CN111333503A - Dicofol hapten, artificial antigen and antibody as well as preparation methods and applications thereof - Google Patents
Dicofol hapten, artificial antigen and antibody as well as preparation methods and applications thereof Download PDFInfo
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- CN111333503A CN111333503A CN202010146446.0A CN202010146446A CN111333503A CN 111333503 A CN111333503 A CN 111333503A CN 202010146446 A CN202010146446 A CN 202010146446A CN 111333503 A CN111333503 A CN 111333503A
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- dicofol
- hapten
- artificial antigen
- antibody
- carrier protein
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- UOAMTSKGCBMZTC-UHFFFAOYSA-N dicofol Chemical compound C=1C=C(Cl)C=CC=1C(C(Cl)(Cl)Cl)(O)C1=CC=C(Cl)C=C1 UOAMTSKGCBMZTC-UHFFFAOYSA-N 0.000 title claims abstract description 114
- 239000000427 antigen Substances 0.000 title claims abstract description 28
- 102000036639 antigens Human genes 0.000 title claims abstract description 28
- 108091007433 antigens Proteins 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 19
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 7
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 22
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 claims description 20
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 15
- HFFLGKNGCAIQMO-UHFFFAOYSA-N trichloroacetaldehyde Chemical compound ClC(Cl)(Cl)C=O HFFLGKNGCAIQMO-UHFFFAOYSA-N 0.000 claims description 15
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 abstract description 6
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- YEPBJSVHMZUONK-UHFFFAOYSA-N 1,2,2,2-tetrachloro-1-phenylethanol Chemical compound ClC(Cl)(Cl)C(Cl)(O)C1=CC=CC=C1 YEPBJSVHMZUONK-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
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- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
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- 229920000742 Cotton Polymers 0.000 description 1
- YVGGHNCTFXOJCH-UHFFFAOYSA-N DDT Chemical compound C1=CC(Cl)=CC=C1C(C(Cl)(Cl)Cl)C1=CC=C(Cl)C=C1 YVGGHNCTFXOJCH-UHFFFAOYSA-N 0.000 description 1
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- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 241000819999 Nymphes Species 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
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- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Substances C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- LKPLKUMXSAEKID-UHFFFAOYSA-N pentachloronitrobenzene Chemical compound [O-][N+](=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl LKPLKUMXSAEKID-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
- C07C59/56—Unsaturated compounds containing hydroxy or O-metal groups containing halogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
- C07C51/367—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by introduction of functional groups containing oxygen only in singly bound form
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- 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
-
- 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
-
- 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
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
- G01N33/184—Herbicides, pesticides, fungicides, insecticides or the like
-
- 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
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses dicofol hapten, artificial antigen and antibody, and a preparation method and application thereof, wherein the dicofol hapten provided by the invention not only furthest reserves the characteristic structure of dicofol, so that the immunogenicity of the dicofol hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the dicofol artificial antigen obtained by coupling the dicofol hapten and the carrier protein is used for immunizing animals, so that the method is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, the sensitivity of the dicofol antibody can reach 0.1 mu g/L through detection, the cross reaction rate with other organic chlorine pesticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of the dicofol.
Description
Technical Field
The invention belongs to the field of food safety detection. More particularly, the invention relates to dicofol hapten, artificial antigen and antibody, and preparation methods and applications thereof.
Background
Dicofol is an organic chlorine acaricide, can be used for preventing and treating acarid on crops such as cotton, fruit trees, vegetables, etc., has strong contact poisoning and stomach poisoning effects on adult acarid, nymph and ovum, and has long effective period and quick-acting period. The research shows that the medicine has certain toxicity to human and livestock, the main toxic symptoms are headache, dizziness, hyperhidrosis, chest distress, mydriasis, blurred vision, nausea, vomit and diarrhea, and contact dermatitis can be caused by local contact. The dicofol is forbidden in China, and the national standard GB2763-2019 maximum limit of pesticide residue in food safety national standard food also provides that the residue limit of the dicofol in fruits is 1 mg/kg.
At present, the dicofol is detected at home and abroad mainly by adopting analysis methods such as a gas chromatography, a gas chromatography-mass spectrometry, a liquid chromatography-tandem mass spectrometry and the like, and the defects of complicated sample pretreatment, long detection time, expensive instruments and the like exist, so the dicofol can not be widely applied in China and does not meet the requirements of on-site detection on accurate detection and screening of a large number of samples at low cost in a short time. The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with detection methods such as instruments and the like. Therefore, the immunoassay provides a new analysis and detection method for the residual research of the dicofol.
When an immunological detection method is established and the detection method is applied to detecting the residual quantity of the dicofol, the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the aim is to realize the aim under the precondition that a proper dicofol hapten is synthesized and prepared. Research shows that the chlorphenyl trichloroethanol of the dicofol is a characteristic structure and plays an important role in immune response. In the invention patents CN107907697A and CN110607283A, the-OH of the dicofol is derivatized into a spacer arm with-COOH, and then the spacer arm is coupled with a carrier protein, so that the modification site is closer to the characteristic structure of the dicofol molecule, which is not beneficial to the exposure of the characteristic structure of the hapten molecule, thus the difficulty of the immune system of an animal in recognizing the characteristic structure of the hapten molecule is increased, and the affinity and the specificity of an antibody are reduced. Therefore, in order to prepare the antibody with high specificity, the modification site in the dicofol molecule is far away from the characteristic structure of chlorphenyl trichloroethanol, so that the characteristic molecular structure part of the hapten is fully exposed, and the shielding effect of the carrier protein on the characteristic structure of the hapten molecule is avoided.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the hapten which can furthest reserve the characteristic structure of dicofol and has a connecting arm with a certain length and the preparation method of the hapten; the artificial antigen prepared by the hapten and the antibody with high detection sensitivity and strong specificity; and the use of such haptens.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a dicofol hapten, which has the following structural formula:
the dicofol hapten provided by the invention introduces a carboxyl active group at the chlorine ortho-position on the benzene ring of dicofol, so that the dicofol hapten can be coupled with carrier protein to obtain an artificial antigen for immunization; the dicofol hapten reserves all characteristic groups of dicofol, changes the original structural characteristics of dicofol to the minimum, and is coupled with carrier protein to highlight the unique chlorphenyl trichloroethanol structure of the dicofol, thereby laying a foundation for generating antibodies with stronger specificity and higher sensitivity for subsequent stimulation of animal immune response.
In a second aspect, the invention provides a preparation method of the dicofol hapten, which is obtained by reacting o-chlorophenylacetic acid, chlorobenzene and chloral under the catalysis of sulfuric acid.
Further, the preparation method comprises the following steps: dissolving o-chlorophenylacetic acid in carbon tetrachloride, adding chlorobenzene, fully stirring, adding trichloroacetaldehyde, cooling to 10-15 ℃, dropwise adding a 10% sulfuric acid solution, continuously stirring for reacting for 4 hours, and after the reaction is finished, separating and purifying to obtain dicofol hapten; wherein the molar ratio of the o-chlorophenylacetic acid to the chlorobenzene to the chloral is 1:1: 1.
Further, the preparation method comprises the following specific steps: dissolving 1.7g of o-chlorophenylacetic acid in 100mL of carbon tetrachloride, adding 1.12g of chlorobenzene, fully stirring, adding 1.47g of trichloroacetaldehyde, cooling to 10-15 ℃, dropwise adding 10mL of 10% sulfuric acid solution, continuously stirring for reacting for 4 hours, adding 100mL of 0-5 ℃ cold water after the reaction is finished, transferring to a separating funnel, vibrating, standing for layering, extracting the water phase with trichloromethane for three times, 80mL each time, and combining the organic phases; and (3) washing the carbon tetrachloride with water again, combining the carbon tetrachloride and the chloroform extract, evaporating the mixture to dryness to obtain red oily matter, purifying the red oily matter by using a silica gel column, and eluting and separating the red oily matter by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 6:1 to obtain the dicofol hapten.
The preparation method of the hapten mainly comprises the following steps: (1) generating corresponding functional groups by using the existing structure or intermediate of an object to be detected through oxidation, reduction, substitution, hydrolysis and other reactions; (2) transforming the metabolite or structural analogue of the hapten into a required hapten by using the metabolite or structural analogue as a primer; (3) raw materials and reagents are used for re-synthesis, and small molecules with functional groups are introduced at proper positions. The invention synthesizes the dicofol hapten with carboxyl under the catalysis of sulfuric acid by using o-chlorophenylacetic acid, chlorobenzene and chloral, has higher difficulty compared with the former two methods, but has the advantages of easily establishing the optimal coupling site and easily constructing a reasonable spacing arm, thereby increasing the possibility of generating antibodies aiming at characteristic structures.
The method for preparing the dicofol hapten is reasonably designed according to the structural characteristics of dicofol, the used raw materials are easy to obtain, the reaction operation is simple, the reaction conditions are easy to control, and the purity and the yield of the prepared dicofol hapten are high.
In a third aspect, the invention provides a dicofol artificial antigen which is a conjugate obtained by conjugating a carrier protein and the dicofol hapten. The dicofol artificial antigen can be used as an immunogen and also can be used as a coating antigen.
Further, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin; bovine serum albumin and ovalbumin are preferred.
More specifically, the following are: an immunogen formed by dicofol hapten-Bovine Serum Albumin (BSA); dicofol hapten-Ovalbumin (OVA).
The dicofol hapten molecule is only immunoreactive and not immunogenic. Therefore, in order to endow the dicofol hapten molecule with immunogenicity, the dicofol hapten molecule is coupled and combined with a proper carrier protein molecule, so that the dicofol artificial antigen with both immunoreactivity and immunogenicity is generated.
In a fourth aspect, the invention provides a method for preparing the dicofol artificial antigen, wherein a carrier protein is coupled to a carboxyl group of the dicofol hapten by an active ester method.
In a fifth aspect, the invention provides a dicofol antibody, which is obtained by immunizing animals with the dicofol artificial antigen and can perform specific immune reaction with dicofol.
Further, the dicofol antibody is a monoclonal antibody or a polyclonal antibody. In addition, the dicofol antibody can be prepared by a method which is conventional in the art.
In a specific embodiment, the dicofol antibody is a murine monoclonal antibody specific for a dicofol artificial antigen to the above-described dicofol hapten.
The dicofol antibody obtained by using the artificial antigen of dicofol has better titer, specificity and affinity, and has low cross reaction rate with other organic chlorine pesticides.
In a sixth aspect, the invention provides an application of the dicofol antibody in detecting residual dicofol.
The invention induces immune animals to generate antibodies through the dicofol artificial antigen, thereby being used in the immunoassay analysis of dicofol.
The dicofol immunoassay comprises but is not limited to a dicofol ELISA kit, a dicofol colloidal gold test strip and a dicofol time-resolved fluorescence test strip.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the dicofol hapten provided by the invention not only retains the characteristic structure of dicofol to the maximum extent, so that the immunogenicity of the dicofol hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the dicofol artificial antigen obtained by coupling the dicofol hapten and the carrier protein is used for immunizing animals, so that the method is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, and provides a basis for subsequently establishing various immunoassay methods of the dicofol.
The preparation method of the dicofol hapten has the advantages of easily available raw materials, simple reaction operation, easily controlled reaction conditions and high purity and yield of the prepared dicofol hapten.
The dicofol antibody obtained by using the artificial antigen of dicofol has good titer, specificity and affinity, the sensitivity can reach 0.1 mu g/L, and the cross reaction rate with other organic chlorine pesticides is low.
Drawings
FIG. 1 is a synthetic route of dicofol hapten of the invention
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.
Example 1
A preparation method of dicofol hapten comprises the following steps:
dissolving 1.7g of o-chlorophenylacetic acid in 100mL of carbon tetrachloride, adding 1.12g of chlorobenzene, fully stirring, adding 1.47g of trichloroacetaldehyde, cooling to 10-15 ℃, dropwise adding 10mL of 10% sulfuric acid solution, continuously stirring for reacting for 4 hours, adding 100mL of 0-5 ℃ cold water after the reaction is finished, transferring to a separating funnel, vibrating, standing for layering, extracting the water phase with trichloromethane for three times, 80mL each time, and combining the organic phases; and (3) washing the carbon tetrachloride with water again, combining the carbon tetrachloride and the chloroform extract, evaporating the mixture to dryness to obtain red oily matter, purifying the red oily matter by using a silica gel column, and eluting and separating the red oily matter by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 6:1 to obtain the dicofol hapten.
Example 2
A preparation method of dicofol artificial antigen comprises the following steps:
taking 14mg of dicofol hapten prepared in example 1, adding 1mL of dimethyl sulfoxide (DMSO) to dissolve, adding 8.2mg of N-hydroxysuccinimide (NHS) and 15.7mg of carbodiimide (EDC), and reacting at room temperature for 3h to obtain a hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), and adding 0.05mol/L PB buffer solution for dissolving to obtain solution B; dripping the A solution into the B solution, reacting at 4 deg.C for 12h, dialyzing and purifying with 0.02mol/L PBS for 3 days, changing solution 3 times per day to obtain dicofol artificial antigen coupled with bovine serum albumin, packaging, and storing at-20 deg.C.
Example 3
A preparation method of dicofol artificial antigen comprises the following steps:
taking 8.1mg of dicofol hapten prepared in example 1, adding 1mL of 1, 4-dioxane for dissolving, adding 6.1mg of NHSj and 9.7mg of EDC, and reacting at room temperature for 3h to obtain a hapten solution A; dissolving Ovalbumin (OVA)50mg in 0.05mol/LPB buffer solution to obtain solution B; dripping the A solution into the B solution, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution 3 times every day to obtain the dicofol artificial antigen coupled with the ovalbumin, subpackaging and storing at-20 ℃.
Example 4
A dicofol antibody is prepared through the following steps:
1. animal immunization
Taking 10 healthy female Balb/c mice (divided into two groups of A and B, and 5 mice in each group) in 6-8 weeks, emulsifying the mice by Freund complete adjuvant for primary immunization, and injecting the mice subcutaneously at the back of the neck and the back at multiple points, wherein the immunization dose of each mouse is 200 mu g of dicofol artificial antigen coupled with bovine serum albumin; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the last immunization uses normal saline to replace Freund's incomplete adjuvant, and is injected in the abdominal cavity, and the injection dosage is the same as the previous times. The specific immunization procedure is shown in table 1.
Table 1 mouse immunization procedure
And (3) for the third time, the fourth time and 7d after the boosting immunization, the tail of the mouse is cut off, blood is taken, and the serum titer of the mouse is measured by an ELISA method, wherein the specific steps are as follows:
(1) diluting dicofol artificial antigen coupled with ovalbumin with 0.05mol/L of carbonate buffer solution with pH of 9.6 at a ratio of 1:1000, coating an enzyme label plate with 100 mu L of each hole, incubating at 37 ℃ for 2h, throwing off coating solution, washing for 1 time by PBST, and patting to dry;
(2) adding 150 mu L of sealing liquid into each hole, reacting at 37 ℃ for 2h, then pouring off the sealing liquid, and patting to dry;
(3) adding 50 mu L of antiserum diluted by PBS in each hole, reacting at 25 ℃ for 30min, then removing the reaction liquid, washing 3-5 times by PBST, and patting dry at intervals of 30s each time;
(4) adding 100 mu L/hole of horseradish peroxidase-labeled goat anti-mouse anti-antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST (PBST), and patting dry at intervals of 30s each time;
(5) adding 50 μ L of substrate developing solution A and B into each well, reacting at 25 deg.C in dark for 15min, adding 50 μ L of 2mol/L H into each well2SO4Terminating the reaction by the solution;
(6) measuring OD value with wavelength of 450nm by enzyme-labeling instrument, and determining OD of sample hole450The titer of positive sera was determined as a dilution factor close to 1.
2. Cell fusion
(1) Preparing feeder cells: the Balb/c mice of 8-10 weeks old are killed after neck breakage, soaked in 75% alcohol for 5min, immediately placed in an ultra-clean bench with the abdomen facing upwards in a plate or fixed on a dissecting plate. The skin of the abdomen of the mouse is clamped by an ophthalmic forceps, a small opening is cut by scissors, and the peritoneum is not cut to avoid the outflow and pollution of the abdominal cavity fluid. Then blunt dissection was performed up and down with scissors to fully expose the peritoneum. Wiping peritoneum with alcohol cotton ball for sterilization. 5mL of RPMI-1640 basic culture solution was aspirated by a syringe, injected into the abdominal cavity of the mouse, the syringe was gently withdrawn, and the leg and tail of the mouse were shaken several times. The liquid in the abdominal cavity is pumped back by the original syringe and is injected into the centrifuge tube. The operation is repeated for 3-4 times. Centrifuging at 1000r/min for 10min, and discarding the supernatant. Resuspending the cells with 20-50 mL of complete culture medium, adding 100 μ L/well dropwise to the culture plate, and placing in an incubator for later use.
(2) Preparation of splenocytes: 3d after enhancing the immunity, taking an immune Balb/c mouse, collecting blood from an orbit, dislocating and killing the mouse, disinfecting the mouse in 75% alcohol, taking the spleen, removing connective tissues, preparing a spleen cell suspension, transferring the spleen cell suspension into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging the spleen cell suspension at 1500-2000 r/min for 5min, removing a supernatant, adding RPMI-1640 to 30mL, and counting the number for later use.
(3) Myeloma cell preparation: taking 3 bottles of myeloma cells with good growth state (the number of living cells is more than 95 percent), completely blowing down the myeloma cells, transferring the myeloma cells into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging at 1500-2000 r/min for 5min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(4) Cell mixing: spleen cells and myeloma cells are mixed and centrifuged at 1500-2000 r/min for 5min, wherein the ratio of the spleen cells to the myeloma cells is 8: 1.
(5) Cell fusion: centrifuging the mixed cells, pouring out the supernatant, making the precipitated cell mass into paste, placing the paste in a water bath at 37 ℃, adding 1mL of fusion agent which is polyethylene glycol (PEG)4000 within 1min, acting for 2min, slightly stirring the cells, adding 20mL of serum-free PEG nutrient solution within 4min, centrifuging at 1000r/min for 10min, and discarding the supernatant. The cells were resuspended in 20-50 mL of complete medium, plated on 96-well feeder cells-containing cell culture plates at 100. mu.L per well, and placed in an incubator.
3. Cell line selection
And (3) when the cells grow to 1/3-1/2 of the bottom of the hole, carrying out antibody detection. Screening culture wells with hybridoma cell growth by adopting an ELISA method, wherein the screening comprises two steps: in the first step, positive cell holes are screened by indirect ELISA, and in the second step, dicofol is used as a standard substance to perform inhibition effect determination on positive cells by indirect competition ELISA. And selecting a hole with better inhibition on the dicofol standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the dicofol monoclonal antibody.
4. Preparation of ascites
Injecting liquid paraffin into Balb/c mice for 6-8 weeks, collecting hybridoma cells in logarithmic growth phase after 10 days by using RPMI-1640 basic culture medium, counting by using a blood counting plate and a microscope, wherein the cell concentration is 1.0 × 106～1.5×106In the size per mL range. Each mouse was injected with 0.5mL hybridoma cells into the abdominal cavity. Note that after one week the abdomen of the mouse was enlarged, ascites was collected in the abdomen of the mouse with a sterile syringe once every one to two days, and this was repeated until the mouse died naturally. Centrifuging at 4 deg.C for 5min at 5000r/min, collecting supernatant, and removing fat and protein membrane floating on the upper layer of abdominal water.
5. Antibody purification
The monoclonal antibody is purified by an octanoic acid-ammonium sulfate method.
6. Antibody titer determination
And (3) measuring the antibody titer by adopting an indirect ELISA method, and referring to the step 1, measuring the serum titer of animal immunity. The result shows that the titer of the dicofol monoclonal antibody is more than or equal to 100000.
7. Antibody cross-reactivity assay
The indirect competitive ELISA method is adopted for determination, and the result shows that the cross reaction rate of the dicofol monoclonal antibody to dicofol and other organic chlorine pesticides is as follows: the dicofol is 100%, and the dichlorodiphenyl trichloroethane, the hexachloro-hexa-benzene, the dicofol and the quintozene are all less than 1%. Therefore, the prepared antibody has better specificity.
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (9)
1. A dicofol hapten, which is characterized by the following structural formula:
2. a process for the preparation of a dicofol hapten according to claim 1 which is obtained by reacting o-chlorophenylacetic acid, chlorobenzene and chloral under the catalysis of sulphuric acid.
3. The method for preparing dicofol hapten according to claim 2, which comprises: dissolving o-chlorophenylacetic acid in carbon tetrachloride, adding chlorobenzene, fully stirring, adding trichloroacetaldehyde, cooling to 10-15 ℃, dropwise adding a 10% sulfuric acid solution, continuously stirring for reacting for 4 hours, and after the reaction is finished, separating and purifying to obtain dicofol hapten; wherein the molar ratio of the o-chlorophenylacetic acid to the chlorobenzene to the chloral is 1:1: 1.
4. The method for preparing dicofol hapten according to claim 2, which comprises the following steps: dissolving 1.7g of o-chlorophenylacetic acid in 100mL of carbon tetrachloride, adding 1.12g of chlorobenzene, fully stirring, adding 1.47g of trichloroacetaldehyde, cooling to 10-15 ℃, dropwise adding 10mL of 10% sulfuric acid solution, continuously stirring for reacting for 4 hours, adding 100mL of 0-5 ℃ cold water after the reaction is finished, transferring to a separating funnel, vibrating, standing for layering, extracting the water phase with trichloromethane for three times, 80mL each time, and combining the organic phases; and (3) washing the carbon tetrachloride with water again, combining the carbon tetrachloride and the chloroform extract, evaporating the mixture to dryness to obtain red oily matter, purifying the red oily matter by using a silica gel column, and eluting and separating the red oily matter by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 6:1 to obtain the dicofol hapten.
5. An artificial antigen for dicofol, which is a conjugate obtained by conjugating a carrier protein to the dicofol hapten according to claim 1.
6. The dicofol artificial antigen of claim 5, wherein the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
7. A process for preparing an artificial antigen of dicofol as claimed in claim 5 or 6, wherein the carrier protein is coupled to the carboxyl group of the hapten of dicofol as claimed in claim 1 by active ester method.
8. A dicofol antibody produced by immunizing an animal with the artificial antigen for dicofol according to claim 5, which specifically immunoreacts with dicofol.
9. Use of the dicofol antibody according to claim 8 for detecting residual dicofol.
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