CN110607283A

CN110607283A - Hybridoma cell strain CBC for secreting monoclonal antibody of dicofol and application thereof

Info

Publication number: CN110607283A
Application number: CN201910965997.7A
Authority: CN
Inventors: 胥传来; 张荣荣; 章晓萍; 匡华; 徐丽广; 刘丽强; 宋珊珊; 吴晓玲; 胡拥明; 曹玉朋
Original assignee: Jiangsu Quanzheng Inspection & Testing Co ltd; Jiangnan University
Current assignee: Jiangsu Quanzheng Inspection & Testing Co ltd; Jiangnan University
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2019-12-24

Abstract

A hybridoma cell strain CBC secreting monoclonal antibodies to dicofol and application thereof belong to the field of food safety immunodetection. The cell strain CBC of the invention is preserved in China general microbiological culture Collection center, and the address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17398. The complete antigen of dicofol is mixed and emulsified with Freund's adjuvant in equal amount, and BALB/c mouse is immunized by subcutaneous multi-point injection to neck and back. The first immunization, multiple boosting immunization and sprint immunization. High potency, low IC₅₀Splenocytes from mice, by PEG method with mouse myelomaCell fusion, selective culture, screening cell by indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to obtain cell strain CBC, its secreted monoclonal antibody has good detection sensitivity (IC) for dicofol₅₀The value is 19.09 ng/mL), can be used for detecting the residue of the dicofol in the food.

Description

Hybridoma cell strain CBC for secreting monoclonal antibody of dicofol and application thereof

Technical Field

The invention relates to a hybridoma cell strain CBC secreting dicofol monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.

Background

Dicofol (hereinafter abbreviated as DCF) is a broad-spectrum and low-toxicity organic chlorine acaricide commonly used in agriculture and animal husbandry, has the effects of contact poisoning and stomach poisoning on mites, and is effective on adult mites, young mites and eggs. The production and synthesis of the dicofol take the dichlorodiphenyl trichloroethane as a raw material, the conversion rate of a chlorination section in the production process is about 70 percent generally, and if the dichlorodiphenyl trichloroethane is not purified or is not purified completely, the dichlorodiphenyl trichloroethane contains dichlorodiphenyl trichloroethane to different degrees in a finished product.

The use of dicofol in large quantities leads to excessive residual levels, and in recent years there has been increasing evidence that its exposure to the environment is toxic and estrogenic to fish, reptiles, birds, mammals and humans, and extremely toxic to aquatic organisms. Bringing harm to human health. The use of dicofol pesticide on tea plants has been banned in some countries of the world, and the use of high residue dicofol pesticide is also severely restricted in some countries. The maximum residual limit of the dicofol in the fruits is 1mg/kg and the maximum residual limit of the tea is 0.2mg/kg, which are specified in the national standard GB 2763-2016.

At present, the dicofol detection method is mainly instrument detection, and gas chromatography, liquid chromatography and gas chromatography-mass spectrometry are commonly used. Although these chromatographic-based methods have high sensitivity and specificity, they suffer from drawbacks such as the need for thorough sample purification, high solvent consumption, expensive equipment and skilled technicians. Therefore, a fast and simple method for analyzing the residue of dicofol is needed.

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 the enzyme linked immunosorbent assay to detect the dicofol is to obtain the monoclonal antibody with high specificity and high sensitivity to the dicofol, so that the key point is to find a method for preparing the monoclonal antibody with high specificity and high sensitivity to the dicofol. The inventors tried to prepare a monoclonal antibody against dicofol from hybridoma cells, but in the course of preparing hybridoma cell lines capable of secreting monoclonal antibody against dicofol, further studies were required on how to prepare a hapten against dicofol and a complete antigen against dicofol and how to generate strong immunity in mice; further research is needed on how to successfully secrete the dicofol monoclonal antibody from the prepared hybridoma cell strain; further research is needed to ensure that the secreted monoclonal antibody of dicofol has strong specificity and high sensitivity.

Disclosure of Invention

The invention aims to overcome the defects and provides a hybridoma cell strain CBC for secreting the dicofol monoclonal antibody and application thereof, wherein the dicofol monoclonal antibody secreted by the hybridoma cell strain has good detection sensitivity (IC) on dicofol₅₀The value is 19.09 ng/mL), can be used for establishing an immunological detection method of the dicofol and detecting the residue of the dicofol in the food.

The technical scheme of the invention is that a hybridoma cell strain CBC secreting dicofol monoclonal antibody is preserved in China general microbiological culture Collection center, and the address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17398.

The invention provides a preparation method of a hybridoma cell strain CBC secreting dicofol monoclonal antibody, which comprises the following steps:

(1) preparing dicofol hapten and dicofol complete antigen, and preparing the obtained dicofol complete antigen into complete Freund's adjuvant and incomplete Freund's 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 dicofol antibody in the serum to obtain the 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 using dicofol complete antigen without Freund's adjuvant;

(5) fusing splenocytes and myeloma cells of a BALB/c mouse after the sprint 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 dicofol monoclonal antibody;

the molecular formula of the dicofol hapten in the step (1) is as follows:

；

the molecular formula of the dicofol complete antigen in the step (1) is as follows:

。

in one embodiment of the present invention, the first immunization and the booster immunization in the steps (2) and (4) are separated by one month, the booster immunization is separated by 21 days, and the booster immunization and the sprint immunization are separated by 18 ~ 21 days.

In one embodiment of the present invention, the primary immune dose in the steps (2) and (4) is 100 μ g/mouse, the booster immune dose is 50 μ g/mouse, and the sprint immune dose is 25 μ g/mouse.

In one embodiment of the present invention, the immunization process in the 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 provides application of the hybridoma cell strain CBC secreting the dicofol monoclonal antibody or a preparation method of the hybridoma cell strain CBC secreting the dicofol monoclonal antibody in preparation of the dicofol monoclonal antibody.

The invention provides a dicofol monoclonal antibody, which is obtained by secreting hybridoma cell strain CBC with the preservation number of CGMCC No. 17398.

The invention provides a preparation method of the dicofol monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting a hybridoma cell strain CBC with the preservation number of CGMCC No.17398 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 1X 10 paraffin oil into the abdominal cavity of each mouse 7 days later⁶Collecting ascites from 7 days, purifying the ascites by an octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.

The invention provides an application of the dicofol monoclonal antibody or the preparation method of the dicofol monoclonal antibody in recognition of dicofol.

The invention has the beneficial effects that: the dicofol monoclonal antibody obtained by the invention has good detection sensitivity (IC) on dicofol₅₀A value of 19.09 ng/mL); the dicofol monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection.

Biological material sample preservation: a hybridoma cell strain CBC for secreting monoclonal antibodies of dicofol is preserved in China general microbiological culture Collection center, and the address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17398.

Drawings

FIG. 1 shows the standard curve of the dicofol inhibition by the monoclonal antibody of the 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 Na₂CO₃ 1.59 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 containing 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 dicofol inhibition rate detection method 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 0.01, 0.03, 0.1, 0.3. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03, 0.1, 0.3, 1. mu.g/mL with antibody diluent. After selecting the optimal working point, the dicofol standard was diluted to 8 concentrations (0, 1, 3, 10, 30, 90, 270, 810 ng/mL), and plotted according to the IC-ELISA protocol using originPro 8.5 (results are shown in FIG. 1), to obtain a standard inhibition curve of dicofol, and IC was calculated₅₀。

Example 1: synthesis of dicofol hapten

Since the dicofol small molecule has no immunogenicity and can not stimulate mice to generate immune response so as to generate antibodies, the dicofol needs to be coupled to protein by a protein coupling technology so as to obtain the immunogenicity; active groups commonly used in protein coupling technology comprise amino, carboxyl, hydroxyl, sulfydryl and the like, and since the dicofol does not contain the active groups in the molecular structural formula, derivatization is needed.

Dicofol (250 mg, 0.67 mmol), ethyl cyanoacetate (1 mL) and acetic acid (1 mL) were placed together in a round bottom flask and 96% sulfuric acid (4 mL) was slowly added. After the addition was complete, the mixture was taken up at 50Heating in water bath at deg.C for 15 min. The mixture turned dark red and was then stirred at room temperature for 20 h. The mixture was extracted with diethyl ether. The ether phase was washed with water and sodium bicarbonate solution, dried and concentrated. Use of Hexane/CH on silica gel preparation plates₂Cl₂(1: 1) purifying the crude product by using an eluent, collecting a product part, and spin-drying the collected liquid to obtain a product, namely the dicofol hapten, wherein the chemical structural formula of the product is shown as the following formula:

。

example 2: synthesis of dicofol complete antigen

Weighing 5.5mg dicofol hapten (DCF-COOH) and 4.2mg N-hydroxysuccinimide (NHS), dissolving in 300 μ L N, N-Dimethylformamide (DMF), and stirring at room temperature for 10 min; 6.9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved in 100. mu.L of DMF, and then added to the DCF-COOH solution, followed by reaction for 6 to 8 hours with stirring at room temperature (referred to as solution A). Diluting 8mg of KLH to 4mg/mL (called as solution B) by using 0.01M Carbonate Buffer Solution (CBS), 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 DCF-COOH-KLH, and identifying by ultraviolet absorption scanning method.

Example 3: synthesis of dicofol coating antigen

Dissolving 3.0mg dicofol hapten (DCF-COOH) and 2.3mg N-hydroxysuccinimide (NHS) in 300 μ L anhydrous N, N-Dimethylformamide (DMF), and stirring at room temperature for 10min to obtain dicofol hapten (DCF-COOH) solution; dissolving 3.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 100 mu L of anhydrous DMF, adding the solution into a DCF-COOH solution, and stirring at room temperature to react for 6-8h to obtain a solution A; diluting 10mg of egg albumin (OVA) with 1mL 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 (DCF-COOH-OVA).

Example 4: preparation of hybridoma cell strain CBC secreting monoclonal antibody of dicofol

(1) Obtaining animal immunity: mixing and emulsifying the dicofol complete antigen and equivalent Freund's adjuvant, and performing neck and back subcutaneous multipoint injection immunization (except puncture immunization) on BALB/c mice; complete Freund adjuvant is used for the first immunization, and the dosage is 100 ug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 ug/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 ug/mouse; 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, SP2/0 tumor cell number was required to reach (1 ~ 4) x 10 before fusion⁷Ensuring 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 7 min: at 1min, 1mL of PEG 1500 was added dropwise from slow to fastIn a cell; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dripping 2mLRPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then carrying out warm bath at 37 ℃ for 5 min; centrifuging (800 rpm, 8 min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum, 2% 50 × HAT, adding to 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: in the first step, positive cell holes are screened by an ic-ELISA method, and in the second step, dicofol is used as a standard substance to measure the inhibition effect of positive cells by the ic-ELISA method.

And selecting cell holes with good inhibition on the dicofol standard substance, performing subcloning by using a limiting dilution method, and detecting by using the same method after seven days.

And subcloning for three times according to the method to finally obtain the dicofol monoclonal antibody cell strain CBC.

Example 5: preparation and identification of dicofol 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 10⁶Dicofol hybridoma cells were obtained from the seventh day, and ascites fluid was purified 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 ℃.

Using indirect competitionELISA, IC of dicofol monoclonal antibody is measured₅₀The value is 19.09ng/mL, which shows that the dicofol has good sensitivity and can be used for the immunoassay detection of the dicofol.

Example 6: application of dicofol monoclonal antibody

The monoclonal antibody prepared from the hybridoma cell strain CBC through in vivo ascites is applied to an ELISA addition recovery test of dicofol, and the method specifically comprises the following 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, 1, 3, 10, 30, 90, 270 and 810ng/mL dicofol standard solution by using Phosphate Buffer Solution (PBS), respectively adding the standard solution and a sample extracting solution to be detected into a closed enzyme label plate, wherein each hole has 50 mu L, repeating each sample for 3 holes, adding 50 mu L of anti-DCF monoclonal antibody diluted to 0.3 mu g/mL into each hole, reacting at 37 ℃ for 0.5h, and washing and drying the plate;

(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;

(6) adding and recovering and sample pretreatment: apple was selected as the test sample. Cleaning and cutting a sample to be tested, and stirring and mixing the sample to be tested in a mashing machine. Three samples were weighed out separately and homogenized, 5g each, and 20 ppb, 100 ppb and 250 ppb of dicofol standards (based on antibody linearity range and IC) were added to the samples₅₀Set the addition concentration), shake vigorously for 2 min. 10mL of acetone, 1g of NaCl, 5g of anhydrous NaSO were added₄Ultrasonic vibration for 15min, centrifuging at 4000rpm for 10min, collecting supernatant, taking 1mL of supernatant, drying with nitrogen, and redissolving with 5mL of PBS (namely diluting five times to reduce the influence of the sample matrix).

The additive recovery tests were performed using indirect competitive ELISA with recovery rates of 92%, 93%, and 89%, respectively.

Claims

1. A hybridoma cell strain CBC for secreting monoclonal antibodies of dicofol is preserved in China general microbiological culture Collection center, and the address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain with the preservation date of 2019, 3 months and 7 days and the preservation number of CGMCC No. 17398.

2. The dicofol monoclonal antibody is secreted and produced by the hybridoma cell strain CBC with the preservation number of CGMCC No.17398 as claimed in claim 1.

3. The use of monoclonal antibodies to dicofol as claimed in claim 2, characterized in that: an ELISA detection method of dicofol is established, which is used for identifying dicofol in food detection.

4. A method for preparing a monoclonal antibody against dicofol according to claim 2, which comprises the steps of: injecting paraffin oil into the abdominal cavity of a BALB/c mouse, then injecting the hybridoma cell strain CBC with the preservation number of CGMCC No.17398 of claim 1 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and preserving the obtained monoclonal antibody at low temperature.

5. The dicofol complete antigen for preparing the hybridoma cell strain CBC according to claim 1, wherein the complete antigen DCF-COOH-KLH has the following molecular formula:

。

6. a process for preparing a dicofol complete antigen as claimed in claim 5, which comprises the steps of: dissolving dicofol hapten DCF-COOH, N-hydroxysuccinimide NHS and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in anhydrous N, N-dimethylformamide DMF, and stirring for reaction to obtain a dicofol hapten DCF-COOH solution, namely 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 PBS to obtain the complete antigen DCF-COOH-KLH.

7. The method for preparing the dicofol complete antigen according to claim 6, wherein the dicofol hapten DCF-COOH has the following formula:

。