CN111763657A - Hybridoma cell strain secreting amitraz monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting amitraz monoclonal antibody and application thereof Download PDFInfo
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- CN111763657A CN111763657A CN202010650620.5A CN202010650620A CN111763657A CN 111763657 A CN111763657 A CN 111763657A CN 202010650620 A CN202010650620 A CN 202010650620A CN 111763657 A CN111763657 A CN 111763657A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
Abstract
A hybridoma cell strain secreting amitraz monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain RC for secreting the amitraz monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 19166. The amitraz monoclonal antibody secreted by the strain is used for analyzing and detecting amitraz residues in food safety detection. The monoclonal antibody cell strain RC of amitraz obtained by the invention can be used for immunoassay detection, and has better detection sensitivity to amitrazSensitivity and specificity (IC)50Value of 0.3ng/mL, crossover to amitraz analogue of less than 10%, crossover rate = (IC of amitraz)50IC of/analogue50）×100%。
Description
Technical Field
The invention relates to a hybridoma cell strain secreting amitraz monoclonal antibody and application thereof, belonging to the field of food safety immunodetection.
Background
Amitraz is also called mite, is a broad-spectrum amitraz insecticide and acaricide, has multiple poisoning and killing mechanisms, is mainly used for killing mites on crops such as fruit trees, vegetables, tea, cotton, soybeans and the like, is effective on partial lepidoptera pest eggs, has contact killing and fumigating effects on pest mites, can be used for preventing and treating acarids of livestock such as cattle and sheep and can be widely used for removing mites in honeycombs because amitraz can effectively prevent and treat bee diseases caused by varroa destructor and has low toxicity on bees. The amitraz has toxicity to human and livestock, irritation to skin and mucosa, and inhibition to central nervous system, and has no specific drug after poisoning, and its main hydrolysis metabolite 2, 4-dimethylaniline has high toxicity, and has various potential hazards such as carcinogenesis and mutation. Therefore, countries and regions such as australia, japan, korea, etc. have made strict regulations on the amount of residual amitraz in foods. For example, the European Union, Japan and United states stipulate that the limit of amitraz in bee products is 0.2 mg/kg; the Chinese national standards also stipulate the maximum residual limit of amitraz in food: 0.05-0.5 mg/kg.
For detecting the amitraz pesticide residue, a high performance liquid chromatography, a gas chromatography-mass spectrometry or a liquid chromatography-mass spectrometry combined method is usually adopted. However, these methods have the disadvantages of complicated sample pretreatment and long detection time, and are not suitable for rapid detection of a large number of samples, and in order to maintain the benefits of consumers, it is necessary to establish an efficient and rapid detection method for amitraz. The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, has the advantages of simple pretreatment of a sample during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for field rapid detection of a large number of samples, so the ELISA is widely applied to pesticide residue analysis. The precondition of using an enzyme linked immunosorbent assay to detect the amitraz is that a monoclonal antibody with high specificity and high sensitivity to the amitraz is obtained, so that the key is to find a method for preparing the monoclonal antibody with high specificity and high sensitivity to the amitraz.
Disclosure of Invention
The invention aims to overcome the defects and provide a hybridoma cell strain RC secreting the monoclonal antibody of amitraz and application thereof. The monoclonal antibody of amitraz secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC) to amitraz50A value of 0.3 ng/mL) can be used for establishing an immunological detection method of amitraz and detecting the amitraz residue in food.
The technical scheme of the invention is as follows: a hybridoma cell strain RC secreting amitraz monoclonal antibodies is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences (China institute of microbiology, institute of sciences, No. 3, Xilu No.1, North Cheng, south China, Beijing, and the like, and is classified and named as a monoclonal cell strain, wherein the preservation date is 2019, 11 and 28 days, and the preservation number is CGMCC No. 19166.
The amitraz monoclonal antibody is secreted and produced by the hybridoma cell strain RC with the preservation number of CGMCC number 19166.
The amitraz monoclonal antibody is applied to analysis and detection of amitraz residues in food safety detection.
The invention provides a preparation method of a hybridoma cell strain RC secreting a amitraz monoclonal antibody, which comprises the following steps:
(1) using amitraz hapten to prepare amitraz complete antigen, and preparing the obtained amitraz complete antigen into antigen-containing Freund adjuvant and antigen-containing incomplete Freund adjuvant;
(2) injecting the Freund's adjuvant into BALB/c mouse via back subcutaneous injection for multiple times of immunization, wherein complete Freund's adjuvant is adopted for the first immunization, and incomplete Freund's adjuvant is adopted for boosting immunization;
(3) collecting blood from the mice subjected to the immunization process, detecting the serum immune titer and the immune suppression capacity of the mice through indirect ELISA, and screening the mice with high content of the amitraz antibody in the serum to obtain immunized mice;
(4) carrying out the last boosting immunization on the screened mice by using incomplete Freund's adjuvant, and then carrying out the sprint immunization by intraperitoneal injection, wherein the sprint immunization is carried out by adopting amitraz complete antigen without Freund's adjuvant;
(5) fusing splenocytes and myeloma cells of a BALB/c mouse after the spurt immunization, culturing the fused cells through a culture medium, detecting positive cell holes by using indirect ELISA, further determining the inhibition effect of the positive cell holes by using an indirect competitive ELISA method, subcloning the positive cell holes with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain capable of secreting a amitraz monoclonal antibody;
the molecular formula of the amitraz hapten in the step (1) is as follows:
the molecular formula of the amitraz complete antigen in the step (1) is as follows:
in one embodiment of the invention, the interval between the first immunization and the booster immunization in the steps (2) and (4) is one month, the interval between the booster immunization is 21 days, and the interval between the booster immunization and the sprint immunization is 18-21 days.
In one embodiment of the present invention, the first immunization dose of the steps (2) and (4) is 100. mu.g/mouse, the boosting immunization dose is 50. mu.g/mouse, and the sprint immunization dose is 25. mu.g/mouse.
In one embodiment of the present invention, the immunization process of steps (2) and (4) comprises 1 first immunization, 4 booster immunizations and 1 sprint immunizations;
in one embodiment of the present invention, the blood collection in step (3) is performed on day 7 after the 3 rd immunization course.
In one embodiment of the present invention, the cell fusion in step (5) is performed 3 days after the completion of the sprint immunization.
In one embodiment of the present invention, the cell fusion in step (5) is performed by a polyethylene glycol (PEG 4000) method.
In one embodiment of the present invention, the medium in the step (5) is RPMI-1640 medium.
In one embodiment of the present invention, the number of subclones in step (5) is 3.
The invention also provides application of the hybridoma cell strain RC secreting the amitraz monoclonal antibody or the preparation method of the hybridoma cell strain RC secreting the amitraz monoclonal antibody in preparation of the amitraz monoclonal antibody.
The invention provides a amitraz monoclonal antibody, which is secreted and produced by a hybridoma cell strain RC with the preservation number of CGMCC number 19166.
The invention provides a preparation method of the amitraz monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting a hybridoma cell strain RC with the preservation number of CGMCC number 19166 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and storing the obtained monoclonal antibody at low temperature.
In one embodiment of the invention, the method is to take BALB/c mice 8-10 weeks old, inject 1mL paraffin oil into the abdominal cavity of each mouse, and inject 1 × 10 into the abdominal cavity of each mouse 7 days later6Collecting ascites from 7 days of hybridoma cell strain RC with preservation number of CGMCC number 19166, purifying the ascites by an octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.
The invention provides application of the amitraz monoclonal antibody or the preparation method of the amitraz monoclonal antibody in identifying amitraz.
The invention has the beneficial effects that: the monoclonal antibody cell strain of amitraz obtained by the invention can be used for immunoassay detection, and has good detection sensitivity and specificity (IC) for amitraz50Value of 0.3ng/mL, crossover to amitraz analogue of less than 10%, crossover rate = (IC of amitraz)50IC of/analogue50）×100%）。
The invention has the beneficial effects that: (1) the monoclonal antibody for resisting amitraz has good detection sensitivity and affinity for amitraz; (2) a new method for synthesizing amitraz immunogen has more simplified and effective synthesis steps, and provides the idea and method for synthesizing immunogen for the research of people in the future.
Biological material sample preservation: a hybridoma cell strain RC secreting amitraz monoclonal antibodies is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences (China institute of microbiology, institute of sciences, No. 3, Xilu No.1, North Cheng, south China, Beijing, and the like, and is classified and named as a monoclonal cell strain, wherein the preservation date is 2019, 11 and 28 days, and the preservation number is CGMCC No. 19166.
Drawings
FIG. 1 is a standard curve of inhibition of the monoclonal antibody against amitraz according to the present invention.
Detailed Description
The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.
The media involved in the following examples are as follows:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, calcium D-pantothenate 0.25, Folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, and sodium bicarbonate 2000.
The reagents involved in the following examples are as follows:
carbonate Buffer (CBS): weighing Na2CO31.59 g，NaHCO32.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000mL, and storing at 4 ℃ for later use.
Phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12 H2Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;
PBST: PBS containing 0.05% tween 20;
antibody dilution: PBS was added with 0.1% gelatin.
TMB color development liquid: solution A: na (Na)2HPO4 .12H218.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. And the volume ratio of the solution B to the solution B is 5: 1 to obtain the TMB color developing solution which is mixed at present.
The detection methods referred to in the following examples are as follows:
the method for detecting the inhibition rate of amitraz comprises the following steps: the most suitable antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.03,0.1,0.3 and 1. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03,0.1,0.3 and 1. mu.g/mL with antibody diluent. After selecting the optimal working point, amitraz standards were diluted to 8 concentrations (0, 3, 6, 12, 24, 48, 96, 192 ng/mL), and plotted against originPro 8.5 (results are shown in FIG. 1) following the IC-ELISA protocol to obtain amitraz standard inhibition curves and IC was calculated50。
Example 1: synthesis of amitraz hapten
Because the amitraz micromolecules have no immunogenicity and can not stimulate mice to generate immune response so as to generate antibodies, the amitraz is coupled to the protein by a protein connection technology so as to obtain the immunogenicity; the active groups commonly used in protein coupling technology include amino, carboxyl, hydroxyl, sulfydryl and the like, and the diamidine does not contain the active groups in the molecular structural formula, so the diamidine needs to be derived.
The structure of the derivatized amitraz hapten is as follows:
example 2: synthesis of amitraz complete antigen
Weighing 2.3mg amitraz hapten and 2.2mg N-hydroxysuccinimide (NHS), dissolving in 200 μ L N, N-Dimethylformamide (DMF), and stirring at room temperature for 10 min; then, 3.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved sufficiently in 100. mu.L of DMF, and then added to the formamidine hapten solution, followed by stirring at room temperature for 6 to 8 hours (referred to as solution A). Taking 7.5mg KLH, diluting to 3mg/mL (called as solution B) by using 0.01M Carbonate Buffer Solution (CBS), then slowly adding the solution A into the solution B dropwise, and reacting at room temperature overnight; then dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen of amitraz, and identifying by ultraviolet absorption scanning method.
Example 3: synthesis of amitraz coatingen
Dissolving 1.5mg amitraz hapten and 2.3mg N-hydroxysuccinimide (NHS) in 200 μ L anhydrous N, N-Dimethylformamide (DMF), and stirring at room temperature for 10min to obtain amitraz hapten solution; dissolving 3.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 100 mu L of anhydrous DMF, adding the solution into a formamidine hapten solution, and stirring at room temperature to react for 6-8h to obtain a solution A; dissolving 10mg of egg albumin (OVA) with 3mL of Carbonate Buffer Solution (CBS) with the concentration of 0.01mmol/L to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction to obtain a reaction solution; and dialyzing the reaction solution by using a PBS solution to remove unreacted small molecule hapten to obtain the coating antigen of the amitraz.
Example 4: preparation of hybridoma cell strain secreting amitraz monoclonal antibody
1. Obtaining animal immunity: mixing and emulsifying a amitraz complete antigen and an equivalent amount of Freund's adjuvant, and performing neck-back subcutaneous multipoint injection immunization (except puncture immunization) on a BALB/c mouse; complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g of the Chinese medicinal preparation; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; the immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;
2. cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:
a. cutting the tail, taking blood, immediately putting the mouse into 75% alcohol for disinfection after the mouse is killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mouse through aseptic operation, properly grinding the spleen by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200 rpm, 8 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO2In the incubator, before fusion, SP2/0 tumor cell number is required to reach (1-4) × 107Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;
c. the fusion process is carried out for 7min, at 1min, dropping 1mL PEG4000 into cells from slow to fast, at 2min, standing, at 3min and 4min, dropping 1mL RPMI-1640 culture medium within 1min, at 5min and 6min, dropping 2mLRPMI-1640 culture medium within 1min, at 7min, dropping 1mL RPMI-1640 culture medium per 10s, then bathing at 37 deg.C for 5min, centrifuging (800 rpm, 8 min), discarding supernatant, re-suspending in RPMI-1640 screening culture solution containing 20% fetal calf serum and 2% 50 × HAT, adding into 96-well cell plate at 200 μ L/well, standing at 37 deg.C, and 5% CO2Culturing in an incubator;
3. cell screening and cell strain establishment: on day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected for screening.
The screening is divided into two steps: firstly, screening out positive cell holes by using an ic-ELISA method, secondly, selecting amitraz as a standard substance, and determining the inhibition effect of positive cells by using the ic-ELISA method;
selecting a cell hole with good inhibition on the amitraz standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days;
subcloning for three times according to the method to finally obtain the cell strain secreting the amitraz monoclonal antibody.
Example 5: preparation and identification of amitraz monoclonal antibody
Injecting sterile paraffin oil 1mL into the abdominal cavity of 8-10 week-old BALB/c mouse, and injecting 1 × 10 into the abdominal cavity of 7 days later6Amitraz hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method.
Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
IC determination of monoclonal antibodies to amitraz Using an Indirect competitive ELISA50The value is 0.3ng/mL, the crossover to the amitraz analogue is less than 10%, which shows that the amitraz has good sensitivity and can be used for the immunoassay detection of the amitraz.
(Cross-over Rate = (IC of amitraz)50IC of/analogue50）×100%）。
Example 6: application of amitraz monoclonal antibody
The monoclonal antibody prepared from the hybridoma cell strain through in-vivo ascites is applied to an ELISA addition recovery test of amitraz, and the method comprises the following specific steps:
(1) coating a 96-well enzyme label plate with coating antigen with the concentration of 0.3 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the enzyme label plate in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor for three times, wherein 200 mu L of the washing liquor in each well is used for 3min, and drying by beating;
(2) sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;
(3) respectively preparing 0, 3, 6, 12, 24, 48, 96 and 192ng/mL amitraz standard solution by using Phosphate Buffer Solution (PBS), respectively adding the standard solution and the extract of a sample to be detected into a closed enzyme label plate, respectively adding 50 mu L of the extract of the sample to be detected into each well, repeating 3 wells of each sample, adding 50 mu L of an amitraz-resistant monoclonal antibody diluted by 1:32000 into each well, reacting at 37 ℃ for 0.5h, washing the plate, and drying;
(4) adding 100 μ L of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at a ratio of 1:3000 into each well, reacting at 37 ℃ for 0.5h, washing and drying;
(5) adding 100 μ L of TMB developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 2M H into each well2SO4Stopping solution, measuring the light absorption value at 450 nm;
the standard curve for inhibition of amitraz by amitraz monoclonal antibody is shown in FIG. 1, and IC of amitraz monoclonal antibody is determined by IC-ELISA50The value is 0.3ng/mL, which indicates that the antibody has better sensitivity to amitraz and can be used for immunoassay detection of amitraz.
Claims (6)
1. A hybridoma cell strain RC secreting amitraz monoclonal antibodies is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences (China institute of microbiology, institute of sciences, No. 3, Xilu No.1, North Cheng, south China, Beijing, and the like, and is classified and named as a monoclonal cell strain, wherein the preservation date is 2019, 11 and 28 days, and the preservation number is CGMCC No. 19166.
2. The amitraz monoclonal antibody is characterized in that: it is secreted and produced by hybridoma cell strain RC with the preservation number of CGMCC number 19166 as claimed in claim 1.
3. A amitraz hapten, which is characterized in that: the molecular formula is as follows:
4. a amitraz complete antigen, which is characterized in that: the molecular formula is as follows:
5. the process for the preparation of the amitraz complete antigen of claim 4, characterized by the following steps: dissolving the amitraz hapten and N-hydroxysuccinimide NHS as defined in claim 3 in anhydrous N, N-dimethylformamide DMF, and stirring for reaction to obtain an amitraz hapten solution; dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in anhydrous DMF, adding the solution into a formamidine hapten solution, and stirring the solution to react to obtain a solution A; diluting keyhole limpet hemocyanin KLH with carbonate buffer solution CBS to obtain solution B; adding the solution A into the solution B for reaction to obtain a reaction solution; and dialyzing the reaction solution by phosphate buffer solution PBS to obtain the complete antigen of the amitraz.
6. The use of the monoclonal antibodies to amitraz according to claim 2, wherein: the method is applied to the analysis and detection of amitraz residue in food safety detection.
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