Hybridoma cell line, monoclonal antibody and use thereof

Abstract

The application provides a hybridoma cell strain, a monoclonal antibody and application, and belongs to the technical field of food safety immune detection. The hybridoma cell strain is named 11D8, is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No.45937. The monoclonal antibody secreted by the hybridoma cell strain can simultaneously and specifically recognize fluroxypyr and fluroxypyr-meptyl, has good detection sensitivity for fluroxypyr and fluroxypyr-meptyl, can realize detection of the residual amount of fluroxypyr and fluroxypyr-meptyl in food, and provides a new means for establishing a rapid, simple, inexpensive, sensitive and specific fluroxypyr and fluroxypyr-meptyl detection method.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety immune detection, and particularly relates to a hybridoma cell strain, a monoclonal antibody and application. BACKGROUND

[0002] Clofencet and clofencet-isoctyl are pyridyloxyacetate herbicides, have internal conductive effect, have typical hormone herbicide reaction, and are widely used due to large production. Clofencet-isoctyl can be rapidly hydrolyzed into the parent substance clofencet in the plant body after application.

[0003] At present, the methods for detecting the residues of clofencet and clofencet-isoctyl mainly focus on instrument detection, such as high performance liquid chromatography (LC), high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), gas chromatography (GC), gas chromatography-tandem mass spectrometry (GC-MS / MS) and the like. However, the above methods need professional operators, large instruments and equipment, complex sample pretreatment and time-consuming analysis process, and have high detection cost, which is not conducive to the detection of clofencet and clofencet-isoctyl. The immune analysis method has the advantages of rapidness, simplicity, real-time, easy on-site detection, simple sample pretreatment, high sensitivity, strong selectivity, suitability for high-throughput analysis and the like due to the specific recognition of antigens and antibodies. However, there is currently a lack of monoclonal antibodies that can specifically recognize the broad spectrum of clofencet and clofencet-isoctyl. SUMMARY

[0004] The application aims to provide a hybridoma cell strain, a monoclonal antibody and application, and the monoclonal antibody of the application can specifically recognize the broad spectrum of clofencet and clofencet-isoctyl.

[0005] The application provides a hybridoma cell strain, which is named 11D8, is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No.45937.

[0006] The application also provides application of the hybridoma cell strain in the preparation of a monoclonal antibody that can specifically recognize clofencet and clofencet-isoctyl.

[0007] The application also provides a monoclonal antibody, which is secreted by the hybridoma cell strain or a subculture strain thereof.

[0008] The application further provides a monoclonal antibody, wherein an amino acid sequence of a heavy chain variable region of the monoclonal antibody comprises: a heavy chain variable region CDR1 as shown in SEQ ID NO. 1, a heavy chain variable region CDR2 as shown in SEQ ID NO. 2 and a heavy chain variable region CDR3 as shown in SEQ ID NO. 3; and an amino acid sequence of a light chain variable region of the monoclonal antibody comprises: a light chain variable region CDR1 as shown in SEQ ID NO. 4, a light chain variable region CDR2 as shown in SEQ ID NO. 5 and a light chain variable region CDR3 as shown in SEQ ID NO. 6.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 7; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 8.

[0010] The application further provides a coding gene of the monoclonal antibody, wherein a nucleotide sequence of a heavy chain variable region of the coding gene of the monoclonal antibody comprises: a heavy chain variable region CDR1 as shown in SEQ ID NO. 9, a heavy chain variable region CDR2 as shown in SEQ ID NO. 10 and a heavy chain variable region CDR3 as shown in SEQ ID NO. 11; and a nucleotide sequence of a light chain variable region of the coding gene of the monoclonal antibody comprises: a light chain variable region CDR1 as shown in SEQ ID NO. 12, a light chain variable region CDR2 as shown in SEQ ID NO. 13 and a light chain variable region CDR3 as shown in SEQ ID NO. 14.

[0011] Preferably, the gene sequence of the heavy chain variable region of the coding gene of the monoclonal antibody is shown in SEQ ID NO. 15; and the gene sequence of the light chain variable region of the coding gene of the monoclonal antibody is shown in SEQ ID NO. 16.

[0012] The application further provides a kit or test strip comprising the monoclonal antibody.

[0013] The application further provides application of the hybridoma cell strain, the monoclonal antibody, the coding gene or the kit or test strip in detection of chlorofenoxyfop and / or chlorofenoxyfop-isooctyl.

[0014] The application further provides a method for detecting chlorofenoxyfop and / or chlorofenoxyfop-isooctyl by indirect competitive ELISA, comprising the following steps:

[0015] The sample to be detected is used as a coating antigen, and the monoclonal antibody is used as a primary antibody for indirect competitive ELISA detection.

[0016] The application provides a hybridoma cell strain, which is named 11D8, and is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No.45937. The monoclonal antibody secreted by the hybridoma cell strain of the application can specifically recognize both fluroxypyr and fluroxypyr-meptyl and has good detection sensitivity for fluroxypyr and fluroxypyr-meptyl, and can realize the detection of the residual amount of fluroxypyr and fluroxypyr-meptyl in food, and provides a new method for establishing a rapid, simple, inexpensive, sensitive and specific fluroxypyr and fluroxypyr-meptyl detection method.

[0017] Biological preservation instructions

[0018] The mouse hybridoma cell strain 11D8 is preserved in the China General Microbiological Culture Collection Center, has a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing, a preservation number of CGMCC No.45937, and a preservation date of June 4, 2024. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 is a MALDI-TOF-MS spectrum of an ovalbumin (OVA) standard and OVA with a molar ratio (coupling ratio) of fluroxypyr and fluroxypyr-meptyl hapten B of 1:30;

[0021] Figure 2 is a MALDI-TOF-MS spectrum of an ovalbumin (OVA) standard and OVA with a molar ratio (coupling ratio) of fluroxypyr and fluroxypyr-meptyl hapten B of 1:40;

[0022] Figure 3 is a MALDI-TOF-MS spectrum of an ovalbumin (OVA) standard and OVA with a molar ratio (coupling ratio) of fluroxypyr and fluroxypyr-meptyl hapten B of 1:50;

[0023] Figure 4 is a MALDI-TOF-MS spectrum of an ovalbumin (OVA) standard;

[0024] Figure 5MALDI-TOF-MS spectrum of a bovine serum albumin (BSA) standard and BSA with a molar ratio (coupling ratio) of 1:30 of chlorofenoxyfop and chlorofenoxyfop-ethyl hapten B;

[0025] Figure 6 MALDI-TOF-MS spectrum of a bovine serum albumin (BSA) standard and BSA with a molar ratio (coupling ratio) of 1:40 of chlorofenoxyfop and chlorofenoxyfop-ethyl hapten B;

[0026] Figure 7 MALDI-TOF-MS spectrum of a bovine serum albumin (BSA) standard and BSA with a molar ratio (coupling ratio) of 1:50 of chlorofenoxyfop and chlorofenoxyfop-ethyl hapten B;

[0027] Figure 8 MALDI-TOF-MS spectrum of a bovine serum albumin (BSA) standard;

[0028] Figures 1-8 The abscissa is m / z (mass-to-charge ratio), and the ordinate is signal intensity (arbitrary unit);

[0029] Figure 9 Detection results of subtypes of monoclonal antibodies produced by hybridoma cell strain 11D8;

[0030] Figure 10 Standard curve of broad-spectrum specific monoclonal antibodies produced by hybridoma cell strain 11D8. DETAILED DESCRIPTION

[0031] The present application provides a hybridoma cell strain, which is named 11D8, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.45937.

[0032] The hybridoma cell strain of the present application can secrete monoclonal antibodies capable of simultaneously recognizing chlorofenoxyfop and chlorofenoxyfop-ethyl.

[0033] In the present application, chlorofenoxyfop, also known as fluroxypyr, fluroxypyr-meptyl, mefliodol and fentrazamide, has a chemical name of 4-amino-3,5-dichloro-6-fluoro-pyridine-2-oxoacetic acid, a CAS number of 69377-81-7, a molecular formula of C7H5O3N2FCL2, a relative molecular weight of 255, and a chemical structural formula I:

[0034]

[0035] In the present application, the chlorofenpyr is clofencet, 1-methylheptyl ester, ((4-amino-3,5-dichloro-6-fluoro-2-pyridinyl)oxy)-acetic acid 1-methylheptyl ester, or fluroxypyr-meptyl, with CAS number 81406-37-3, molecular formula C 15 H 21 Cl2FN2O3, relative molecular weight 367.24, and chemical structure shown in formula II:

[0036]

[0037] In the present application, the hybridoma cell strain is preferably prepared by the following method:

[0038] The chlorofenpyr is used as a hapten; the hapten is coupled with a carrier protein to obtain a complete antigen; the complete antigen is mixed with Freund's adjuvant for emulsification, and mice are immunized subcutaneously to screen mice with good immunization effect; and the spleen cells and myeloma cells of the mice with good immunization effect are fused to obtain a hybridoma cell strain.

[0039] In the present application, the chlorofenpyr is used as a hapten (hereinafter referred to as hapten A), with the structure shown in formula A:

[0040]

[0041] In the present application, as a hapten, an active group or an active group with a connecting arm group must be provided, and the active group is preferably a carboxyl group, an amino group, a hydroxyl group or an ester group; the selected chlorofenpyr structure itself has a carboxyl group structure.

[0042] In the present application, the hapten is coupled with a carrier protein to obtain a complete antigen. In the present application, the carrier protein is preferably bovine serum albumin or egg white albumin.

[0043] In the present application, the chlorofenpyr is preferably connected to the amino group of the carrier protein by an activated ester method, and after the reaction is completed, the complete antigen and the uncoupled small molecule hapten are separated by dialysis; and the complete antigen is identified by MALDI-TOF-MS method.

[0044] In the present application, the -COOH on formula A is activated to form an activated intermediate of hapten A by using N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide hydrochloride (EDC) under the action of N,N-dimethylformamide, so as to facilitate the coupling with the carrier protein and improve the success rate of coupling; and the chemical structure of the activated intermediate of hapten A is shown in formula B:

[0045]

[0046] In the present application, when the carrier protein is OVA, the complete antigen of the fluroxypyr and fluroxypyr-hexylester has the structure shown in formula C1 (denoted as fluroxypyr and fluroxypyr-hexylester complete antigen C1); when the carrier protein is BSA, the fluroxypyr and fluroxypyr-hexylester has the structure shown in formula C2 (denoted as fluroxypyr and fluroxypyr-hexylester complete antigen C2):

[0047]

[0048] In the present application, the molar ratio of the activated intermediate of hapten A to carrier protein is preferably (10-80):1, and more preferably (30-60):1. In the present application, the buffer preferably comprises carbonate buffer, phosphate buffer (PBS buffer), borate buffer or 4-hydroxyethylpiperazineethanesulfonic acid buffer, and more preferably PBS buffer. The amount of the buffer used as solvent can be determined according to the need of ensuring the smooth progress of the coupling reaction, and the present application does not make special limitations on this. In the present application, the mixing method of the activated intermediate of hapten A, carrier protein and buffer is preferably as follows: mixing the carrier protein and buffer to obtain a carrier protein buffer solution, and then adding the activated intermediate of hapten A. The pH value of the carrier protein buffer solution is preferably 5-9, and more preferably 7.4. In the present application, the temperature of the coupling reaction is preferably 0-50°C, and more preferably 4-25°C; the time is preferably 4-36 h, and more preferably 4-12 h; and the coupling reaction is preferably carried out under stirring. After the coupling reaction, the present application preferably performs dialysis on the obtained product system to obtain the fluroxypyr and fluroxypyr-hexylester complete antigen having the structure shown in formula C. In the embodiments of the present application, PBS buffer is used as the solvent for the coupling reaction, and the dialysis liquid used after the coupling reaction is preferably PBS buffer, the pH value of the PBS buffer is preferably 7-10, and more preferably 7.4; the concentration of the PBS buffer is preferably 0.01-0.2 mol / L, and more preferably 0.01 mol / L; the number of dialysis is preferably 5-7 times, and more preferably 6 times; and the time of each dialysis is preferably 3-5 h, and more preferably 4 h.

[0049] After obtaining the complete antigen, the present application mixes and emulsifies the complete antigen with Freund's complete adjuvant, and immunizes mice subcutaneously to screen mice with good immune effect. In the specific implementation process of the present application, the first immunization is mixed and emulsified with immunogen and complete Freund's adjuvant, the multiple booster immunization is mixed and emulsified with immunogen and incomplete Freund's adjuvant, and finally the immunogen is diluted with PBS and injected intraperitoneally for priming immunization.

[0050] In the embodiment of the present application, the mice are preferably 6-8 weeks old BALB / c mice. After the immunogen is completely emulsified with Freund's complete adjuvant, the mice are immunized by subcutaneous injection at multiple points, the first immunization is performed with Freund's complete adjuvant, the booster immunization is performed with Freund's incomplete adjuvant, and the priming immunization is performed without adjuvant, and the mice are directly injected intraperitoneally after being mixed with normal saline; the interval between each immunization is two weeks. After the third immunization, the serum titer and inhibition are detected by blood sampling after one week.

[0051] After the mice with good immunization effect are obtained, the spleen cells and myeloma cells of the mice with good immunization effect are fused to obtain a hybridoma cell strain.

[0052] In the embodiment of the present application, the method for fusing the spleen cells and myeloma cells is preferably a polyethylene glycol (PEG 2000) method; after the fusion, the positive cell wells are detected by HAT medium culture and indirect ELISA, and the inhibition effect of the positive cell wells is further determined by indirect competitive ELISA method, and the positive cell wells with the best inhibition are subcloned three times by limited dilution method, and finally the hybridoma cell strain 11D8 is screened.

[0053] After the hybridoma cell strain is obtained, the hybridoma cell strain is preferably identified; the method for identification preferably comprises: determining the antibody subtype, IC 50 value, cross-reactivity and affinity; the antibody subtype is preferably determined by using Sigma's mouse monoclonal antibody typing test strip; the determination of the IC 50 value, cross-reactivity and affinity is preferably performed by indirect competitive ELISA method (ic-ELISA method).

[0054] The present application also provides a simultaneous anti-fluroxypyr and fluroxypyr-hexyl ester antibody, which is prepared from the fluroxypyr and fluroxypyr-hexyl ester complete antigen in the above technical solution. In the present application, the anti-fluroxypyr antibody is preferably anti-fluroxypyr serum.

[0055] The fluroxypyr and fluroxypyr-hexyl ester complete antigen is used to immunize a host animal, the spleen cells are separated from the host animal, the spleen cells are fused with SP2 / 0 tumor cells in vitro, a hybridoma cell strain capable of secreting a simultaneous anti-fluroxypyr and fluroxypyr-hexyl ester antibody is screened, and a monoclonal antibody against fluroxypyr and fluroxypyr-hexyl ester is prepared based on the hybridoma cell strain; or, the mRNA transcribed by B lymphocytes in the blood of the host animal is extracted, reverse transcribed into cDNA, and a monoclonal antibody against fluroxypyr and fluroxypyr-hexyl ester is screened by using a phage or yeast antibody expression library constructed by biological engineering technology.

[0056] The application also provides the use of the hybridoma cell strain described in the above scheme in the preparation of a monoclonal antibody that can simultaneously and specifically recognize both fluroxypyr and fluroxypyr-meptyl ester.

[0057] The application also provides a monoclonal antibody secreted by the hybridoma cell strain or its subculture cell strain described in the above scheme. The monoclonal antibody secreted by the hybridoma cell strain 11D8 of the application exhibits excellent affinity and sensitivity to fluroxypyr and fluroxypyr-meptyl ester, with IC 50 values of 1.95 ng / mL and 1.36 ng / mL, respectively.

[0058] The application also provides a monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody comprises: a heavy chain variable region CDR1 as shown in SEQ ID NO. 1, a heavy chain variable region CDR2 as shown in SEQ ID NO. 2, and a heavy chain variable region CDR3 as shown in SEQ ID NO. 3; and the amino acid sequence of the light chain variable region of the monoclonal antibody comprises: a light chain variable region CDR1 as shown in SEQ ID NO. 4, a light chain variable region CDR2 as shown in SEQ ID NO. 5, and a light chain variable region CDR3 as shown in SEQ ID NO. 6.

[0059] In the application, the amino acid sequence shown in SEQ ID NO. 1 is specifically RYWMH; the amino acid sequence shown in SEQ ID NO. 2 is specifically YINPSTGYTEYNQKFRD; the amino acid sequence shown in SEQ ID NO. 3 is specifically PKDDY; the amino acid sequence shown in SEQ ID NO. 4 is specifically SATSSVSSSYLH; the amino acid sequence shown in SEQ ID NO. 5 is specifically GTSNLAS; and the amino acid sequence shown in SEQ ID NO. 6 is specifically QQRSGYPLT.

[0060] In the application, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is preferably as shown in SEQ ID NO. 7; and the amino acid sequence of the light chain variable region of the monoclonal antibody is preferably as shown in SEQ ID NO. 8.

[0061] In the application, the amino acid sequence of the heavy chain variable region (FR1+CDR1+FR2+CDR2+FR3+CDR3+FR4) of the monoclonal antibody is as shown in SEQ ID NO. 7, and is specifically:

[0062] QVQLQQSGAELAKPGASVKMSCKASGYTIN RYWMH WVKQRPGQGLEWIG YINPSTGYTEYNQKFRD KATLTADKSSSTAYMQLSSLTSEDSAVYYCAI PKDDY WGQGTTLTVSS.

[0063] In the application, the amino acid sequence of (FR1+CDR1+FR2+CDR2+FR3+CDR3+FR4) of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 8, in particular:

[0064] ENVLTQSPAIMAASLGEKVPMTC SATSSVSSSYLH WYQQKSGTSPKLWIY GTSNLAS GV PARFSGSGAGISYSLTISSMEAENDATYYR QQRSGYPLT FGAGTKLELK.

[0065] In the application, the monoclonal antibody is preferably prepared by the following method:

[0066] Take BALB / c mice, inject paraffin oil intraperitoneally, then inject hybridoma cell strain 11D8 intraperitoneally, collect ascites after injection, purify the ascites, and store the obtained monoclonal antibody against chlorofenoxyfop and chlorofenoxyfop isooctyl at low temperature.

[0067] In the application, the monoclonal antibody of the above-mentioned scheme can specifically recognize chlorofenoxyfop and chlorofenoxyfop isooctyl at the same time. The monoclonal antibody of the application can be used for developing a combined immunoassay method for chlorofenoxyfop and / or chlorofenoxyfop isooctyl, and establishing a colloidal gold immunochromatographic test strip rapid detection method, which lays a foundation for the research and popularization of indirect competitive ELISA kit and colloidal gold test strip.

[0068] The application also provides a coding gene of the monoclonal antibody of the above-mentioned scheme, the nucleotide sequence of the heavy chain variable region of the coding gene of the monoclonal antibody comprises: the heavy chain variable region CDR1 shown in SEQ ID NO. 9, the heavy chain variable region CDR2 shown in SEQ ID NO. 10 and the heavy chain variable region CDR3 shown in SEQ ID NO. 11; the nucleotide sequence of the light chain variable region of the coding gene of the monoclonal antibody comprises: the light chain variable region CDR1 shown in SEQ ID NO. 12, the light chain variable region CDR2 shown in SEQ ID NO. 13 and the light chain variable region CDR3 shown in SEQ ID NO. 14.

[0069] In the application, the nucleotide sequence shown in SEQ ID NO. 9 is specifically: aggtactggatgcac ; the nucleotide sequence shown in SEQ ID NO. 10 is specifically:tacattaatcctagcactggttatactgagtacaatcagaagttcagg gac ; The nucleotide sequence shown in SEQ ID NO.11 is specifically: cctaaggatgactac ; The nucleotide sequence shown in SEQ ID NO.12 is specifically: agtgccacctcaagtgtaagttccagctacttgcac ; The nucleotide sequence shown in SEQ ID NO.13 is specifically: ggcacatccaacctggcttct ; The nucleotide sequence shown in SEQ ID NO.14 is specifically: cagcagcggagtggttatccgctcacg .

[0070] In the present invention, the gene sequence of the heavy chain variable region of the gene encoding the monoclonal antibody is preferably shown as SEQ ID NO.15; the gene sequence of the light chain variable region of the gene encoding the monoclonal antibody is preferably shown as SEQ ID NO.16.

[0071] In the present invention, the nucleotide sequence shown in SEQ ID NO.15 is specifically:

[0072] caggtccaacttcagcagtctggggctgaactggcaaaacctggggcctcagtgaagatgtcctgcaaggcttctggctacaccattaat aggtactggatgcac tgggtaaaacagaggcctggacagggtctggaatggattgga tacattaatcctagcactggttatactgagtacaatcagaagttcagggac aaggccacattgactgcagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaatt cc taaggatgactac tggggccaaggcaccactctcacagtctcctca.

[0073] In the present invention, the nucleotide sequence shown in SEQ ID NO.16 is specifically:

[0074] gaaaatgtgctcacccagtctccagcaataatggctgcctctctggggggagaaggtccccatgacctgc agtgccacctcaagtgtaagttccagctacttgcac tggtaccagcagaagtcaggcacttcccccaaactctggatttat ggcacatccaacctggcttctggagtcccag ctcgcttcag tggcagtggg gctgggatct cttactctct cacaatcagc agcatggagg ctgaaaatga tgcaacttat taccgc cagcagcggagtggttatccgctcacg ttcggtgctg ggaccaagct ggagctgaaa.

[0075] The application further provides a kit or test strip, comprising the monoclonal antibody described in the above scheme.

[0076] In the application, the kit is preferably an indirect competitive ELISA kit; and the test strip is preferably a colloidal gold test strip.

[0077] In the application, the kit preferably further comprises an enzyme-labeled plate, a coating antigen, a fluroxypyr standard, an enzyme-labeled secondary antibody and a substrate reaction solution.

[0078] In the application, the enzyme-labeled secondary antibody is preferably a goat anti-mouse IgG horseradish peroxidase; and the substrate reaction solution is preferably o-phenylenediamine.

[0079] The application further provides application of the hybridoma cell strain, the monoclonal antibody, the coding gene or the kit or test strip described in the above scheme in detection of fluroxypyr and / or fluroxypyr-octylester.

[0080] In the application, the sample to be detected preferably comprises food; and the fluroxypyr and / or fluroxypyr-octylester to be detected preferably comprises fluroxypyr and / or fluroxypyr-octylester residues in food.

[0081] The application further provides an indirect competitive ELISA method for detecting fluroxypyr and / or fluroxypyr-octylester, comprising the following steps:

[0082] The sample to be detected is used as a coating antigen, and the monoclonal antibody described in the above scheme is used as a primary antibody to perform indirect competitive ELISA detection.

[0083] The application provides a method for efficiently and sensitively detecting fluroxypyr and / or fluroxypyr-octylester residues, which has important application value.

[0084] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application and the discovered hapten or the developed complete antigen by using the method, those skilled in the art can easily think of using a suitable antibody preparation method to prepare the antibody against both the chlorofenoxyfuran and the chlorofenoxyfuran isooctyl ester, regardless of the animal used for immunization, regardless of the setting or change of the immunization conditions or parameters, as long as the discovered hapten or the developed complete antigen in the present application is used, which belongs to the protection scope of the present application

[0085] To make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be purchased in the market.

[0086] Sources of some materials in the examples: Sodium bicarbonate, ethanol and dichloromethane are purchased from Beijing Chemical Plant; N, N-diisopropyl ethyl amine (DIPEA) and 3-aminobutyric acid are purchased from Beijing Coupling Technology Co., Ltd.; Hydrochloric acid, sodium dihydrogen phosphate dodecahydrate, sodium chloride, gelatin, citric acid monohydrate and Tween-20 are purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; Anhydrous N, N dimethyl formamide (DMF) is purchased from Aladdin; 1-ethyl-(3-dimethyl aminopropyl) carbonyl diimide hydrochloride (EDC), N-hydroxy succinimide (NHS), Freund's complete adjuvant, Freund's incomplete adjuvant, bovine serum albumin (BSA) and egg white albumin (OVA) are purchased from Sigma Company; Goat anti-mouse IgG-HRP is purchased from Jackson Company.

[0087] Embodiment 1

[0088] The activated intermediate of the hapten A is prepared by selecting chlorofenoxyfuran (haptene A), and the reaction formula is as follows:

[0089]

[0090] Chlorofenoxyfuran (24.34 mg, 0.096 mmol), N-hydroxy succinimide (NHS, 22.10 mg, 0.192 mmol) and 1-ethyl-(3-dimethyl aminopropyl) carbonyl diimide hydrochloride (EDC, 36.81 mg, 0.237 mmol) are fully dissolved in anhydrous N, N-dimethyl formamide (DMF, 2 mL), and the reaction is magnetically stirred in a 4℃ refrigerator for 10 h; after the reaction is completed, the product system containing the activated intermediate of the hapten A is obtained, which can be directly used for the coupling of the subsequent carrier protein without post-treatment.

[0091] Example 2

[0092] Preparation of the complete antigen C1 of the chlorofenoxyfuranic acid and chlorofenoxyfuranic acid isooctyl ester, the reaction formula is as follows:

[0093]

[0094] Take four groups of ovalbumin (OVA), 10 mg each, and set the molar ratio (coupling ratio) of OVA to the activated intermediate of hapten A prepared in Example 1 to be 1:30, 1:40, 1:50 respectively, dissolve in phosphate buffer (PBS buffer, pH value is 7.4, 1 mL) to obtain a carrier protein solution; under stirring conditions, the product system containing the activated intermediate of hapten A prepared in Example 1 is added dropwise to the carrier protein solution, and the reaction is stirred at 25°C for 4h; after the reaction is completed, the obtained product system is dialyzed with PBS buffer (pH value is 7.4, concentration is 0.01 mol / L) for 6 times, and each dialysis time is 4h, to obtain a solution containing the complete antigen C1 of the chlorofenoxyfuranic acid and chlorofenoxyfuranic acid isooctyl ester, and the obtained solution is dialyzed with pure water and then subjected to MALDI-TOF-MS determination, and according to the results, the appropriate chlorofenoxyfuranic acid and chlorofenoxyfuranic acid isooctyl ester complete antigen C1 solution is selected and diluted with PBS buffer (pH value is 7.4, concentration is 0.01 mol / L) to a chlorofenoxyfuranic acid and chlorofenoxyfuranic acid isooctyl ester complete antigen C1 concentration of 1 mg / mL, and the obtained diluent is subjected to liquid nitrogen quick freezing and stored at -20°C for use.

[0095] Figure 4 The MALDI-TOF-MS spectrum of the OVA standard is shown in the figure, and the result shows that the single-charge ion peak of OVA is 44578.958; Figures 1-3 The MADLI-TOF-MS spectrum of the coupling complete antigen C1 of the chlorofenoxyfuranic acid and chlorofenoxyfuranic acid isooctyl ester hapten with different feeding ratios is shown in the figure, and the result shows (see Table 1) that the coupling of the chlorofenoxyfuranic acid and chlorofenoxyfuranic acid isooctyl ester complete antigen C1 with a hapten feeding ratio of 40:1 has the best effect, and the single-charge ion peak is 46717.288. The coupling ratio of carrier protein to hapten = (molecular weight of complete antigen - molecular weight of carrier protein) / molecular weight of hapten, and the coupling ratio of the complete antigen C1 is calculated to be 1:8.38 according to the formula.

[0096] Example 3

[0097] Preparation of the complete antigen C2 of the chlorofenoxyfuranic acid and chlorofenoxyfuranic acid isooctyl ester, the reaction formula is as follows:

[0098]

[0099] Take four groups of bovine serum albumin (OVA, 10 mg each group), and set the molar ratio (coupling ratio) of OVA to the activated intermediate of hapten A prepared in Example 1 to be 1:30, 1:40, 1:50, respectively, to dissolve in PBS buffer (pH 7.4, 5 mL) to obtain a carrier protein solution; under stirring, dropwise add the product system containing the activated intermediate of hapten A prepared in Example 1 to the carrier protein solution, and stir at 25°C for 4 h; after the reaction is completed, dialyze the obtained product system with PBS buffer (pH 7.4, 0.01 mol / L) for 6 times, and each time for 4 h to obtain a solution containing chlorofenoxyfop and chlorofenoxyfop-isooctyl ester complete antigen C1, and then dialyze the obtained solution with pure water, and then perform MALDI-TOF-MS determination, and according to the results, select a suitable chlorofenoxyfop and chlorofenoxyfop-isooctyl ester complete antigen C1 solution, dilute with PBS buffer (pH 7.4, 0.01 mol / L) to a chlorofenoxyfop and chlorofenoxyfop-isooctyl ester complete antigen C1 concentration of 1 mg / mL, and then perform liquid nitrogen quick freezing on the obtained diluent, and store at -20°C for use.

[0100] Figure 8 The MALDI-TOF-MS spectrum of the BSA standard is shown in the figure, and the results show that the single-charge ion peak of BSA is 67208.345; Figures 5-7 The MADLI-TOF-MS spectrum of the chlorofenoxyfop and chlorofenoxyfop-isooctyl ester hapten different feeding ratio coupled complete antigen C2 is shown in the figure, and the results show (see Table 1) that the chlorofenoxyfop and chlorofenoxyfop-isooctyl ester complete antigen C2 obtained by coupling the hapten at a feeding ratio of 50:1 has the best effect, and the single-charge ion peak is 74119.262. The coupling ratio of carrier protein to hapten = (molecular weight of complete antigen - molecular weight of carrier protein) / molecular weight of hapten, and thus the coupling ratio of complete antigen C2 is calculated to be 1:27.10.

[0101] Table 1 Synthesis results of immunogen and coating source

[0102]

[0103] The MADLI-TOF-MS spectrum is abnormal, and the results are not calculated.

[0104] Example 4 Preparation of monoclonal antibody.

[0105] 1. Mouse immunization

[0106] (1) Take 6-8 week old Balb / C mice as experimental animals (8 week old Balb / c mice weigh 23-25 g).

[0107] (2) Primary immunization: Take the diluted solution of complete antigen C2 of clofencet and clofencet isooctyl ester (concentration of 1 mg / mL) in Example 3 as immunogen, filter through a sterile filter, add an equal volume of Freund's complete adjuvant, emulsify thoroughly until it does not spread when dropped into water, to obtain the emulsion of complete antigen C2 of clofencet and clofencet isooctyl ester; the immunization strategy of the emulsion of complete antigen C2 of clofencet and clofencet isooctyl ester is to inject 0.05 mg into the abdominal cavity of a mouse and inject 0.05 mg into the back subcutaneously in multiple points, and the total injection dose is 0.1 mg of the emulsion of complete antigen C2 of clofencet and clofencet isooctyl ester per mouse.

[0108] (3) Booster immunization: two weeks after the primary immunization, take 1 mL of the diluted solution of complete antigen C2 of clofencet and clofencet isooctyl ester (concentration of 1 mg / mL) in Example 3, then add an equal volume of Freund's incomplete adjuvant, emulsify thoroughly until it does not spread when dropped into water, to obtain the emulsion of complete antigen C2 of clofencet and clofencet isooctyl ester; inject the emulsion of complete antigen C2 of clofencet and clofencet isooctyl ester into the abdominal cavity of a mouse at 0.05 mg and inject into the back subcutaneously in multiple points at 0.05 mg, and the total injection dose is 0.1 mg of the emulsion of complete antigen C2 of clofencet and clofencet isooctyl ester per mouse; booster immunization is performed once every 14 days, and after each immunization for 3 days, blood is collected from the eye orbit of the mouse to detect the immunization performance of the complete antigen by measuring the antibody titer and the inhibition rate to clofencet; the coating antigen is 1 mg / mL of complete antigen C1 of clofencet and clofencet isooctyl ester, which is diluted in four gradients, i.e. 1000 times, 2000 times and 4000 times, and 8000 times; when the titer is equal to or greater than 1:64000 (the definition of titer is that when the OD value of the control well is 1.0 or more, the dilution multiple of the serum is 1:64000), and the inhibition rate reaches 50%, it indicates that the hapten structure is reasonable, the complete antigen is successfully synthesized, and the immunization performance is excellent; and the complete antigen can be used for the preparation of monoclonal antibodies.

[0109] 2. Screening of monoclonal cells.

[0110] (1) Resuscitation and expansion culture of myeloma cells: one week before cell fusion, the SP2 / 0 myeloma cells are resuscitated. The cells are taken out from the liquid nitrogen tank and quickly transferred to a 37℃ water bath for melting, and then transferred to a centrifuge tube containing preheated DMEM culture solution. After centrifugation and discarding the supernatant, the cells are resuspended and cultured at 37℃ and 5% CO2 until the cells grow to a suitable state for fusion.

[0111] (2) Preparation of feeder cells: One day before fusion, feeder cells were collected from the abdominal cavity of mice of the same strain. After the mice were properly euthanized, the abdomen was carefully cut open using sterilized tools, and DMEM culture solution was injected into the abdominal cavity and collected. After centrifugation and resuspension, the collected cells were cultured in a 96-well culture plate.

[0112] (3) Preparation of spleen cells: 24 h before fusion, the mice were stopped from feeding to reduce peripheral fat. After the mice were euthanized, blood was collected and the spleen was removed, and the fat and connective tissue were removed, and then the spleen cells were dispersed in DMEM.

[0113] (4) Cell fusion: PEG 2000 was used as a fusion agent, and spleen cells and myeloma cells were mixed in a certain proportion, and after adjusting the pH value, fusion was performed. The fused cells were resuspended in 2% HAT complete culture solution and cultured at 37°C, 5% CO2.

[0114] (5) Screening of specific hybridoma cells: The positive and specific hybridoma cells were detected by icELISA method. First, the positive cells were screened by indirect ELSIA method, and then the inhibition rate of chlorofenapyr was detected by indirect competitive ELISA method, and the high-specificity hybridoma cells were screened.

[0115] (6) Cloning of hybridoma cells: The high-specificity hybridoma cells were cloned by limited gradient dilution method. The diluted cells were cultured in the 96-well plate with feeder cells, and single clone cells were selected by microscope observation, and were cultured in large scale.

[0116] 3. Purification of antibodies.

[0117] (1) Preparation of monoclonal antibodies: The best monoclonal cell strain 11D8 was selected, and ascites antibodies were prepared by in vivo preparation of ascites tumor. BALB / c female mice were selected, and after pretreatment with paraffin oil, monoclonal cell suspension was injected into the abdominal cavity.

[0118] (2) Ascites collection and preliminary treatment: After the mouse abdomen swelled, the ascites was collected and centrifuged, and the clear liquid was used for subsequent purification.

[0119] (3) Purification by salting out: Salting out was used to remove non-specific impurities in ascites. First, the ascites was mixed with PBS, and saturated ammonium sulfate was added for precipitation, and then the precipitate was dissolved and precipitated again.

[0120] (4) Dialysis and freeze-drying: The purified antibody protein was dialyzed to remove excess salt and impurities, and then vacuum freeze-dried.

[0121] (5) Antibody storage: The antibody powder after lyophilization was dissolved with PBS, 50% glycerol was added, and a 1 mg / mL mother liquor was prepared and stored at -20°C.

[0122] 4. Fusion mouse titer and inhibition rate detection.

[0123] The blood of the fusion mouse was incubated in a 37°C constant temperature incubator for 30 min, then incubated in a 4°C refrigerator for 2 h, then centrifuged at 4°C, 10000 r / min for 5 min in a centrifuge, and the polyclonal antibody serum of propanil was separated and used in the following experiments.

[0124] The various buffers used in the following experiments are as follows:

[0125] (1) Coating buffer (pH = 9.6, 0.05M carbonate buffer): weigh Na2CO3 1.5 g and NaHCO3 2.94 g, and dilute to 1000 mL with ultrapure water;

[0126] (2) Phosphate buffer (0.01M, pH = 7.4): weigh KH2PO4 0.2 g, NaCl 8 g, and NaH2PO4·12H2O 2.92 g, and dilute to 1000 mL with ultrapure water;

[0127] (3) Washing buffer: add Tween-20 to the prepared phosphate buffer to make the volume fraction of Tween-20 0.1%;

[0128] (4) Sample diluent: add 10 mL of Tween-20 and 1 g of gelatin to the prepared phosphate buffer, heat in a microwave oven to melt, and dilute to 1 L with ultrapure water;

[0129] (5) Substrate buffer (pH = 5.5): weigh Na2HPO4·12H2O 9.22 g, citric acid monohydrate 2.55 g, and measure 0.5 mL of Tween-20, and dilute to 1 L with ultrapure water;

[0130] (6) Stop solution (2M H2SO4): measure 445.6 mL of distilled water, and add 54.4 mL of 98% (volume / volume) concentrated sulfuric acid dropwise under stirring conditions.

[0131] 5. Preparation of fluroxypyr standard:

[0132] (1) Weigh 10 mg of fluroxypyr standard separately, and dissolve in 10 mL of methanol to obtain a 1 mg / mL fluroxypyr standard stock solution;

[0133] (2) Use the sample diluent to dilute the 1 mg / mL fluroxypyr standard stock solution in step (1) to a final concentration of 1 μg / mL fluroxypyr standard solution.

[0134] Preparation of fluroxypyr coating stock solution:

[0135] After the prepared complete antigen is freeze-dried, the powder is dissolved in PBS, 50% glycerol is added, and a 1 mg / mL stock solution is prepared. The stock solution is gradient diluted to a suitable concentration in a 1:1000, 1:2000, 1:4000 pattern for determination.

[0136] Preparation of antiserum and antibody diluent:

[0137] The antiserum prepared in the above step is diluted with the sample and antibody stock solution, and the antiserum and antibody stock solution are gradient diluted to a suitable concentration in a 1:1000, 1:2000, 1:4000 pattern for determination.

[0138] 6. Checkerboard experiment of antigen and antiserum:

[0139] (1) Coating: Add 100 μL of the fluroxypyr complete antigen C1 coating antigen solution prepared in step 1 to each well of a 96-well enzyme-labeled plate, incubate at 37°C for 3 h, wash 3 times with washing buffer, and spin dry.

[0140] (2) Competition: In the control wells, add 50 μL of sample diluent to each well; in the inhibition wells, add 50 μL of the fluroxypyr standard solution prepared in step 2 to each well; add the antiserum diluent prepared in step 3 to the enzyme-labeled plate (50 μL / well), incubate at 37°C for 30 min, wash the plate 3 times with washing buffer, and spin dry.

[0141] Add enzyme-labeled secondary antibody: Dilute the goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson Company) 1000-fold with the sample diluent (0.1 M, pH 9.6), add 100 μL to each well, incubate at 37°C for 30 min, wash the plate 3 times with washing buffer, and spin dry.

[0142] (3) Color development: Prepare the color developing solution immediately before use. Add 15-20 mg of o-phenylenediamine (OPD) and 4 μL of 30 wt% hydrogen peroxide to 10 mL of substrate buffer, and add 100 μL of the resulting mixture to each well. Develop the color at room temperature in the dark for 15 min.

[0143] (4) Termination: Add 50 μL of termination solution to each well, and measure the OD value of each well at 492 nm with an enzyme-labeled instrument.

[0144] The formula for calculating the inhibition rate is: IR = (C - I) / C x 100%, IR represents the inhibition rate, I represents the inhibition well in the enzyme-labeled plate, and C represents the control well in the enzyme-labeled plate. The results of the third, fourth, and fusion mouse booster immunization antiserum titer are shown in Table 2.

[0145] Table 2 Anti-chlorofenprop-methyl mouse serum titer and inhibition rate detection

[0146]

[0147]

[0148] Note: In Table 2, I represents the inhibition well in the enzyme-labeled plate, C represents the control well in the enzyme-labeled plate, IR represents the inhibition rate, K represents 1000 times, the coating source concentration is 1 mg / mL, and the inhibition concentration is 1000 ng / mL (chlorofenprop-methyl).

[0149] The results in Table 2 show that after the fourth immunization, when the coating antigen is diluted 16000 times and the antibody is diluted 64000 times, the antiserum inhibition rate is best, reaching 55.94%. It is shown that the chlorofenprop-methyl and chlorofenprop-methyl isooctyl complete antigen C2 prepared in Example 3 above can be used as an immunogen to prepare anti-chlorofenprop-methyl antibodies.

[0150] The results of the titer and inhibition rate of the purified antibody are shown in Table 3.

[0151] Table 3 Monoclonal hybridoma 11D8 ascites titer and inhibition rate

[0152]

[0153]

[0154] Note: In Table 3, I represents the inhibition well in the enzyme-labeled plate, C represents the control well in the enzyme-labeled plate, IR represents the inhibition rate, K represents 1000 times, the coating source concentration is 1 mg / mL, and the inhibition concentration is 100 ng / mL (chlorofenprop-methyl).

[0155] The monoclonal antibody obtained by ascites purification was identified for the light and heavy chain subtypes of immunoglobulin using a mouse monoclonal antibody subtype identification test strip, and the heavy chain subtype was IgG3 type and the light chain subtype was κ type, as shown in Table 4. Figure 9The nucleotide sequence and amino acid sequence of the antibody light chain and heavy chain were determined according to the subtypes of the light chain and heavy chain of the antibody. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 7; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 8. In the present application, the genetic sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 15; the genetic sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 16.

[0156] The optimal coating concentration and antibody dilution concentration were selected, and the concentrations of chlorofenoxyfop and chlorofenoxyfop-isooctyl ester were diluted by gradient dilution method to 0.001, 0.003, 0.010, 0.030, 0.0915, 0.274, 0.823, 2.469, 7.407, 22.222, 66.667 and 200 ng / mL, and then the IC50 of the monoclonal antibody against chlorofenoxyfop and chlorofenoxyfop-isooctyl ester was determined by ic-ELISA. 50 The results were 1.95 ng / mL and 1.36 ng / mL, respectively. The regression curve equation for chlorofenoxyfop was Y=0.099+0.940 / (1+(X / 1.543)), R=0.998; and the regression curve equation for chlorofenoxyfop-isooctyl ester was Y=0.052+0.965 / (1+(X / 1.1778)), R=0.997, as shown in Table 2. 1.278 2 2 Figure 10 Further verification work showed that the cross-reactivity of the monoclonal antibody to the same pesticides such as picloram, dicamba, etc. and structurally similar compounds was less than 0.2%, and the specific data are shown in Table 4. These results confirmed that the monoclonal antibody produced by the cell strain not only has high sensitivity, but also has strong specificity, which can meet the current national pesticide residue limit detection regulation requirements.

[0157] Table 4 Specific detection of chlorofenoxyfop 11D8 monoclonal antibody

[0158]

[0159]

[0160] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments. Other embodiments can be obtained according to the present embodiment without creativity, which are within the protection scope of the present application.​​​

Claims

1. A hybridoma cell line, characterized in that: The hybridoma cell line was named 11D8 and deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC No.45937.

2. Use of the hybridoma cell line according to claim 1 in the preparation of monoclonal antibodies that specifically recognize both lofluanid and lofluanid ethyl ester.

3. A monoclonal antibody, characterized in that The monoclonal antibody is secreted and produced by the hybridoma cell line or its successive cell line according to claim 1.

4. A monoclonal antibody, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody includes: the heavy chain variable region CDR1 shown in SEQ ID NO.1, the heavy chain variable region CDR2 shown in SEQ ID NO.2, and the heavy chain variable region CDR3 shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region of the monoclonal antibody includes: the light chain variable region CDR1 shown in SEQ ID NO.4, the light chain variable region CDR2 shown in SEQ ID NO.5, and the light chain variable region CDR3 shown in SEQ ID NO.

6.

5. The monoclonal antibody according to claim 4, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

8.

6. The gene encoding the monoclonal antibody according to claim 4 or 5, characterized in that The nucleotide sequence of the heavy chain variable region of the gene encoding the monoclonal antibody includes: the heavy chain variable region CDR1 as shown in SEQ ID NO.9, the heavy chain variable region CDR2 as shown in SEQ ID NO.10, and the heavy chain variable region CDR3 as shown in SEQ ID NO.11; the nucleotide sequence of the light chain variable region of the gene encoding the monoclonal antibody includes: the light chain variable region CDR1 as shown in SEQ ID NO.12, the light chain variable region CDR2 as shown in SEQ ID NO.13, and the light chain variable region CDR3 as shown in SEQ ID NO.

14.

7. The monoclonal antibody encoding gene according to claim 6, characterized in that The gene sequence of the heavy chain variable region of the gene encoding the monoclonal antibody is shown in SEQ ID NO.15; the gene sequence of the light chain variable region of the gene encoding the monoclonal antibody is shown in SEQ ID NO.

16.

8. A kit or test strip, characterized in that: The monoclonal antibody comprises the monoclonal antibody according to any one of claims 3 to 5.

9. Use of the hybridoma cell line according to claim 1, the monoclonal antibody according to any one of claims 3 to 5, the encoding gene according to claim 6 or 7, or the kit or test strip according to claim 8 in the detection of lofluanid and / or lofluanid ethyl ester.

10. A method for detecting lofluanid and / or lofluanid 2-ethylhexyl ester by indirect competitive ELISA, characterized in that: The following steps are involved: The indirect competitive ELISA test is performed using the sample to be tested as the coating antigen and the monoclonal antibody according to any one of claims 3 to 5 as the primary antibody.

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