CN116675619A - Ether-pyrethrin artificial hapten, artificial antigen, antibody and application thereof - Google Patents
Ether-pyrethrin artificial hapten, artificial antigen, antibody and application thereof Download PDFInfo
- Publication number
- CN116675619A CN116675619A CN202310269791.7A CN202310269791A CN116675619A CN 116675619 A CN116675619 A CN 116675619A CN 202310269791 A CN202310269791 A CN 202310269791A CN 116675619 A CN116675619 A CN 116675619A
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- China
- Prior art keywords
- ethofenprox
- hapten
- artificial
- antibody
- amide
- Prior art date
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- Pending
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—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
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C201/00—Preparation of esters of nitric or nitrous acid or of compounds containing nitro or nitroso groups bound to a carbon skeleton
- C07C201/06—Preparation of nitro compounds
- C07C201/08—Preparation of nitro compounds by substitution of hydrogen atoms by nitro groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C213/02—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton by reactions involving the formation of amino groups from compounds containing hydroxy groups or etherified or esterified hydroxy groups
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
- C07C233/24—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
- C07C233/25—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses hapten design and synthesis of ethofenprox, development of ethofenprox artificial antigen and antibody and application thereof in the field of rapid detection and affinity purification columns. The preparation method comprises the steps of designing the ethofenprox hapten, introducing a connecting arm, introducing a carrier, synthesizing, purifying and the like to prepare the artificial antigen with immunogenicity, and obtaining the ethofenprox hapten product modified by the amide carboxylic acid. The ether pyrethrin hapten with the structure is respectively coupled with keyhole limpet hemocyanin, bovine serum albumin or chicken ovalbumin to synthesize an immunogen and a coating antigen, and the artificial hapten has higher immune activity through test, can stimulate an animal immune system to generate an anti-ether pyrethrin antibody, and has the serum antibody titer of an immunized Blab/c mouse of 3200. The invention fills the gap that the current ethofenprox has no proper artificial antigen for preparing specific and high-sensitivity antibodies, and establishes a rapid and sensitive immunoassay method for the ethofenprox.
Description
Technical Field
The invention belongs to the field of immunoassay detection of small molecular compounds, and particularly relates to the technologies of organic synthesis, immunochemistry, biochemistry, physicochemical determination and the like. In particular to the design and synthesis of hapten and artificial antigen of an insecticidal ethofenprox small molecular compound with functional carboxylic acid groups, the immunization of mice, the screening of hybridoma cells, the preparation of specific monoclonal antibodies and the establishment of an immunoassay method thereof, which are particularly shown in the fields of ELISA detection methods, immunochromatography test paper rapid detection methods and immunoaffinity purification columns, agricultural products and ecological environment.
Background
The etofenprox (etofenprox) is called a pyrethroid-like pesticide, has the advantages of high insecticidal activity and high knockdown speed, and has no chrysanthemic acid in the structure, but has a spatial structure similar to that of pyrethroid, but in fact, the etofenprox belongs to ethers instead of pyrethroid pesticides. The insecticidal composition is suitable for preventing and controlling rice, vegetables and cotton, has special effects on homoptera planthoppers, has good effects on various pests such as lepidoptera, hemiptera, orthoptera, coleoptera, diptera, isoptera and the like, and has remarkable prevention and control effects on rice planthoppers. The ethofenprox is used as a low-toxicity safe pesticide, is also a specified product of the national prohibition of the application of high-toxicity pesticides on rice, is the only pyrethroid pesticide allowed to be registered on the rice, and has quick acting and lasting effects superior to those of pymetrozine and nitenpyram. Since 2009, ethofenprox has been listed as a major popularization product by agricultural technology popularization centers nationwide, and plant protection stations in Anhui, jiangsu, hubei, hunan, guangxi and other places have listed agents as major popularization varieties of plant protection stations. The toxicity of the ethofenprox is comprehensively evaluated, belongs to a low-toxicity pesticide, but has high toxicity to fishes and bees and has certain environmental risk. Therefore, we need to analyze the advantages and disadvantages of ethofenprox correctly, and the supervision of ethofenprox should be enhanced while the ethofenprox is promoted and used mainly, and the effective supervision method mainly is to strictly manage and control the medication and to monitor and control on site.
The invention provides technical support for on-site detection supervision of ethofenprox, and develops a method for rapidly detecting ethofenprox and extracting the ethofenprox by pretreatment. At present, the quantitative detection method of the ethofenprox residue is mainly an instrument analysis method such as high performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and the like, and has high sensitivity and good accuracy, but has high equipment condition requirements, complex pretreatment and high technical requirements for analysts, and is difficult to meet the requirements of rapid on-site detection of mass samples. The invention develops an immunochemistry analysis technology taking the ethofenprox antigen antibody as a core, overcomes the defects, and has the advantages of high sensitivity, high specificity, quick and convenient use, environmental friendliness and the like. To date, there is no disclosure of a monoclonal antibody hybridoma cell technology reporting ethofenprox in China, and application of a specific antibody generated by the monoclonal antibody to rapid detection and analysis of ethofenprox, and development of an ethofenprox immunoaffinity purification column for rapid sample pretreatment.
Disclosure of Invention
The invention discloses the design and synthesis of ethofenprox hapten, the preparation of ethofenprox artificial antigen and the preparation of monoclonal antibody thereof. The preparation method aims to provide a preparation method of the ethofenprox hapten, the corresponding artificial antigen and the corresponding antibody, and is applied to the immune rapid detection analysis of the ethofenprox in a sample, and an immune affinity purification column is used for the pretreatment of the ethofenprox rapid sample.
The invention selects ether represented by ethofenprox or similar pyrethroid compounds, carries out hapten design and structure transformation to obtain the ethofenprox hapten molecules containing amide carboxylic acid groups, and the ethofenprox hapten molecules have no immunogenicity and can obtain the immunogenicity only by taking macromolecular proteins as carriers, and then a certain number of hapten molecules are connected with protein macromolecules to respectively obtain coating antigen and immunogen.
The invention is characterized in that the chemical synthesis difficulty of the ethofenprox hapten is overcome, and a 1-set ethofenprox hapten synthesis technical route is formed. When the method is used for immunizing animals, specific antibodies with high affinity can be generated, and an ELISA method, namely an ELISA method, is established, so that the content of the ethofenprox can be accurately detected. The agarose immunoaffinity purification column constructed by the depending on the specific antibody can be used for the rapid extraction pretreatment of the trace etofenprox in the sample.
The invention is characterized in that the keyhole limpet hemocyanin coupled small molecule is selected as an immunogen, so that the coupling ratio of hapten molecules to protein molecules is improved in the coupling reaction, and the immunogenicity is enhanced.
The structure of ethofenprox is shown below:
in order to solve the technical problems, the invention adopts the following technical scheme: the ethofenprox hapten is an amide carboxylic acid group substituted by an R site on a benzene ring, R can be one or more of amide propionic acid, amide butyric acid, amide valeric acid and amide caproic acid derivative groups, and the molecular structural formula is as follows:
further, the ethofenprox hapten is prepared by carrying out nitration, amination, acylation and hydrolysis reactions on ethofenprox, wherein the ethofenprox hapten is prepared by introducing amide carboxylic acid to react with ethofenprox under the condition of strong base catalysis, and the molecular structural formula is as follows:
the invention also discloses an ethofenprox immunogen (MJZ-KLH or MJZ-BSA), which is synthesized by coupling hapten modified by ethofenprox R site structure and KLH protein or BSA protein, wherein the molecular structural formulas of the MJZ-KLH and the MJZ-BSA are as follows:
4. the ether pyrethroid artificial coating antigen (MJZ-OVA) is synthesized by coupling an artificial hapten with the modified ether pyrethroid R locus structure and OVA protein, and the molecular structural formula of the MJZ-OVA is as follows:
the invention also discloses an ethofenprox antibody, which is a monoclonal antibody capable of carrying out specific immunoreaction with an ethofenprox immunogen or an ethofenprox artificial coating antigen.
The anti-ethofenprox monoclonal antibody is a monoclonal antibody which can generate specific immune reaction with MJZ-KLH, MJZ-BSA or MJZ-OVA and ethofenprox compounds.
The ethofenprox antibody can be used for rapidly and sensitively detecting the ethofenprox content in agricultural products and environmental samples, and is particularly applied to ELISA immunoassay methods, immunochromatography test paper rapid detection methods and affinity purification columns. The application fields comprise agricultural products such as vegetables, fruits, meats and the like, and environmental samples such as water, soil, atmosphere and the like.
The ethofenprox is prepared by a series of reactions such as nitrating, amination, acylation, hydrolysis and the like, and the synthetic route is as follows:
the preparation method of the ethofenprox hapten comprises the following steps:
(1) Dissolving ethofenprox in 10-20 times of MeOH water solution, adding 1-2 times of HNO 3 Heating to 60-80 ℃ under stirring, and reacting to obtain nitroetherpyrethrin;
(2) Adding a proper amount of Fe and HCl for catalytic reaction to obtain the amino ether pyrethrin;
(3) Further adding 1-2 times of equivalent methyl acyl chloride n-acid, stirring and reacting to obtain the ethofenprox-methyl amide n-acid;
(4) And adding NaOH with the equivalent weight of 1-2 times, reacting for 1-2 hours to obtain ethofenprox-amide n carboxylic acid, cooling to 5-10 ℃, carrying out suction filtration, recrystallizing a solid sample obtained by filtration by using 1, 4-dioxane, and drying to obtain the ethofenprox hapten crystal.
The preparation method of the MJZ-KLH or MJZ-BSA is a carbodiimide method, and comprises the following steps:
(1) 0.004mmoL of ethofenprox hapten is weighed and dissolved in 0.5mL of DMF, 0.008 mmoL-0.02 mmoL of N-hydroxysuccinimide (NHS) is added, the mixture is stirred and reacted for 15min, and then 0.004 mmoL-0.016 mmoL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is added, and the mixture is stirred and reacted overnight at room temperature. The reaction liquid is the ethofenprox hapten activating liquid.
(3) 5-10mg/mL KLH or BSA solution was prepared with 0.01M carbonic acid buffer (pH 9.0), and the above-mentioned HaptonA-activated solution was added dropwise under stirring at room temperature, followed by slow magnetic stirring for 3.5h. The reaction solution was placed in a pretreated dialysis bag and dialyzed 3 times against 100 volumes of 0.01M Phosphate Buffer (PBS), changing the solution every 6 hours.
(4) And taking out the dialyzate after the dialysis is finished, uniformly mixing, sub-packaging into a centrifuge tube with low adsorptivity to obtain MJZ-KLH or MJZ-BSA, and storing at the temperature of minus 20 ℃.
The preparation method of the MJZ-OVA is carbonyl diimidazole method, and comprises the following steps:
(1) The 0.01mmoL ethofenprox hapten was weighed into 0.5mL DMF. Adding 0.02-0.1mmoL of carbonyl diimidazole under stirring, and stirring at room temperature for reacting for 3h to obtain the ethofenprox hapten activating solution.
(2) 10-20mg/mL OVA solution is prepared by using 0.01M carbonic acid buffer solution (pH 9.0), the ethofenprox hapten activating solution is added dropwise under the condition of stirring at room temperature, the stirring reaction is continued for 3 hours, the reaction solution is filled into a pretreated dialysis bag, and the solution is dialyzed for 3 times by 100 times of 0.01M PBS, and is changed every 6 hours.
(3) And taking out the dialyzate after the dialysis is finished, uniformly mixing, sub-packaging into a centrifuge tube with low adsorptivity to obtain MJZ-OVA solution, and storing at the temperature of minus 20 ℃.
The preparation method of the anti-ethofenprox monoclonal antibody comprises animal immunization, hybridoma cell screening and antibody preparation, and comprises the following steps:
(1) Immunization: the immunized animals are BALB/c female mice of 6 weeks old, the immunization method adopts a subcutaneous multipoint Freund adjuvant injection mode, the booster immunization is carried out 21 days after the primary immunization, the booster immunization is carried out every 14 days, 3 times of booster immunization and 1 time of final immunization are carried out, and the specific test method is as follows:
primary immunization: dissolving 0.1-0.2mg MJZ-KLH or MJZ-BSA in 0.9% physiological saline, adding equal volume of Freund's complete adjuvant into syringes, and emulsifying immunogen MJZ-KLH with emulsifying tube. The mice were subcutaneously injected at 7 points, and immunized by subcutaneous injections in the neck, back, limbs, and abdomen, respectively.
Boosting: dissolving 0.05-0.1mg of MJZ-KLH or MJZ-BSA in 0.9% physiological saline, taking equal volumes of Freund's complete adjuvant, respectively filling into syringes, and docking the syringes with an emulsifying tube to fully emulsify the immunogen MJZ-KLH or MJZ-BSA. Mice were immunized by intraperitoneal injection.
Last immunization: 0.05-0.1mg of MJZ-KLH or MJZ-BSA is dissolved in 0.9% physiological saline, and the mixture is directly subjected to intraperitoneal injection immunization.
(2) Hybridoma cell selection: the mouse antibody titer is monitored periodically, the mouse tail blood titer is measured at intervals of one week after 2 times of booster immunization, and the absorbance value OD is obtained 450nm And is more than or equal to 1 positive. Taking MJZ-OVA as a coating source, performing final immunization when the blood titer of the tail of the test mouse reaches 1:1000-1:4000, taking out the spleen after the final immunization for 3d, performing hybridoma cell fusion, and screening monoclonal hybridoma cells. Coating 5-10 mug/mL MJZ-OVA by adopting a homologous indirect competition ELISA method, taking 100-500ng/mL of ethofenprox standard solution as a competition medicament, and picking out strong positive OD 450nm And (3) continuously subcloning the cell strain with the inhibition rate of more than or equal to 0.5 and more than or equal to 50 percent for 2-3 times to obtain a stable monoclonal hybridoma cell strain.
(3) Antibody preparation: 7-week-old F1 mice were selected and were first intraperitoneally injected with pristane 500. Mu.L/mouse. After 7 days, the stable monoclonal hybridoma cell strain obtained by the screening in the step (2) (cell density about 2X 10) 6 Per mL) 500 μl/mouse peritoneal cavity. And after 7-10 days, collecting ascites after the abdominal cavity of the mouse swells, and purifying by adopting an octanoic acid-ammonium sulfate precipitation method. The purified ascites is coated with MJZ-OVA, and the potency of the antibody and the inhibition rate of the ethofenprox are measured by an indirect method.
The method for detecting and analyzing the ethofenprox ELISA comprises the following steps:
the ethofenprox monoclonal antibody is applied to the rapid detection of the ethofenprox content in a sample, and the immune detection is enzyme-linked immunosorbent assay (ELISA). The application of the ethofenprox antibody in the immunodetection of the ethofenprox content comprises the following steps:
(1) Coating: MJZ-OVA was dissolved in 0.01mol/L carbonate buffer (pH 9.0), coated on an ELISA plate at 100. Mu.l/Kong Baobei, chilled at 4℃overnight, and washed 3 times with 0.01M PBST after removal.
(2) Closing: after the coating was completed, 2% nonfat milk powder was prepared with 0.01mol/L PBS (pH 7.4), the ELISA plate was blocked with 300. Mu.l/well, the plate was blocked in a 37℃incubator for 30 minutes, and the plate was washed 2 times with 0.01mol/L PBST after removal.
(3) Competing reaction: the sample to be tested is dissolved in 0.01mol/L PBS solution of 30% methanol, 50 μl/hole of the ELISA plate is added, 50 μl/hole of the ethofenprox antibody solution is added, the reaction is carried out for 0.5 hour in a 37 ℃ incubator, and the plate is washed 3 times after being taken out.
(4) Enzyme-labeled secondary antibody reaction: the rabbit anti-mouse enzyme-labeled secondary antibody was diluted 20000 times with 0.01mol/L PBS (pH 7.4), 100. Mu.l/well was added, reacted in an incubator at 37℃for 0.5 hours, and the plate was washed 4 times after removal.
(5) Color development and termination: the chromogenic substrate is tetramethyl benzidine (TMB) solution, 100 μl/well is added during chromogenic process, and 2mol/L H is added after 15min of chromogenic process 2 SO 4 50 μl/Kong Zhongzhi.
(6) After the color development is terminated, the ELISA plate is placed on an ELISA reader for reading, and OD is read 450nm Numerical values, calculation and analysis data.
The method for rapidly detecting the ethofenprox immunochromatography test paper comprises the following steps:
the ethofenprox coating antigen and the ethofenprox monoclonal antibody are applied to rapid detection of the ethofenprox content in agricultural products and environmental samples, and the immunodetection is a colloidal gold immunochromatography test strip. The method for rapidly detecting the ethofenprox antibody by using the immunochromatography test paper comprises the following steps:
(1) The coating antigen MJZ-OVA is streaked on a chromatographic membrane with the concentration of 0.05-0.5mg/mL to be used as a detection line (T line), the goat anti-mouse secondary antibody is streaked on the chromatographic membrane with the concentration of 0.05-0.4mg/mL to be used as a quality control line (C line), and the mixture is placed in a blast drying box for drying.
(2) And (3) assembling the chromatographic membrane, a sample pad and a water absorption pad to prepare chromatographic test paper, and cutting the chromatographic test paper into test strips with the thickness of 3-4 mm.
(3) Labeling the ethofenprox monoclonal antibody on 30nm colloidal gold particles with the labeling concentration of 2-10 mug/mL to prepare the Cheng Jinbiao ethofenprox antibody. The prepared gold-labeled antibody is added into 96 micro-well plates in 10-50 mu L, and the mixture is freeze-dried or air-dried.
(4) Preparing an ethofenprox standard solution, wherein the concentration range is 0.01-10mg/L, taking the test strip in the step 2 and the gold-labeled microwell in the step 3 for detection, and the sample volume is 100-200 mu L.
(5) The ethofenprox colloidal gold immunochromatographic test strip is used for detecting the ethofenprox standard solution, positive is displayed when the concentration is 0.5mg/L or more, the T line does not develop color, and the C line develops color; when the concentration is lower than 0.5mg/L, the color is negative, the T line is developed, and the C line is developed.
The preparation method of the ethofenprox immunoaffinity purification column comprises the following steps:
the ethofenprox monoclonal antibody is applied to the preparation of an agarose immunoaffinity purification column, the ethofenprox immunoaffinity purification column is applied to the rapid extraction of ethofenprox in a sample, and the preparation of the affinity purification column comprises the following steps:
(1) Agarose gel was prepared and cyanogen bromide activated agarose gel 4B was dissolved in 0.1M carbonate buffer (pH 9.2) at a concentration of 100-300mg/mL and vortexed.
(2) Adding ethofenprox monoclonal antibody into the agarose gel solution, mixing the monoclonal antibody with vortex of 0.2-1.0mg/mL, and standing for reaction for 1h.
(3) Loading the monoclonal antibody-labeled agarose gel into an empty column of an affinity chromatography column, sealing the column head, sealing the uncombined sites with 2% skimmed milk, filling the column with 2-4 column volumes of skimmed milk, and sealing for 1h.
(4) After the closure is completed, the sealing cover is opened, and 2-4 column volumes are eluted by using 2% skimmed milk.
(5) Unbound and poorly bound proteins were removed and 10-20 column volumes were eluted with 0.1M carbonate buffer (pH 9.2).
(6) The nonspecific proteins were removed and 5-10 column volumes were eluted with 0.2M glycine-HCl buffer (pH 2.8).
(7) Agarose immune affinity chromatographic column preservation, eluting 4-8 column volumes by 0.01M phosphoric acid buffer (pH 7.2) containing 0.02-0.1% proclin300 as preservative, and preserving 1-2mm solution above the affinity chromatographic column, respectively adding sealing caps at column head and column tail, and preserving at 4deg.C.
Compared with the prior art, the invention has the following advantages and effects:
(1) The designed and synthesized ethofenprox hapten has high similarity with a target object to be detected, remains complete for the characteristic structure of the ethofenprox, and lays a foundation for preparing the ethofenprox antibody with good specificity;
(2) Experiments prove that the hapten has simple and convenient synthesis method, high synthesis efficiency and few reaction steps, and the ethofenprox hapten can be synthesized by only 4 steps of reactions, so that the controllability and timeliness of the reactions are improved.
(3) The ethofenprox antibody has good detection sensitivity, the MJZ-OVA coating concentration is 1 mug/mL, the antibody concentration is 1 mug/mL, the methanol content of the ethofenprox standard solution is 30%, and an ELISA method standard curve is established to obtain a linear equation: y= 0.1816ln (x) +0.7153, r 2 Estimate ic= 0.9712 50 306ng/mL was reached.
(4) The ethofenprox monoclonal antibody is applied to the agarose immunoaffinity purification column for the first time, the ethofenprox monoclonal antibody is used for sample pretreatment, the recovery rate is high, the operation is rapid and simple, the whole operation flow can be completed within 30min, and the pretreatment efficiency is improved.
Therefore, compared with other methods, the method for synthesizing the hapten is easier to popularize. The antibody related by the invention has high activity, is successfully applied to an ELISA detection method, has simple and rapid operation, only needs 1 hour and 15 minutes in the detection process, and can reach more than 95 percent of detection accuracy. The immunoaffinity purification column developed by the invention can be applied to rapid pretreatment, can be reused, and has a recovery rate of more than 85%. Therefore, the invention not only has good detection effect in laboratory environment, but also lays a foundation for developing an immunity rapid detection tool with low cost, high efficiency and rapid operation, is suitable for the field rapid detection requirement, has good application prospect, and has economic and social benefits.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a diagram of an ethofenprox hapten 1 H NMR spectrum.
FIG. 2 is an HPLC-MS spectrum of ethofenprox hapten.
FIG. 3 is a standard graph established by the ethofenprox ELISA assay method of the invention.
FIG. 4 is a chart showing the detection color of the ethofenprox gold-labeled immunochromatographic test paper of the present invention.
Detailed Description
The following examples of the present invention are merely further illustrative of the present invention and are not intended to limit the scope or spirit of the present invention. The invention will now be further illustrated by means of examples.
Example 1
(1) Synthesis of ethofenprox hapten
In the immune system of animals, antigens must be immunogenic in order to obtain antibodies. Ethofenprox as a small molecule compound is not naturally immunogenic, i.e., does not stimulate the immune system of animals to produce antibodies. It is necessary to artificially structurally modify and couple proteins to render them immunogenic. The main active groups in the protein coupling method are carboxyl, amino, hydroxyl and sulfhydryl, and the carboxylic acid group is introduced into the ethofenprox structure in the invention, so that the ethofenprox can be coupled with protein in a condensation way.
The method for synthesizing the ethofenprox hapten comprises the following steps: 2g of ethofenprox is weighed and dissolved in a 15-time volume of MeOH aqueous solution, 1.5 times of molar equivalent HNO3 is added, the temperature is raised to 80 ℃ under stirring, and the nitroethofenprox is obtained by reaction. Adding a proper amount of Fe and HCl for catalytic reaction to obtain the amino ether pyrethrin. Further, methyl acyl chloride valerate with the equivalent weight of 1.5 times is stirred for reaction to obtain the ethofenprox-methyl amide valerate. Adding NaOH with the equivalent weight of 1.5 times, hydrolyzing for 2 hours to obtain ethofenprox-amide valeric acid, cooling to 10 ℃, carrying out suction filtration, recrystallizing the obtained solid sample by using 1, 4-dioxane, and drying to obtain the ethofenprox hapten solid0.532g of body with a purity of 96.2%. Characterized by 1 H NMR(500MHz,CDCl 3 )δ8.47(d,J＝2.3Hz,1H),7.78(s,1H),7.34(m,2H),7.27(q,J＝2.9,2.1Hz,1H),7.11(m,1H),7.01(dd,J＝8.6,2.7Hz,4H),6.94(s,1H),6.90(dd,J＝8.2,2.6Hz,1H),6.77(d,J＝8.6Hz,1H),4.47(s,2H),4.07(dd,J＝11.1,6.9Hz,2H),3.46(s,2H),2.42(t,J＝6.6Hz,4H),1.77(m,4H),1.45(d,J＝7.2Hz,4H),1.33(s,6H).ESI-MS C 31 H 37 NO 6 ,calc.519.2,found:[M-H] - 518.2.
(2) Preparation of Ether-pyrethrin artificial antigens MJZ-KLH and MJZ-BSA
0.004mmoL of ethofenprox hapten is weighed and dissolved in 0.5mL of DMF, 0.012mmoL of N-hydroxysuccinimide (NHS) is added, the mixture is stirred and reacted for 15min, and then 0.006mmoL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is added, and the mixture is stirred and reacted at room temperature overnight. The reaction liquid is the ethofenprox hapten activating liquid. 5mg/mL KLH solution was prepared with 0.01M carbonic acid buffer (pH 9.0), and the above-mentioned HaptonA-activated solution was added dropwise under stirring at room temperature, followed by a slow magnetic stirring reaction for 3.5h. The reaction solution was placed in a pretreated dialysis bag and dialyzed 3 times against 100 volumes of 0.01M Phosphate Buffer (PBS), changing the solution every 6 hours. And taking out the dialyzate after the dialysis is finished, uniformly mixing, sub-packaging into a centrifuge tube with low adsorptivity to obtain the ethofenprox immunogen MJZ-KLH, and storing at the temperature of minus 20 ℃.
The 0.01mmoL ethofenprox hapten was weighed into 0.5mL DMF. Adding 0.02mmoL of carbonyl diimidazole under stirring, and stirring at room temperature for reaction for 3 hours to obtain the ethofenprox hapten activating solution. A10 mg/mL BSA solution was prepared in 0.01M carbonate buffer (pH 9.0), the hapten-activated solution was added dropwise under stirring at room temperature, the stirring was continued for 3 hours, the reaction solution was placed in a pretreated dialysis bag, dialyzed 3 times with 100 volumes of 0.01M PBS, and the solution was changed every 6 hours. And taking out the dialyzate after the dialysis is finished, uniformly mixing, sub-packaging into a centrifuge tube with low adsorptivity to obtain the ethofenprox coating original MJZ-OVA solution, and storing at the temperature of minus 20 ℃.
(2) Preparation of ethofenprox monoclonal antibody hybridoma cell strain
The immunized animals are BALB/c female mice of 6 weeks old, the immunization method adopts subcutaneous multipoint Freund adjuvant injection mode, the boost is carried out 21 days after the primary immunization, the boost is carried out every 14 days, and 3 times of boost and 1 time of final immunization are carried out. The primary immunization is carried out by dissolving 0.2mg MJZ-KLH in 0.9% physiological saline, taking equal volume of Freund's complete adjuvant, respectively loading into syringes, fully emulsifying immunogen MJZ-KLH by the emulsifying tube docking syringe, carrying out subcutaneous injection at 7 points of mice, and carrying out subcutaneous injection immunization at neck, back, limbs and abdomen respectively. Boosting, dissolving 0.1mg of MJZ-KLH in 0.9% physiological saline, taking equal volumes of Freund's complete adjuvant, respectively filling into syringes, and fully emulsifying the immunogen MJZ-KLH by using an emulsifying tube to butt joint the syringes to perform intraperitoneal injection immunization on mice. The final immunization was performed by dissolving 0.1mg of MJZ-KLH in 0.9% physiological saline and directly performing intraperitoneal injection.
The antibody titer is periodically monitored during the immunization process of the mice, the mouse tail blood titer is measured every other week after the 2 times of booster immunization, and the absorbance value OD is used 450nm And is more than or equal to 1 positive. Taking MJZ-OVA as a coating source, performing final immunization when the blood titer of the tail of the test mouse reaches 1:10000-1:20000, taking out the spleen after the final immunization for 3d, performing hybridoma cell fusion, and screening monoclonal hybridoma cells. Coating 5 mug/mL MJZ-OVA by adopting a homologous indirect competition ELISA method, taking 100ng/mL of ethofenprox standard solution as a competition medicament, and picking out strong positive OD 450nm And (3) continuously subcloning the cell strain with the inhibition rate of more than or equal to 0.5 and more than or equal to 50 percent for 2-3 times to obtain a stable monoclonal hybridoma cell strain.
(3) Preparation of Ether-chrysanthemate monoclonal antibody
F1 generation mice 7 weeks old are selected, first, 500 mu L of pristane is used for intraperitoneal injection, and after 7 days, the ether pyrethrin stable monoclonal hybridoma cell strain (cell density about 2X 10) 6 Per mL) was inoculated into the peritoneal cavity of mice at a dose of 500 μl/mouse. After one week, the abdominal cavity of the mice begins to expand, the ascites of the mice are collected, and the ethofenprox monoclonal antibody is obtained by purifying by adopting an octanoic acid-ammonium sulfate precipitation method.
Example 2
An ELISA rapid detection method was established using the ethofenprox monoclonal antibody obtained in example 1.
MJZ-OVA was dissolved in 0.01mol/L carbonate buffer (pH 9.0), coated on an ELISA plate at 100. Mu.l/Kong Baobei, chilled at 4℃overnight, and washed 3 times with 0.01M PBST after removal. After the coating was completed, 2% nonfat milk powder was prepared with 0.01mol/L PBS (pH 7.4), the ELISA plate was blocked with 300. Mu.l/well, the plate was blocked in an incubator at 37℃for 30 minutes, and the plate was taken out and washed 2 times with 0.01mol/L PBST. The sample to be tested is dissolved in 0.01mol/L PBS solution of 30% methanol, 50 μl/hole of the ELISA plate is added, 50 μl/hole of the ethofenprox antibody solution is added, the reaction is carried out for 0.5 hour in a 37 ℃ incubator, and the plate is washed 3 times after being taken out. The rabbit anti-mouse enzyme-labeled secondary antibody was diluted 20000 times with 0.01mol/L PBS (pH 7.4), 100. Mu.l/well was added, reacted in an incubator at 37℃for 0.5 hours, and the plate was washed 4 times after removal. The chromogenic substrate is tetramethyl benzidine (TMB) solution, 100 μl/well is added during chromogenic process, and 2mol/L H is added after 15min of chromogenic process 2 SO 4 50 μl/Kong Zhongzhi. After the color development is terminated, the ELISA plate is placed on an ELISA reader for reading, and OD is read 450nm Numerical values, calculation and analysis data.
Inhibition of antibody by target analyte concentration i= ((Amax-Amin) - (Ai-Amin)/(Amax-Amin)) ×100, binding rate of antibody to antigen B/b0= (Ai-Amin)/(Amax-Amin), where: amax is the mean absorbance of blank wells, amin is the mean absorbance of preimmune BLAB/c mouse serum control wells, ai is the mean absorbance of loaded wells. A standard curve is drawn by taking the inhibition rate I or the binding rate B/B0 as an ordinate and the analyte concentration C as an abscissa, as shown in figure 3, the detection limit can reach 306 mug/L stably, and the detection range is 26 mug/L-3642 mug/L by taking the target analyte concentrations corresponding to 5% and 95% inhibition rates as the minimum detection concentration and the maximum detection concentration respectively.
The limit standard of the ethofenprox in vegetables, fruits and meat crops is 0.5-5mg/kg specified in GB2763-2021 maximum residue limit of pesticides in food safety national Standard food, and the sensitivity of the method is 0.306mg/kg, so that the Maximum Residue Limit (MRL) standard detection requirement can be met. The actual sample detection adopts leeks (MRL=1 mg/kg), celery (MRL=1 mg/kg), radishes (MRL=1 mg/kg), pears (MRL=0.6 mg/kg), grapes (MRL=4 mg/kg), beef (MRL=0.5 mg/kg), water and soil, negative samples and ethofenprox positive addition samples are respectively arranged, the addition concentration is 0.02mg/kg, 0.2mg/kg and 2mg/kg, the sample extraction is carried out by adopting an extracting solution, and the final sample to be detected is diluted 10 times. And calculating the concentrations of the ethofenprox in the blank sample and the added sample according to an ELISA standard curve, wherein the result accords with the added concentration. The specific data results are shown in Table 1.
TABLE 1 Ether-pyrethrin ELISA detection results in actual samples
Example 3
The ethofenprox coated antigen and the ethofenprox antibody obtained in the example 1 are used for preparing immunochromatography rapid detection test paper, the detection limit of the rapid detection test paper method on the ethofenprox is 0.5mg/L, and the method is applied to actual sample detection.
The actual sample detection adopts leek, celery, radish, pear, grape, beef, water and soil, blank samples and ethofenprox positive addition samples are respectively arranged, and the sample extraction is carried out by adopting an extracting solution. The blank sample and the added sample with the concentration lower than the detection limit concentration of the ethofenprox are false positive except that the test paper detection result of the added sample with the concentration group of 0.2mg/kg of the ethofenprox in the leek and beef samples is false positive, and the rest samples are negative; the test paper of the added sample with the concentration higher than the detection limit concentration of the ethofenprox is positive. The specific results are shown in Table 2.
TABLE 2 test results of ethofenprox paper in actual samples
Example 4
The ethofenprox antibody obtained in the example 1 is used for preparing an agarose immunoaffinity purification column, and the ethofenprox affinity purification column is mainly applied to the rapid extraction of the ethofenprox in the pretreatment of a sample.
Cyanogen bromide activated sepharose 4B was dissolved in 0.1M carbonate buffer (pH 9.2) at a concentration of 200mg/mL and vortexed. Adding the ethofenprox monoclonal antibody into the agarose gel solution, mixing uniformly by vortex with the adding amount of the monoclonal antibody of 0.2-1.0mg/mL, standing for reaction for 1h, and labeling the ethofenprox antibody on the agarose gel. Loading the monoclonal antibody marked agarose gel into an empty column of an affinity chromatography column, sealing the column head, sealing the unbound sites by using 2% skimmed milk, filling the column by using 2 column volumes of skimmed milk, and sealing for 1h. After the closure was completed, the seal cap was opened and 2 column volumes were eluted with 2% skimmed milk. The unbound and poorly bound proteins were removed by eluting 10-20 column volumes with 0.1M carbonate buffer (pH 9.2). The nonspecific proteins were removed by eluting 5-10 column volumes with 0.2M glycine-HCl buffer (pH 2.8). And (3) preserving the agarose immune affinity chromatographic column, eluting 5 column volumes by using 0.01M phosphoric acid buffer (pH 7.2) containing preservative 0.02-0.1% proclin300, reserving 1-2mm solution above the affinity chromatographic column, and respectively adding sealing covers on the column head and the column tail to obtain the ethofenprox immune affinity purification column.
The ethofenprox immunoaffinity purification column in the embodiment has application prospect in the aspect of rapid sample pretreatment, is used for sample pretreatment, and has the advantages of high recovery rate, environmental protection, safety, rapid and simple operation and the like.
Finally, it should be noted that the present invention is not limited to the above 4 embodiments, but may have other derivative applications. Other variations that can be directly derived or suggested by those skilled in the art from the present disclosure are considered to be within the scope of the present invention.
Claims (7)
1. The ethofenprox artificial hapten is an amide carboxylic acid group substituted by an R site on a benzene ring, and R can be one or more of amide propionic acid, amide butyric acid, amide valeric acid and amide caproic acid derivative groups, and is characterized by having a molecular structural formula:
2. the ethofenprox artificial hapten of claim 1, wherein the ethofenprox artificial hapten is selected from the group consisting of: the ethofenprox hapten is prepared by introducing amide carboxylic acid into ethofenprox for reaction under the catalysis of strong alkali, and the molecular structural formula is as follows:
3. an ethofenprox immunogen characterized by: the artificial hapten structurally modified by the R site of ethofenprox as claimed in claim 1 or 2 is coupled with Keyhole Limpet Hemocyanin (KLH) to synthesize the hapten, and the molecular structural formula is as follows:
4. an ethofenprox immunogen characterized by: the synthetic method comprises the steps of coupling an artificial hapten with the R site structure modified by ethofenprox as claimed in claim 1 or 2 with Bovine Serum Albumin (BSA), wherein the molecular structural formula is as follows:
5. the ethofenprox artificial coating antigen is characterized in that: the synthetic method comprises the steps of coupling an artificial hapten with the structural modification of an R site of the ethofenprox and chicken Ovalbumin (OVA), wherein the molecular structural formula of the ethofenprox artificial coating antigen is as follows:
6. an ethofenprox antibody, characterized in that: a monoclonal antibody which specifically immunoreacts with the ethofenprox immunogen of claim 3 or 4 or the ethofenprox artificial coating antigen of claim 5.
7. Use of the ethofenprox antibody of claim 6, wherein: the ethofenprox antibody can be used for rapidly and sensitively detecting the ethofenprox content in a sample, and is particularly applied to an ELISA detection method, an immunochromatography test paper rapid detection method and an immunoaffinity purification column.
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