CN110845444B - Dimethomorph hapten, artificial antigen and antibody, and preparation method and application thereof - Google Patents
Dimethomorph hapten, artificial antigen and antibody, and preparation method and application thereof Download PDFInfo
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- CN110845444B CN110845444B CN201911132184.6A CN201911132184A CN110845444B CN 110845444 B CN110845444 B CN 110845444B CN 201911132184 A CN201911132184 A CN 201911132184A CN 110845444 B CN110845444 B CN 110845444B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
- C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
- C07D295/182—Radicals derived from carboxylic acids
- C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—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
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- 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
Abstract
The invention discloses a dimethomorph hapten, an artificial antigen and an antibody as well as a preparation method and application thereof, the dimethomorph hapten provided by the invention not only furthest reserves the characteristic structure of dimethomorph, so that the immunogenicity of the dimethomorph hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the dimethomorph hapten is coupled with the carrier protein to obtain the dimethomorph immune antigen to immunize animals, so that the dimethomorph immune antigen is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, the sensitivity of the dimethomorph antibodies can reach 0.1 mu g/L by detection, the cross reaction rate with other pesticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of dimethomorph.
Description
Technical Field
The invention belongs to the field of food safety detection. More specifically, the invention relates to dimethomorph haptens, artificial antigens and antibodies, and preparation methods and applications thereof.
Background
Dimethomorph is a high-efficiency bactericide for oomycetes, has the characteristics of systemic absorption, treatment, anti-sporulation and the like, and is effective for all stages of the life cycle of the oomycetes. Dimethomorph affects the rearrangement of the molecular structure of pathogenic bacteria cell walls, interfering with the assembly of cell wall polymers and further interfering with the formation of cell walls. The bactericidal composition can be used for preventing and treating diseases caused by peronospora and phytophthora on various crops, and has no cross resistance with phenylamide bactericides such as metalaxyl, benalaxyl and the like. Dimethomorph is widely used in vegetables and fruits, and therefore, the residue has a high detectable rate.
At present, methods for measuring dimethomorph mainly comprise gas chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry and the like. The instrument detection method has the defects of complicated sample pretreatment, long detection time, expensive instruments and the like, so the instrument detection method cannot be widely applied in China and does not meet the requirements of on-site detection on accurately detecting and screening 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 research of the residue of the dimethomorph.
When establishing an immunological detection method and applying the detection method to detect the residual quantity of the dimethomorph, the key technology is to obtain an antibody with strong specificity and high sensitivity, and the precondition is to synthesize and prepare a proper dimethomorph hapten to achieve the aim.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a hapten which can furthest reserve the characteristic structure of dimethomorph and has a connecting arm with a certain length and a 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.
The technical problem to be solved by the invention is realized by the following technical scheme:
the first purpose of the invention is to provide a dimethomorph hapten which has the following structural formula:
the second purpose of the invention is to provide a preparation method of dimethomorph hapten, which is obtained by reacting 3- (4-chlorphenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid with 3-piperazine-1-propionic acid.
Further, the preparation method comprises the following steps: dissolving 3- (4-chlorphenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid in dichloromethane, adding N, N-dimethylformamide, fully stirring, adding oxalyl chloride, stirring at 0-5 ℃ for reacting for 40min, after the reaction is finished, adding 3-piperazine-1-propionic acid and triethylamine, stirring at room temperature for reacting for 3h, and after the reaction is finished, separating and purifying to obtain the dimethomorph hapten; wherein the mass ratio of the 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid, oxalyl chloride and 3-piperazine-1-propionic acid is 1:0.1: 1.
Further, the preparation method comprises the following specific steps: dissolving 3.18g of 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid in 150mL of dichloromethane, adding 0.6mL of N, N-dimethylformamide, fully stirring, adding 0.13g of oxalyl chloride, stirring at 0-5 ℃ for reaction for 40min, after the reaction is finished, adding 1.59g of 3-piperazine-1-propionic acid and 0.8mL of triethylamine, stirring at room temperature for reaction for 3h, after the reaction is finished, adding 100mL of water, oscillating, standing, removing an aqueous phase, evaporating an organic phase to dryness, loading on a silica gel column, and eluting and separating by using petroleum ether-ethyl acetate with a volume ratio of 1:1 to obtain the dimethomorph hapten.
The third purpose of the invention is to provide a dimethomorph artificial antigen, which comprises a dimethomorph immune antigen and a dimethomorph coating antigen, and the dimethomorph artificial antigen is a conjugate obtained by coupling a carrier protein and the dimethomorph hapten.
Further, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
The fourth purpose of the invention is to provide a preparation method of the dimethomorph artificial antigen, wherein the preparation method of the dimethomorph immune antigen comprises the following steps: dissolving 50mg of bovine serum albumin by using 0.05mol/L PB buffer solution to obtain protein A solution; dissolving 17mg of dimethomorph hapten by using 1mL of N, N-dimethylformamide, adding 13.2mg of carbodiimide, uniformly mixing, reacting for 10min, adding 9.1mg of N-hydroxysuccinimide, blowing, uniformly mixing, reacting at room temperature for 2h, then dropwise adding into the protein A solution, reacting for 12h at 4 ℃, dialyzing and purifying by using 0.02mol/L PBS buffer solution for 3 days, subpackaging and freezing;
the preparation method of the dimethomorph-coated antigen comprises the following steps: dissolving 50mg of ovalbumin by using 0.05mol/L PB buffer solution to obtain protein B solution; dissolving 9.8mg of dimethomorph hapten in 1mL of N, N-dimethylformamide, adding 0.2mL of triethylamine, mixing uniformly, adding 0.21mL of isobutyl chloroformate, reacting at 0-5 ℃ for 2h, then dropwise adding the mixture into the protein B solution, reacting at 4 ℃ for 12h, dialyzing and purifying by using 0.02mol/L PBS buffer solution for 3 days, and subpackaging and freezing.
The fifth object of the present invention is to provide a dimethomorph antibody which is obtained by immunizing an animal with the dimethomorph immunizing antigen and can specifically react with dimethomorph.
Further, the dimethomorph antibody is a monoclonal antibody or a polyclonal antibody.
The sixth purpose of the invention is to provide the application of the dimethomorph antibody in detecting dimethomorph residues.
The invention has the following beneficial effects:
the dimethomorph hapten provided by the invention not only retains the characteristic structure of dimethomorph to the greatest extent, so that the immunogenicity of the dimethomorph hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the dimethomorph hapten is coupled with the carrier protein to obtain the dimethomorph immune antigen for immunizing animals, which 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 dimethomorph.
According to the invention, the 3- (4-chlorphenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid and 3-piperazine-1-propionic acid are adopted to react to prepare the dimethomorph hapten, the reaction steps are simple, the required experimental conditions are mild and easy to control, and the prepared dimethomorph hapten has higher purity and yield.
The dimethomorph antibody obtained by adopting the dimethomorph immune antigen has better titer, specificity and affinity, the sensitivity can reach 0.1 mu g/L, and the cross reaction rate with other pesticides is low.
Drawings
FIG. 1 is a scheme for the synthesis of the dimethomorph hapten according to the invention
Detailed Description
In a first aspect, the present invention provides a dimethomorph hapten having the following structural formula:
the dimethomorph hapten provided by the invention not only retains the characteristic structure of dimethomorph to the greatest extent, so that the immunogenicity of the dimethomorph hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the dimethomorph hapten is coupled with the carrier protein to obtain the dimethomorph immune antigen for immunizing animals, which is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity.
In a second aspect, the present invention provides a method for preparing the aforesaid dimethomorph hapten, which is obtained by reacting 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid with 3-piperazine-1-propionic acid.
Preferably, the preparation method of the dimethomorph hapten comprises the following steps: dissolving 3- (4-chlorphenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid in dichloromethane, adding N, N-dimethylformamide, fully stirring, adding oxalyl chloride, stirring at 0-5 ℃ for reacting for 40min, after the reaction is finished, adding 3-piperazine-1-propionic acid and triethylamine, stirring at room temperature for reacting for 3h, and after the reaction is finished, separating and purifying to obtain the dimethomorph hapten;
wherein the mass ratio of the 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid, oxalyl chloride and 3-piperazine-1-propionic acid is 1:0.1: 1.
According to the invention, the 3- (4-chlorphenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid and 3-piperazine-1-propionic acid are adopted to react to prepare the dimethomorph hapten, the reaction steps are simple, the required experimental conditions are mild and easy to control, and the prepared dimethomorph hapten has higher purity and yield.
In a third aspect, the invention also provides a dimethomorph artificial antigen, which comprises a dimethomorph immune antigen and a dimethomorph coating antigen, and the dimethomorph artificial antigen is a conjugate obtained by coupling a carrier protein and the dimethomorph hapten.
Preferably, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
The dimethomorph hapten molecule is only immunoreactive and not immunogenic. Therefore, in order to confer immunogenicity on the dimethomorph hapten molecules, it is also necessary to couple and bind the dimethomorph hapten molecules to suitable carrier protein molecules, thereby generating an immunoreactive and immunogenic dimethomorph artificial antigen.
In a fourth aspect, the present invention also provides a method for preparing the above-mentioned dimethomorph artificial antigen, wherein the preparation of the dimethomorph immune antigen comprises the following steps: dissolving 50mg of bovine serum albumin by using 0.05mol/L PB buffer solution to obtain protein A solution; dissolving 17mg of dimethomorph hapten by using 1mL of N, N-dimethylformamide, adding 13.2mg of carbodiimide, uniformly mixing, reacting for 10min, adding 9.1mg of N-hydroxysuccinimide, blowing, uniformly mixing, reacting at room temperature for 2h, then dropwise adding into the protein A solution, reacting for 12h at 4 ℃, dialyzing and purifying by using 0.02mol/L PBS buffer solution for 3 days, subpackaging and freezing;
the preparation method of the dimethomorph-coated antigen comprises the following steps: dissolving 50mg of ovalbumin by using 0.05mol/L PB buffer solution to obtain protein B solution; dissolving 9.8mg of dimethomorph hapten in 1mL of N, N-dimethylformamide, adding 0.2mL of triethylamine, mixing uniformly, adding 0.21mL of isobutyl chloroformate, reacting at 0-5 ℃ for 2h, then dropwise adding the mixture into the protein B solution, reacting at 4 ℃ for 12h, dialyzing and purifying by using 0.02mol/L PBS buffer solution for 3 days, and subpackaging and freezing.
In a fifth aspect, the invention also provides a dimethomorph antibody which is obtained by immunizing animals with the dimethomorph immunizing antigen and can perform specific immune reaction with dimethomorph.
The dimethomorph antibody may be a monoclonal antibody or a polyclonal antibody. In addition, the dimethomorph antibody can be prepared by a method conventional in the art.
In a specific embodiment, the dimethomorph antibody is a murine monoclonal antibody specific for a dimethomorph immunizing antigen against the dimethomorph hapten described above.
The dimethomorph antibody obtained by adopting the dimethomorph immune antigen has better titer, specificity and affinity, and low cross reaction rate with other pesticides.
In a sixth aspect, the invention also provides the use of the dimethomorph antibody in detecting dimethomorph residues.
The invention induces immune animals to generate antibodies through the dimethomorph artificial antigen, thereby being used in dimethomorph immunodetection analysis.
The dimethomorph immunodetection comprises but is not limited to a dimethomorph ELISA kit and a dimethomorph colloidal gold test strip.
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 dimethomorph hapten comprises the following steps: dissolving 3.18g of 3- (4-chlorphenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid in 150mL of dichloromethane, adding 0.6mL of N, N-dimethylformamide, fully stirring, adding 0.13g of oxalyl chloride, stirring at 0-5 ℃ for reaction for 40min, after the reaction is finished, adding 1.59g of 3-piperazine-1 propionic acid and 0.8mL of triethylamine, stirring at room temperature for reaction for 3h, after the reaction is finished, adding 100mL of water, oscillating, standing, removing an aqueous phase, evaporating an organic phase to dryness, loading on a silica gel column, and eluting and separating by using petroleum ether-ethyl acetate with a volume ratio of 1:1 to obtain the dimethomorph hapten.
Example 2
A preparation method of dimethomorph immunizing antigen comprises the following steps:
dissolving 50mg of bovine serum albumin by using 0.05mol/L PB buffer solution to obtain protein A solution; dissolving 17mg of the dimethomorph hapten obtained in the example 1 in 1mL of N, N-dimethylformamide, adding 13.2mg of carbodiimide, uniformly mixing, reacting for 10min, adding 9.1mg of N-hydroxysuccinimide, uniformly blowing, reacting at room temperature for 2h, then dropwise adding into the protein A solution, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, and subpackaging and freezing.
Example 3
A preparation method of dimethomorph-coated antigen comprises the following steps:
dissolving 50mg of ovalbumin by using 0.05mol/L PB buffer solution to obtain protein B solution; 9.8mg of the dimethomorph hapten obtained in example 1 was dissolved in 1mL of N, N-dimethylformamide, 0.2mL of triethylamine was added, the mixture was mixed, 0.21mL of isobutyl chloroformate was added, the mixture was reacted at 0-5 ℃ for 2 hours, and then the mixture was added dropwise to the protein B solution, reacted at 4 ℃ for 12 hours, dialyzed and purified with 0.02mol/L of PBS buffer for 3 days, and the mixture was stored in a frozen state.
Example 4
A dimethomorph antibody is prepared by the following steps:
1. animal immunization
Taking 10 healthy female Balb/c mice (divided into two groups A and B, and 5 mice in each group) in 6-8 weeks, emulsifying the mice by Freund's complete adjuvant for primary immunization, and injecting the mice subcutaneously at the back of the neck and at multiple points, wherein the immunization dose of each mouse is 200 mu g of dimethomorph immunization antigen; 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 the dimethomorph-coated antigen 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 for 2h at 37 ℃, throwing off the coating solution, washing for 1 time by PBST, and patting 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 1/2-1/3 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, dimethomorph is selected as a standard substance, and the inhibition effect of positive cells is measured by indirect competition ELISA. And selecting a well with better inhibition on the dimethomorph standard, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by adopting the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the dimethomorph monoclonal antibody.
4. Preparation of ascites
Liquid paraffin was injected into Balb/c mice for 6-8 weeks at 500. mu.L/mouse. After 10 days, the hybridoma cells in the logarithmic growth phase were collected in RPMI-1640 basic medium, counted with a hemocytometer and a microscope,the cell concentration is 1.0X 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 dimethomorph monoclonal antibody is more than or equal to 20000.
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 dimethomorph monoclonal antibody to dimethomorph and other carboxylic acid amide bactericides is as follows: 100% of dimethomorph, 11.5% of flumorph, and less than 1% of iprovalicarb, benthiavalicarb-isopropyl and mandipropamid. 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 dimethomorph hapten characterized by the following structural formula:
2. the method of claim 1, wherein the hapten is prepared by reacting 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid with 3-piperazine-1-propionic acid.
3. The method of claim 2, wherein the method comprises: dissolving 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid in dichloromethane, adding N, N-dimethylformamide, fully stirring, adding oxalyl chloride, stirring at 0-5 ℃ for reacting for 40min, after the reaction is finished, adding 3-piperazine-1-propionic acid and triethylamine, stirring at room temperature for reacting for 3h, and after the reaction is finished, separating and purifying to obtain the dimethomorph hapten; wherein the mass ratio of the 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid, oxalyl chloride and 3-piperazine-1-propionic acid is 1:0.1: 1.
4. The method of claim 2, comprising the steps of: dissolving 3.18g of 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) -acrylic acid in 150mL of dichloromethane, adding 0.6mL of N, N-dimethylformamide, fully stirring, adding 0.13g of oxalyl chloride, stirring at 0-5 ℃ for reacting for 40min, after the reaction is finished, adding 1.59g of 3-piperazine-1-propionic acid and 0.8mL of triethylamine, stirring at room temperature for reacting for 3h, after the reaction is finished, adding 100mL of water, oscillating, standing, removing an aqueous phase, evaporating an organic phase, putting on a silica gel column, and eluting and separating by using petroleum ether-ethyl acetate with the volume ratio of 1:1 to obtain the dimethomorph hapten.
5. A dimethomorph artificial antigen comprising a dimethomorph immune antigen and a dimethomorph-coated antigen, wherein the dimethomorph artificial antigen is a conjugate obtained by coupling a carrier protein to the dimethomorph hapten according to claim 1.
6. The dimethomorph artificial antigen of claim 5, wherein the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
7. The method of claim 5, wherein the preparation of the dimethomorph artificial antigen comprises the steps of: dissolving 50mg of bovine serum albumin by using 0.05mol/L PB buffer solution to obtain protein A solution; dissolving 17mg of dimethomorph hapten by using 1mL of N, N-dimethylformamide, adding 13.2mg of carbodiimide, uniformly mixing, reacting for 10min, adding 9.1mg of N-hydroxysuccinimide, blowing, uniformly mixing, reacting at room temperature for 2h, then dropwise adding into the protein A solution, reacting for 12h at 4 ℃, dialyzing and purifying by using 0.02mol/L PBS buffer solution for 3 days, subpackaging and freezing;
the preparation method of the dimethomorph-coated antigen comprises the following steps: dissolving 50mg of ovalbumin by using 0.05mol/L PB buffer solution to obtain protein B solution; dissolving 9.8mg of dimethomorph hapten in 1mL of N, N-dimethylformamide, adding 0.2mL of triethylamine, mixing uniformly, adding 0.21mL of isobutyl chloroformate, reacting at 0-5 ℃ for 2h, then dropwise adding the mixture into the protein B solution, reacting at 4 ℃ for 12h, dialyzing and purifying by using 0.02mol/L PBS buffer solution for 3 days, and subpackaging and freezing.
8. A dimethomorph antibody produced by immunizing an animal with the dimethomorph immunizing antigen of claim 5, which specifically immunoreacts with dimethomorph.
9. Use of the dimethomorph antibody of claim 8 for detecting dimethomorph residues.
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| CN111825562B (en) * | 2020-06-04 | 2022-08-19 | 北京勤邦生物技术有限公司 | Chlordan hapten, artificial antigen and antibody, and preparation method and application thereof |
| CN113354600B (en) * | 2021-03-03 | 2022-03-25 | 华南农业大学 | Dimethomorph hapten, artificial antigen, antibody and preparation method and application thereof |
| CN113621583B (en) * | 2021-09-17 | 2023-06-30 | 江南大学 | Hybridoma cell strain secreting dimethomorph monoclonal antibody and application thereof |
| CN113967212B (en) * | 2021-10-12 | 2024-03-12 | 延边大学 | Disproportionated rosin compound and preparation method and application thereof |
| CN114989038B (en) * | 2022-05-30 | 2023-02-24 | 华南农业大学 | Dimethomorph hapten, artificial antigen, nano antibody and application thereof |
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