CN113637642A - Hybridoma cell strain capable of secreting monoclonal antibody of dicofol and application of hybridoma cell strain - Google Patents
Hybridoma cell strain capable of secreting monoclonal antibody of dicofol and application of hybridoma cell strain Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain secreting dicofol monoclonal antibody and application thereof, and belongs to the technical field of immunodetection. The invention discloses a hybridoma cell strain JHJ for secreting dicofol monoclonal antibody, which is deposited in China general microbiological culture Collection center (CGMCC), is classified and named as monoclonal cell strain JHJ, and has a preservation date of 2021, 05 and 13 months and a preservation number of CGMCC No. 22329. The invention uses dicofol complete antigen to immunize BALB/c mice, takes splenocytes of the mice with high titer and good inhibition, fuses the splenocytes with myeloma cells by a PEG method, and obtains hybridoma cell strains through screening and three times of subcloning by an indirect competitive enzyme linked immunosorbent assay. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity (IC) on dicofol50The value is 2ng/mL), provides raw materials for the immunodetection of residual dicofol in food, and has practical application value.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a hybridoma cell strain secreting dicofol monoclonal antibody and application thereof.
Background
Dicofol (Dicofol), also known as 1, 1-bis (p-chlorophenyl) -2,2, 2-trichloroethanol or clotrimazole, is a common organic chloride acaricide in agricultural production and is commonly used in China to control various mites of cotton, fruit trees and flowers. However, in the production process of dicofol, a certain amount of DDT residues can be contained, and the environment and the human health are harmed. There has been increasing evidence in recent years that its exposure to the environment is toxic and estrogenic to fish, reptiles, birds, mammals and humans, and extremely toxic to aquatic organisms. Therefore, the application of dicofol in agricultural production is forbidden in China. In 2017, 10 and 27, the national institutes of health (world health organization) publishes a carcinogen list for preliminary reference, and dicofol is in a category 3 carcinogen list. The maximum residual limit of dicofol in various foods (MRL) is specified in GB 2763-2021 food maximum residual limit of pesticide), and for example, the MRL in vegetables and fruits is 0.01mg/kg, the MRL in nuts is 0.02mg/kg, and the MRL in rice, wheat and miscellaneous cereals is 0.02 mg/kg.
In order to effectively monitor the using condition of dicofol in food, a determination method with good specificity and high sensitivity is needed, but the existing detection methods such as gas chromatography, high performance liquid chromatography tandem mass spectrometry and the like have the disadvantages of high cost, complex sample pretreatment, high technical requirements on operators, incapability of immediately outputting results and more interferents in food, so the instrumental method is not suitable for field detection. The immunological rapid detection method is a low-cost, rapid, high-sensitivity and high-specificity immunological detection method, is simple and convenient to operate, and is widely applied to the field of pesticide residue detection. Establishing an efficient immunological detection method, and screening a monoclonal antibody with high specificity is an important prerequisite.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a hybridoma cell strain secreting monoclonal antibodies for dicofol and application thereof.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:
the invention aims to provide a hybridoma cell strain JHJ for secreting dicofol monoclonal antibody, which is deposited in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, and is named as a monoclonal cell strain JHJ by classification, wherein the JHJ is deposited at China institute of academy of sciences, No. 1, west way, No. 3, north Chen, south China, the morning area, the date of deposition is 2021 year 05 month 13, and the number of deposition is CGMCC No. 22329.
The dicofol monoclonal antibody is secreted and produced by a hybridoma cell strain JHJ of the dicofol monoclonal antibody with the preservation number of CGMCC No. 22329.
The second purpose of the invention is to provide a dicofol hapten, wherein the structural formula of the dicofol hapten is as follows:
the third purpose of the invention is to provide a method for synthesizing dicofol hapten, which comprises the following steps: adding 4, 4' -dichlorobenzophenone and carboxymethyl hydroxylamine hemihydrochloride solid into a mixed solution consisting of methanol, pyridine and pure water, and continuously stirring in a water bath at the temperature of 60-90 ℃ for reaction for 5-10 hours; taking out, and standing overnight at room temperature; and drying by nitrogen, adding dichloromethane for redissolving, extracting by pure water, discarding a water layer, and drying an organic phase by nitrogen to obtain a viscous solid which is the dicofol hapten.
In one embodiment of the present invention, the molar mass ratio of the 4, 4' -dichlorobenzophenone to the carboxymethyl hydroxylamine hemihydrochloride solid is 1: 3-6, wherein the concentration of the 4, 4' -dichlorobenzophenone in the mixed solution is 5-10 g/L; preferably, the molar mass ratio of the 4, 4' -dichlorobenzophenone to the carboxymethyl hydroxylamine hemihydrochloride solid is 1: 4, the concentration of the 4, 4' -dichlorobenzophenone in the mixed solution is 8.3 g/L.
In one embodiment of the invention, the mixed solution is formed by methanol, pyridine and pure water according to a volume ratio of 2-5: 1-3: 1; preferably, the mixed solution is formed by methanol, pyridine and pure water according to the volume ratio of 4:1: 1.
A fourth object of the present invention is to provide a method for preparing a dicofol complete antigen, which comprises a dicofol immunogen, the method comprising the steps of: dissolving dicofol hapten with N, N-dimethylformamide, adding 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, and continuously stirring at room temperature for reaction to obtain a dicofol hapten activation solution; dissolving carrier protein with boric acid buffer solution to obtain protein A solution; slowly dripping the dicofol hapten activating solution into the protein A solution at room temperature, stirring at room temperature for reacting overnight, and dialyzing to obtain the dicofol complete antigen.
In one embodiment of the invention, the carrier proteins include keyhole limpet hemocyanin, bovine serum albumin, BSA, and other carrier proteins suitable for coupling.
In one embodiment of the present invention, the molar mass ratio of dicofol hapten to 1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1: 3-5: 3-5, wherein the concentration of the dicofol hapten in the N, N-dimethylformamide is 20-30 g/L; preferably, the molar mass ratio of the dicofol hapten to the 1-ethyl carbodiimide hydrochloride to the N-hydroxysuccinimide is 1: 4.1: 3.4, the concentration of the dicofol hapten in the N, N-dimethylformamide is 25 g/L.
In one embodiment of the invention, the concentration of the carrier protein in the boric acid buffer solution is 3-5 g/L; preferably, the concentration of the carrier protein in the boric acid buffer solution is 3.3 g/L.
In one embodiment of the invention, the molar mass ratio of the dicofol hapten to the carrier protein is 6000-8000: 1; preferably, the molar mass ratio of the dicofol hapten to the carrier protein is 6160: 1.
in one embodiment of the present invention, the structural formula of the dicofol hapten is as follows:
the fifth purpose of the invention is to provide a preparation method of dicofol complete antigen, wherein the complete antigen comprises dicofol coating antigen, and the method comprises the following steps: dissolving 2, 2-di-p-chlorophenylacetic acid in anhydrous N, N-dimethylformamide, adding 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, and continuously stirring at room temperature for reaction to obtain dicofol hapten activation solution; dissolving carrier protein in boric acid buffer solution, called B solution; and (3) dropwise adding the dicofol hapten activation solution into the solution B at room temperature, stirring at room temperature for reacting overnight, and dialyzing to obtain the dicofol coating antigen.
In one embodiment of the invention, the carrier proteins include chicken ovalbumin OVA, bovine serum albumin BSA and other carrier proteins suitable for conjugation.
In one embodiment of the present invention, the molar mass ratio of the 2, 2-bis-p-chlorophenylacetic acid, 1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1: 3-5: 3-5, wherein the concentration of the 2, 2-bis-p-chlorophenylacetic acid in the N, N-dimethylformamide is 10-25 g/L; preferably, the molar mass ratio of the 2, 2-di-p-chlorophenylacetic acid to the 1-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1: 3.5: 3.0, the concentration of the 2, 2-di-p-chlorophenylacetic acid in the N, N-dimethylformamide is 20 g/L.
In one embodiment of the invention, the concentration of the carrier protein in the boric acid buffer solution is 3-7 g/L; preferably, the concentration of the carrier protein in the boric acid buffer solution is 5 g/L.
In one embodiment of the present invention, the molar mass ratio of the 2, 2-bis-p-chlorophenylacetic acid to the carrier protein is 50 to 100: 1; preferably, the molar mass ratio of the 2, 2-di-p-chlorophenylacetic acid to the carrier protein is 64: 1.
in one embodiment of the present invention, the basic steps for preparing a hybridoma cell strain JHJ secreting dicofol monoclonal antibody provided by the present invention are:
(1) synthesis of hapten:
the synthetic scheme of the dicofol hapten is shown in the reaction formula. 4, 4' -dichlorobenzophenone (15mg) and carboxymethyl hydroxylamine hemihydrochloride (30mg) were weighed out as solids and charged into a 10mL screw-top brown glass bottle, and a mixed solution of 1.8mL of methanol, pyridine and pure water (v: v: v ═ 4:1:1) was added, followed by placing in a water bath at 70 ℃ and continuously stirring for reaction for 6 hours. After the reaction was completed, the reaction mixture was taken out and allowed to stand at room temperature overnight. And after the solution is dried by nitrogen, adding 2mL of dichloromethane for redissolving, repeatedly extracting by pure water for 3-4 times (1 mL/time), removing a water layer, and drying an organic phase by nitrogen to obtain a viscous solid which is the dicofol hapten.
(2) Preparation of immunogen and coatingen:
preparation of immunogen: 5.0mg of dicofol hapten was weighed, dissolved in 200. mu.L of anhydrous N, N-dimethylformamide, and then 12mg of 1-ethylcarbodiimide hydrochloride and 6.0mg of N-hydroxysuccinimide were added thereto, mixed well, and reacted for 6 hours with continuous stirring at room temperature (referred to as solution A). Dissolving keyhole limpet hemocyanin (10 mg) in boric acid buffer solution (3.0 mL), slowly adding the solution A into the solution A at room temperature, stirring at room temperature for reacting overnight, and dialyzing to obtain immunogen;
preparation of coating antigen: 4.0mg of 2, 2-bis-p-chlorophenylacetic acid was weighed out and dissolved in 200. mu.L of anhydrous N, N-dimethylformamide, followed by addition of 9.6mg of 1-ethylcarbodiimide hydrochloride and 4.8mg of N-hydroxysuccinimide, followed by mixing and reaction for 6 hours at room temperature with continuous stirring (referred to as solution B). Weighing 10mg of chicken ovalbumin OVA, dissolving in 2mL of boric acid buffer solution (called B solution), dropwise adding the B solution into the B solution at room temperature, stirring at room temperature for reaction overnight, and dialyzing to obtain the coating antigen.
(3) Immunization of mice: after the dicofol complete antigen is mixed and emulsified with an equal volume of complete Freund's adjuvant, BALB/c mice are injected subcutaneously through the back and the neck. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used for boosting the immunization. Each boost was 3 weeks apart. The last booster immunization adopts complete antigen (without adjuvant) for intraperitoneal injection; the serum titer and inhibition rate were measured by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).
(4) Cell fusion and cell line establishment: fusing mouse splenocytes and myeloma cells by a PEG method, selectively culturing by HAT culture medium, detecting the positive rate and inhibition rate of cell holes by ic-ELISA, subcloning hybridoma cells with high positive rate and inhibition rate by a limiting dilution method, and finally screening to obtain the dicofol monoclonal antibody hybridoma cell strain JHJ.
(5) And (3) identification of the properties of hybridoma cell strains: the sensitivity was determined by ic-ELISA.
Get high-efficient low IC50The spleen cells of a mouse are fused with myeloma cells of the mouse by a PEG method, and a hybridoma cell strain JHJ is obtained by screening and subcloning by an indirect competitive enzyme-linked immunosorbent assay.
The sixth purpose of the invention is to provide an application of the dicofol monoclonal antibody, which is used for analyzing and detecting the residual dicofol in food safety detection.
In one embodiment of the invention, the dicofol monoclonal antibody is used for preparing a detection main body for analyzing and detecting the residual quantity of dicofol in food safety detection.
In one embodiment of the present invention, the detection main body comprises a reagent, a detection plate, and a kit.
The invention has the beneficial effects that: firstly, the monoclonal antibody secreted by the cell strain JHJ provided by the invention has better specificity and detection sensitivity (IC) on dicofol50The value is 2ng/mL), can realize the detection of the residual quantity of the dicofol in fruits, vegetables and grains, provides raw materials for the immunodetection of the residual quantity of the dicofol in food, and has practical application value. Secondly, the invention provides a preparation method of a hybridoma cell strain capable of secreting a dicofol monoclonal antibody, in particular a novel hapten preparation method, which is obtained by adding 4, 4' -dichlorobenzophenone and carboxymethyl hydroxylamine hemihydrochloride solid into a mixed solution consisting of methanol, pyridine and pure water for reaction,the raw materials are simple and easy to obtain, and the preparation process is precise and simple to operate.
Biological material sample preservation: a trichlorfon monoclonal antibody hybridoma cell strain JHJ 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, Kyoho, and is classified and named as monoclonal cell strain JHJ, the preservation date is 2021 year, 05 month and 13 days, and the preservation number is CGMCC No. 22329.
Drawings
FIG. 1 is a schematic diagram of the result of immunoglobulin subtype identification of a JHJ monoclonal antibody according to an embodiment of the present invention.
FIG. 2 is a standard curve of inhibition of JHJ monoclonal antibody on dicofol in the embodiment of the invention.
Detailed Description
The invention is further described with reference to the following figures and examples. The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results described in the examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention.
Reagents used in the examples of the invention:
solution preparation: carbonate Buffer (CBS): weighing Na2CO3 1.59g，NaHCO32.93g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;
phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12H2Dissolving 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;
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. Liquid B according to volume ratioMixing at the ratio of 1:5 to obtain the TMB color development liquid, and mixing at the moment when the TMB color development liquid is used.
According to the embodiment of the invention, a monoclonal antibody hybridoma cell strain with high sensitivity to dicofol is finally obtained by immunizing a mouse with a dicofol complete antigen, performing cell fusion, culturing in a HAT selective culture medium and screening cell supernatant through ic-ELISA.
Example 1 preparation of hybridoma cell line JHJ
(1) Synthesis of hapten:
the synthetic scheme of the dicofol hapten is shown in the reaction formula. Solid 4, 4' -dichlorobenzophenone (15mg, 0.0597mmol) and carboxymethyl hydroxylamine hemihydrochloride (30mg, 0.235mmol) were weighed into a 10mL screw-top brown glass bottle, and a mixed solution of 1.8mL of methanol, pyridine and pure water (v: v: v ═ 4:1:1) was added, followed by placing in a 70 ℃ water bath and stirring continuously for reaction for 6 hours. After the reaction was completed, the reaction mixture was taken out and allowed to stand at room temperature overnight. And after the solution is dried by nitrogen, adding 2mL of dichloromethane for redissolving, repeatedly extracting by pure water for 3-4 times (1 mL/time), removing a water layer, and drying an organic phase by nitrogen to obtain a viscous solid which is the dicofol hapten.
(2) Preparation of immunogen and coatingen:
preparation of immunogen: 5.0mg (0.0154mmol) of dicofol hapten is weighed out and dissolved in 200. mu.L of anhydrous N, N-dimethylformamide, 12mg (0.0628mmol) of 1-ethylcarbodiimide hydrochloride and 6.0mg (0.0522mmol) of N-hydroxysuccinimide are added and mixed well, and the reaction is continued for 6h at room temperature (referred to as solution A). Dissolving keyhole limpet hemocyanin 10mg (0.0000025mmol) in boric acid buffer solution (3.0 mL), slowly adding solution A into solution A at room temperature, stirring at room temperature for reaction overnight, and dialyzing to obtain immunogen;
preparation of coating antigen: 4.0mg (0.0142mmol) of 2, 2-bis-p-chlorophenylacetic acid was weighed out and dissolved in 200. mu.L of anhydrous N, N-dimethylformamide, followed by addition of 9.6mg (0.0503mmol) of 1-ethylcarbodiimide hydrochloride and 4.8mg (0.0417mmol) of N-hydroxysuccinimide, followed by mixing and stirring at room temperature for 6 hours (referred to as "solution B"). Weighing 10mg (0.000222mmol) of chicken ovalbumin OVA, dissolving in 2mL boric acid buffer solution (called B solution), dropwise adding the B solution into the B solution at room temperature, stirring at room temperature for reaction overnight, and dialyzing to obtain the coating antigen.
(3) Immunization of mice: after the dicofol complete antigen is mixed and emulsified with an equal volume of complete Freund's adjuvant, BALB/c mice are injected subcutaneously through the back and the neck. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used for boosting the immunization. Each boost was 3 weeks apart. The last booster immunization adopts complete antigen (without adjuvant) for intraperitoneal injection; the serum titer and inhibition rate were measured by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).
(4) Cell fusion: after three days of the last booster immunization, cell fusion is carried out according to a conventional PEG method, and the specific steps are as follows:
a. taking eyeballs and blood, immediately putting the mice into 75% alcohol for disinfection after the mice are killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mice by 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 (1200rpm, 8min), 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% CO2An incubator. Before fusion, the number of SP2/0 tumor cells is required to reach (1-4) x 107Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
c. the fusion process is 7 min. 1min, 1mL of PEG 1500 is added to the cells from slow to fast; standing for 2 min. Dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5 min. Centrifugation (800rpm, 8 mi)n), discarding the supernatant, resuspending into RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding to 96-well cell plate at 200. mu.L/well, standing at 37 ℃ with 5% CO2Culturing in an incubator.
(5) 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 positive cell holes by using ic-ELISA, secondly, selecting dicofol as a standard substance, and measuring the inhibition effect of the positive cells by using ic-ELISA. And selecting cell holes which have better inhibition on dicofol standard substances, subcloning by adopting a limiting dilution method, and detecting by using the same method. Repeating the steps for three times to obtain a cell strain JHJ.
(6) Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 106Hybridoma cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by the octanoic acid-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; precipitating IgG type monoclonal antibody with ammonium sulfate solution of equal saturation degree, centrifuging, discarding supernatant, dissolving with 0.01M PBS solution (pH7.4), dialyzing, desalting, and storing at-20 deg.C;
the monoclonal antibody of dicofol obtained by ascites purification was subjected to immunoglobulin subtype identification using a mouse monoclonal antibody subtype identification kit, the subtype was IgG2b type, and the light chain type was KAPPA type as detected by the mouse monoclonal antibody subtype identification kit, as shown in FIG. 1.
Example 2 IC of monoclonal antibodies to dicofol50Measurement of (2)
1 coating: the coating source prepared in step (2) of example 1 was diluted in 0.05M carbonate buffer pH9.6 at a rate of 1. mu.g/mL, 100. mu.L/well, and reacted at 37 ℃ for 2 hours;
2, washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
3, sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. Drying for later use after washing;
4, loading of sample: diluting antiserum from 1:1000 times, adding into coated wells of each dilution, reacting at 37 deg.C for 30min at 100 μ L/well; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;
5, color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
6 termination and determination: the reaction was stopped by adding 50. mu.L of a stop buffer to each well, and the OD 450 value of each well was measured by a microplate reader.
The sensitivity of the assay to dicofol was determined by ic-ELISA, as shown in FIG. 2, according to the standard equation y 0.1159+1.375/(1+ (x/1.240)0.784) Determination of the IC of the monoclonal antibodies to dicofol by IC-ELISA50The antibody is 2ng/mL, which indicates that the antibody has higher sensitivity and can be used for the immunoassay detection of dicofol.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (16)
1. A hybridoma cell strain secreting dicofol monoclonal antibody is deposited in China general microbiological culture collection center (CGMCC), China academy of sciences (institute of microbiology 3, Xilu 1, Beijing, the morning area, Beijing), and is classified and named as monoclonal cell strain JHJ, the preservation date is 2021 year, 05 month and 13 days, and the preservation number is CGMCC No. 22329.
2. The dicofol monoclonal antibody is secreted by the hybridoma cell line JHJ of the dicofol monoclonal antibody with the preservation number of CGMCC No.22329 as claimed in claim 1.
3. A dicofol hapten, wherein the structural formula of the dicofol hapten is as follows:
4. a method for synthesizing dicofol hapten, which is characterized by comprising the following steps: adding 4, 4' -dichlorobenzophenone and carboxymethyl hydroxylamine hemihydrochloride solid into a mixed solution consisting of methanol, pyridine and pure water, and continuously stirring in a water bath at the temperature of 60-90 ℃ for reaction for 5-10 hours; taking out, and standing overnight at room temperature; and drying by nitrogen, adding dichloromethane for redissolving, extracting by pure water, discarding a water layer, and drying an organic phase by nitrogen to obtain a viscous solid which is the dicofol hapten.
5. The method for synthesizing dicofol hapten according to claim 4, wherein the molar mass ratio of the 4,4 '-dichlorobenzophenone to the carboxymethyl hydroxylamine hemihydrochloride solid is 1: 3-6, and the concentration of the 4, 4' -dichlorobenzophenone in the mixed solution is 5-10 g/L; preferably, the molar mass ratio of the 4,4 '-dichlorobenzophenone to the carboxymethyl hydroxylamine hemihydrochloride solid is 1: 4, and the concentration of the 4, 4' -dichlorobenzophenone in the mixed solution is 8.3 g/L.
6. The method for synthesizing dicofol hapten according to claim 4, wherein the mixed solution is formed by methanol, pyridine and pure water according to a volume ratio of 2-5: 1-3: 1; preferably, the mixed solution is formed by methanol, pyridine and pure water according to the volume ratio of 4: 1.
7. A process for the preparation of a dicofol complete antigen, wherein said complete antigen comprises a dicofol immunogen, said process comprising the steps of: dissolving dicofol hapten with N, N-dimethylformamide, adding 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, and continuously stirring at room temperature for reaction to obtain a dicofol hapten activation solution; dissolving carrier protein with boric acid buffer solution to obtain protein A solution; slowly dripping the dicofol hapten activating solution into the protein A solution at room temperature, stirring at room temperature for reacting overnight, and dialyzing to obtain dicofol complete antigen;
the carrier protein comprises keyhole limpet hemocyanin and bovine serum albumin.
8. The method for synthesizing the dicofol complete antigen as claimed in claim 7, wherein the molar mass ratio of the dicofol hapten to 1-ethyl carbodiimide hydrochloride to N-hydroxysuccinimide is 1: 3-5, and the concentration of the dicofol hapten in N, N-dimethylformamide is 20-30 g/L; preferably, the molar mass ratio of the dicofol hapten to the 1-ethyl carbodiimide hydrochloride to the N-hydroxysuccinimide is 1: 4.1: 3.4, and the concentration of the dicofol hapten in the N, N-dimethylformamide is 25 g/L.
9. The method for synthesizing the dicofol complete antigen as claimed in claim 7, wherein the molar mass ratio of the dicofol hapten to the carrier protein is 6000-8000: 1; preferably, the molar mass ratio of the dicofol hapten to the carrier protein is 6160: 1.
10. A dicofol complete antigen according to any one of claims 7 to 9, wherein the dicofol hapten is of the formula:
11. the preparation method of the dicofol complete antigen is characterized in that the complete antigen comprises dicofol coatingen, and the method comprises the following steps: dissolving 2, 2-di-p-chlorophenylacetic acid in anhydrous N, N-dimethylformamide, adding 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide, and continuously stirring at room temperature for reaction to obtain dicofol hapten activation solution; dissolving carrier protein in boric acid buffer solution, called B solution; dropwise adding the dicofol hapten activation solution into the solution B at room temperature, stirring at room temperature for reacting overnight, and dialyzing to obtain dicofol coating antigen;
the carrier protein comprises chicken ovalbumin OVA and bovine serum albumin BSA.
12. The method for synthesizing dicofol complete antigen according to claim 11, wherein the molar mass ratio of 2, 2-bis-p-chlorophenylacetic acid, 1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:3 to 5, and the concentration of 2, 2-bis-p-chlorophenylacetic acid in N, N-dimethylformamide is 10 to 25 g/L; preferably, the molar mass ratio of the 2, 2-di-p-chlorophenylacetic acid to the 1-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1: 3.5: 3, and the concentration of the 2, 2-di-p-chlorophenylacetic acid in the N, N-dimethylformamide is 20 g/L.
13. The method for synthesizing the dicofol complete antigen as claimed in claim 11, wherein the molar mass ratio of the 2, 2-bis-p-chlorophenylacetic acid to the carrier protein is 50-100: 1; preferably, the molar mass ratio of the 2, 2-di-p-chlorophenyl acetic acid to the carrier protein is 64: 1.
14. The use of the monoclonal antibody against dicofol as claimed in claim 2, characterized in that it is used for the analytical detection of residual dicofol in food safety tests.
15. The use of the monoclonal antibody against dicofol according to claim 14, wherein the monoclonal antibody against dicofol is used for preparing a test body for analyzing and detecting residual dicofol in food safety tests.
16. The use of the monoclonal antibody against dicofol as claimed in claim 15, wherein the test body comprises reagents, test panels and kits.
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