4 , i.e. mouse ascites antibody diluted 6.4×10 4 The solution test result was positive when the affinity constant was as high as 4.74×10 10 L / mol.

[0039] Sensitivity determination: The sensitivity of the monoclonal antibody to chlorfenapyr was determined by conventional indirect competitive ELISA. The competitive inhibition standard curve was plotted as shown in Figure 1 .from Figure 1 As can be seen, the IC 50 At the same time, the cross-reaction rate with diuron, chlorotoluron, thidiazuron, liuron and clofentezine analogues was less than 0.1%.

[0040] Example 3: Determination of variable region sequence of anti-chlorfenapyr monoclonal antibody

[0041] First, the total RNA of the hybridoma cell line 14G1 obtained in Example 1 was extracted by the Trizol method. Then, the total RNA was used as a template, oligo (dT)18 (purchased from Shanghai Shenggong Biotechnology Co., Ltd.) was used as a primer, and reverse transcriptase MLV (purchased from Promega Biotechnology Co., Ltd.) was added to synthesize the first chain of cDNA through the reverse transcription system. Then, primers were designed according to the conserved sites of the mouse antibody gene sequence in GenBank, and the antibody light and heavy chain variable region genes were amplified using cDNA as a template. The amplification was performed for 30 cycles. The PCR program for each cycle was: 98° C. 15 s, 55° C. 15 s, and 72° C. 30 s. After the PCR products were separated by 1% (weight percentage) agarose gel electrophoresis, the DNA fragments were recovered using the gel recovery kit of Axygen Company, ligated into the vector pMD19-T, transformed into Escherichia coli DH5α competent cells, and the positive clones were picked and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. Among them, the heavy chain variable region primer was 5'-AGG TSM ARC TGC AGS AGT CWG G-3' (22nt) and 5'-TGA GGAGAC GGT GAC CGT GGT CCC TTG GCC CC-3' (32nt), S, M, R and W are merged bases, M=A / C, R=A / G, S=C / G, W=A / T, the light chain variable region primers are 5'-GAC ATT GAG CTC ACC CAG TCT CCA -3' (24nt) and 5'-CCG TTT CAG CTC CAG CTT GGT GCC-3' (24nt).

[0042] The gene sequence results obtained are as follows: the nucleic acid sequence encoding the heavy chain variable region is 351 bp long, and the sequence is shown in SEQ ID NO: 1; based on the obtained gene sequence, it is deduced that the heavy chain variable region encoded by the gene sequence consists of 127 amino acids, and the sequence is shown in SEQ ID NO: 3. The nucleic acid sequence encoding the light chain variable region is 324 bp long, and the sequence is shown in SEQ ID NO: 2; based on the obtained gene sequence, it is deduced that the light chain variable region encoded by the gene sequence consists of 108 amino acids, and the sequence is shown in SEQ ID NO: 4.

[0043] Example 4: Application of anti-chlorfenapyr monoclonal antibody

[0044] The anti-chlorfenapyr monoclonal antibody obtained in Example 2 was used to establish a magnetic bead ELISA method, comprising the following steps:

[0045] (1) Preparation of biotin-labeled complete antigen

[0046] Biotinylated chlorfenapyr complete antigen CPPU-BSA was prepared using a biotin labeling kit purchased from Beijing Biolong Immunotechnology Co., Ltd. The specific operation is as follows: take 0.5 mg of complete antigen CPPU-BSA, add 200 μL of labeling buffer, calculate the volume of biotin solution added according to the formula in the manual, mix by suction and blow, and place in a 37°C constant temperature incubator for reaction for 30 minutes. Transfer the reactant to an ultrafiltration tube (molecular weight cutoff of 3K) and centrifuge at 4°C and 12000rpm for 12 minutes. Add an appropriate amount of streptavidin magnetic bead buffer, mix well, and centrifuge again under the above conditions. Repeat the operation 3 times, remove the unreacted biotin by centrifugation, and replace the labeling buffer with magnetic bead buffer. After centrifugation, add an appropriate amount of magnetic bead buffer to mix and dilute the biotinylated CPPU-BSA, and measure its concentration with a BCA kit, and store it in a 4°C refrigerator for later use.

[0047] (2) Preparation of biotinylated complete antigen-coupled streptavidin magnetic bead probe

[0048] The magnetic bead-coupled chlorfenapyr antigen probe was prepared using streptavidin magnetic beads purchased from Chongqing Bolanying Biotechnology Co., Ltd. The specific operation is as follows: take 250 μL of streptavidin magnetic bead solution (20 g / L) and add it to a 1.5 mL centrifuge tube, add 1 mL of magnetic bead buffer, place it on a magnetic rack, let it stand for 15 seconds, and remove the supernatant. Repeat the above operation twice, transfer the magnetic beads to a 15 mL centrifuge tube, and then add 4800 μL of magnetic bead buffer and 200 μL of biotinylated CPPU-BSA (2 mg / mL) in sequence to make the final concentration of the magnetic beads 1 mg / mL, and place it at room temperature for 30 minutes of shaking reaction. After the reaction is completed, place it on a magnetic rack, wash it repeatedly with magnetic bead buffer 3 times, and finally resuspend it in 5 mL of magnetic bead buffer, and dispense it into 2 mL centrifuge tubes at 1 mL / tube. The prepared "magnetic bead-antigen probe" is stored at 4 ° C for use.

[0049] (3) Preparation of anti-chlorfenapyr monoclonal antibody labeled with horseradish peroxidase

[0050] The HRP-coupled anti-chlorfenuron monoclonal antibody was prepared using the horseradish peroxidase labeling kit purchased from Proteintech. The specific operation is as follows: 200 μL of anti-chlorfenuron monoclonal antibody (0.5 mg / mL) was added to a 1.5 mL centrifuge tube, 20 μL of reaction initiation solution was added, mixed, and then 100 μg of HRP was added. After fully mixing, the reaction was placed at 37°C for 3 hours. Then 20 μL of reaction termination solution was added, mixed, and placed at 37°C for 1 hour. After termination, an equal volume of product protection solution was added, mixed, and stored at -20°C.

[0051] (4) Establishment of magnetic bead ELISA method

[0052] Block the ELISA microplate with PBST solution containing 5% skimmed milk powder, 200μL / well, and block for 1h. Wash the plate three times with PBST, add 10μL of "magnetic beads-antigen probe" diluted 10 times with PBS, 50μL of chlorfenapyr standard or sample solution and 50μL of HRP-labeled anti-chlorfenapyr monoclonal antibody diluted 3200 times with PBS to each well, and react at 37℃ for 20min. Place the 96-well plate on a magnetic stand and repeat the enrichment and washing three times with 160μL PBST solution. Add 100μL of freshly prepared TMB substrate solution to each well, develop color at 37℃ for 15min, and add 50μL of 2mol / L HCl to terminate the reaction after color development. Detect the absorbance of each well at 450nm on an enzyme reader, and calculate the content of chlorfenapyr in the sample based on the absorbance value measured for the sample. The results are as follows: Figure 2 shown.

[0053] from Figure 2 It can be seen that its IC 50 It is 0.0061ng / mL, with high sensitivity.

[0054] (5) Detection of chlorfenapyr by magnetic bead ELISA method

[0055] Grapes and kiwifruit were selected as test samples. The samples to be tested (without skin removed) were chopped and put into a juicer for homogenization. Three homogenates of each fruit were weighed, 10 g each. 10 μg / kg, 20 μg / kg and 50 μg / kg of chlorfenapyr standards were added to the samples, vortexed for 1 min and left to stand for 1 h. Then 10 mL of acetonitrile was added to each sample, vortexed for 2 min, and placed in an ultrasonic cleaner for 25 min. 4.00 g of anhydrous sodium sulfate and 1.00 g of NaCl were weighed and added to the ultrasonicated fruit samples, vortexed for 2 min, and then centrifuged at 5000 rpm for 8 min at 4 °C. 1 mL of the supernatant of each kiwifruit and grape sample was aspirated, diluted 100 times, and then magnetic bead ELISA was performed.

[0056] Test results: The magnetic bead ELISA method was used to carry out the addition recovery test of the above-mentioned grape and kiwi samples. The recovery rates of grape samples were 120.0%, 101.5% and 93.0%, respectively, and the recovery rates of kiwi samples were 86.0%, 96.5% and 90.2%, respectively.

[0057] In summary, the anti-chlorfenapyr monoclonal antibody secreted by the hybridoma cell 14G1 of the present invention can effectively identify chlorfenapyr, and can be applied to the establishment and actual detection of chlorfenapyr such as ELISA immunoassay methods, which is of great significance for the monitoring of chlorfenapyr in the field of food safety. SEQUENCE LISTING <110> Institute of Precision Measurement Science and Technology Innovation, Chinese Academy of Sciences Hubei Optics Valley Laboratory <120> Hybridoma cell line 14G1 and monoclonal antibodies produced therefrom and their applications <160> 10 <210> 1 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 1 gtccaactgc agcagtcagg acctgagctg gtaaggcctg ggacttcact gaaggtgtcc 60 tgcaaggctt ctggatacgc cttcactaat tactggatag actggataaa acagaggcct 120 ggacagggcc ttgagtggat tggggtgatt aatcctggaa gtggtggcac tcacttcaat 180 gagaagttca ggggcaaggc aacactgact gcagacaaat cctccagcac tgcctacatg 240 cagctcagca gcctgacatc tgatgactct gcggtctatt tctgtgcaag attgagtagc 300 cgtttctact ttgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 2 <211> 324 <212> DNA <213> Mouse (Mus musculus) <400> 2 gacattgagc tcacccagtc tcacaaattc atgtccacat cagtaggaga cagagtcagc 60 atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acagaaacca 120 ggacaatctc ctaaactact gatttactcg gcatcctacc gcttcactgg agtccctgat 180 cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240 gaagacctgg cagtttatta ctgtcagcaa cattatacta ctccgctcgc gttcggtgct 300 ggcaccaagc tggagctgaa acgg 324 <210> 3 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 3 Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20 25 30 Tyr Trp Ile Asp Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu 35 40 45 Trp Ile Gly Val Ile Asn Pro Gly Ser Gly Gly Thr His Phe Asn 50 55 60 Glu Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser 65 70 75 Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser 80 85 90 Ala Val Tyr Phe Cys Ala Arg Leu Ser Ser Arg Phe Tyr Phe Asp 95 100 105 Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 110 115 <210> 4 <211> 108 <212> PRT <213> Mouse (Mus musculus) <400> 4 Asp Ile Glu Leu Thr Gln Ser His Lys Phe Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Phe Thr Gly Val Pro Asp 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile 65 70 75 Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 80 85 90 His Tyr Thr Thr Pro Leu Ala Phe Gly Ala Gly Thr Lys Leu Glu 95 100 105 Leu Lys Arg <210> 5 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 5 Gly Tyr Ala Phe Thr Asn Tyr Trp 1 5 <210> 6 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 6 Ile Asn Pro Gly Ser Gly Gly Thr 1 5 <210> 7 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 7 Ala Arg Leu Ser Ser Arg Phe Tyr Phe Asp Tyr 1 5 10 <210> 8 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 8 Gln Asp Val Ser Thr Ala 1 5 <210> 9 <211> 3 <212> PRT <213> Mouse (Mus musculus) <400> 9 Ser Ala Ser 1 <210> 10 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 10 Gln Gln His Tyr Thr Thr Pro Leu Ala 1 5

Claims

1. Hybridoma cell line 14G1, characterized in that: The deposit number of the hybridoma cell line is CCTCC NO: C2021201.

2. Anti-chlorfenapyr monoclonal antibody, characterized in that: The monoclonal antibody is secreted and produced by the hybridoma cell line according to claim 1.

3. Use of the anti-forchlorfenuron monoclonal antibody according to claim 2 in determining the content of forchlorfenuron.

4. Use of the anti-forchlorfenuron monoclonal antibody according to claim 2 in enrichment or purification of forchlorfenuron, characterized in that: The anti-chlorfenapyr monoclonal antibody is coupled with agarose gel and silica gel microspheres to prepare an immunoaffinity chromatography column, and the chlorfenapyr is enriched or purified by using the immunoaffinity chromatography column.

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