CN110627726B

CN110627726B - Prochloraz hapten, artificial antigen and antibody, and preparation method and application thereof

Info

Publication number: CN110627726B
Application number: CN201910897483.2A
Authority: CN
Inventors: 万宇平; 崔廷婷; 崔海峰; 张瑜; 何方洋; 马玉华; 崔娜; 赵正苗
Original assignee: Beijing Kwinbon Biotechnology Co Ltd
Current assignee: Beijing Kwinbon Biotechnology Co Ltd
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2022-07-08
Anticipated expiration: 2039-09-23
Also published as: CN110627726A

Abstract

The invention discloses a prochloraz hapten, an artificial antigen and an antibody as well as a preparation method and application thereof, the prochloraz hapten provided by the invention not only retains the characteristic structure of the prochloraz to the greatest extent, so that the immunogenicity of the prochloraz hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the prochloraz immunizing antigen obtained by coupling the prochloraz 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 detected prochloraz antibody can reach 0.1 mu g/L, the cross reaction rate with other pesticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of the prochloraz.

Description

Prochloraz hapten, artificial antigen and antibody, and preparation method and application thereof

Technical Field

The invention belongs to the field of food safety detection. More specifically, the invention relates to a prochloraz hapten, an artificial antigen and an antibody as well as a preparation method and application thereof.

Background

Prochloraz (Prochloraz) is a broad-spectrum bactericide, and has control effects on various diseases of field crops, fruits and vegetables, turf and ornamental plants; in addition, prochloraz can be used for preserving the harvested fruits such as oranges and bananas, and has a wide application range. The dosage of the prochloraz is strictly regulated in various countries, for example, CAC, European Union and Japan all regulate the MRL value of the prochloraz in raw milk to be 0.05, 0.4 and 0.05mg/kg respectively, and national standard GB 2763-.

At present, the detection of prochloraz at home and abroad mainly comprises analysis methods such as gas chromatography, high performance liquid chromatography, liquid chromatography-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 prochloraz residue.

When establishing an immunological detection method and applying the detection method to detect the residual amount of the prochloraz, the key technology is to obtain an antibody with strong specificity and high sensitivity, and the aim is to realize the aim on the premise that a proper prochloraz hapten is synthesized and prepared. However, at present, no related report aiming at the prochloraz hapten exists in China.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the hapten which can furthest reserve the characteristic structure of the prochloraz 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.

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 prochloraz hapten which has the following structural formula:

the second purpose of the invention is to provide a preparation method of prochloraz hapten, which comprises the following steps:

1) reacting 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid with 1, 2-dichloroethane to obtain an intermediate 1, wherein the intermediate 1 has a structural formula

2) Reacting the intermediate 1 with n-propylamine to obtain an intermediate 2, wherein the intermediate 2 has a structural formula

3) And reacting the intermediate 2 with bis (trichloromethyl) carbonate and imidazole to obtain the prochloraz hapten.

Further, the step 1) comprises the following steps: dissolving 2.69g of 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid in 60mL of 1, 2-dichloroethane, adding 0.57g of KOH, reacting at 60 ℃ for 6h, stopping the reaction, adding water for washing, and evaporating to dryness to obtain an intermediate 1.

Further, the step 2) comprises the following steps: adding 40mL of n-propylamine, 0.31g of anhydrous sodium iodide and 1.38g of potassium carbonate into the intermediate 1, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the n-propylamine, adding water, shaking, extracting with ethyl acetate, collecting an organic phase, and recrystallizing the organic phase with n-hexane-dichloromethane to obtain an intermediate 2.

Further, the step 3) comprises the following steps: dissolving 21.2 g of the intermediate in 80mL of dichloromethane, adding 1.1g of bis (trichloromethyl) carbonate and 0.3mL of triethylamine, stirring at room temperature for 3h, adding 0.27g of imidazole, continuing stirring for 7h, stopping the reaction, adding water, extracting, washing, evaporating to dryness to obtain red oily matter, loading on a silica gel column, eluting with petroleum ether-ethyl acetate, separating and purifying to obtain the prochloraz hapten.

The third purpose of the invention is to provide a prochloraz artificial antigen which is a conjugate obtained by coupling a carrier protein and the prochloraz 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 prochloraz artificial antigen, which comprises the following steps:

dissolving the prochloraz hapten in an organic solvent, adding carbodiimide and N-hydroxysuccinimide, fully and uniformly mixing, and reacting for 4 hours to obtain solution A, wherein the mass ratio of the prochloraz hapten to the carbodiimide to the N-hydroxysuccinimide is (1), (2-3) to (2-3);

dissolving carrier protein in 0.05mol/L PB buffer solution to obtain solution B; and

and dropwise adding the solution A into the solution B, reacting for 24h at 4 ℃, and purifying to obtain the prochloraz artificial antigen.

The fifth purpose of the invention is to provide a prochloraz antibody which is obtained by immunizing animals with the prochloraz artificial antigen and can generate specific immunoreaction with the prochloraz.

Further, the prochloraz antibody is a monoclonal antibody or a polyclonal antibody.

The sixth purpose of the invention is to provide the application of the prochloraz antibody in detecting prochloraz residue.

The invention has the following beneficial effects:

the prochloraz hapten provided by the invention not only retains the characteristic structure of the prochloraz to the greatest extent, so that the immunogenicity of the prochloraz hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the prochloraz artificial antigen obtained by coupling the prochloraz hapten with 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 prochloraz.

The preparation method of the prochloraz hapten has the advantages of easily available raw materials, simple reaction operation, easily controlled reaction conditions and high purity and yield of the prepared prochloraz hapten.

The prochloraz antibody obtained by adopting the prochloraz artificial antigen has good 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 the synthesis route of the prochloraz hapten

Detailed Description

In a first aspect, the present invention provides a prochloraz hapten having the following structural formula:

the prochloraz hapten provided by the invention not only furthest reserves the characteristic structure of the prochloraz, so that the immunogenicity of the prochloraz hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the prochloraz artificial antigen obtained by coupling the prochloraz hapten and the carrier protein is used for immunizing animals, and is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity. The prochloraz hapten makes up the blank of the technical field of the domestic prochloraz immunological detection method, and lays a foundation for the further development of the prochloraz immunological detection method.

In a second aspect, the present invention provides a method for preparing the above-mentioned prochloraz hapten, which comprises the following steps:

Preferably, the step 1) includes the steps of: dissolving 2.69g of 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid in 60mL of 1, 2-dichloroethane, adding 0.57g of KOH, reacting at 60 ℃ for 6h, stopping the reaction, adding water for washing, and evaporating to dryness to obtain an intermediate 1.

Preferably, the step 2) includes the steps of: adding 40mL of n-propylamine, 0.31g of anhydrous sodium iodide and 1.38g of potassium carbonate into the intermediate 1, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the n-propylamine, adding water, shaking, extracting with ethyl acetate, collecting an organic phase, and recrystallizing the organic phase with n-hexane-dichloromethane to obtain an intermediate 2.

Preferably, the step 3) includes the steps of: dissolving 21.2 g of the intermediate in 80mL of dichloromethane, adding 1.1g of bis (trichloromethyl) carbonate and 0.3mL of triethylamine, stirring at room temperature for 3h, adding 0.27g of imidazole, continuing stirring for 7h, stopping the reaction, adding water, extracting, washing, evaporating to dryness to obtain red oily matter, loading on a silica gel column, eluting with petroleum ether-ethyl acetate, separating and purifying to obtain the prochloraz 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 utilized to synthesize the compound again, and micromolecules with functional groups are introduced into proper positions. The invention uses 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid as the starting material, synthesizes prochloraz hapten with carboxyl through 3 steps of reaction, has higher difficulty compared with the former two methods, but has the advantages of easy establishment of the optimal coupling site and easy establishment of reasonable spacing arms, and increases the possibility of generating antibodies aiming at characteristic structures.

The preparation method of the prochloraz hapten is reasonably designed according to the structural characteristics of the prochloraz, the used raw materials are easy to obtain, the reaction operation is simple, the reaction conditions are easy to control, and the prepared prochloraz hapten is high in purity and yield.

In a third aspect, the invention also provides a prochloraz artificial antigen which is a conjugate obtained by coupling carrier protein and the prochloraz hapten.

Preferably, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

The prochloraz hapten molecule only has immunoreactivity and does not have immunogenicity. Therefore, in order to confer immunogenicity on the prochloraz hapten molecules, the prochloraz hapten molecules are coupled and combined with suitable carrier protein molecules, thereby generating the prochloraz artificial antigen with both immunoreactivity and immunogenicity.

In a fourth aspect, the invention also provides a preparation method of the prochloraz artificial antigen, which comprises the following steps:

In a fifth aspect, the invention also provides a prochloraz antibody which is obtained by immunizing animals with the prochloraz artificial antigen and can perform specific immunoreaction with the prochloraz.

The prochloraz antibody can be a monoclonal antibody or a polyclonal antibody. In addition, the prochloraz antibody can be prepared by a method conventional in the art.

In a specific embodiment, the prochloraz antibody is a murine monoclonal antibody specific for a prochloraz artificial antigen of the above-mentioned prochloraz hapten.

The prochloraz antibody obtained by adopting the prochloraz artificial antigen has better titer, specificity and affinity, and has low cross reaction rate with other pesticides.

In a sixth aspect, the invention also provides an application of the prochloraz antibody in detection of prochloraz residues.

The invention induces immune animals to generate antibodies through the prochloraz artificial antigen, thereby being used in the prochloraz immunodetection analysis.

The prochloraz immunoassay comprises but is not limited to a prochloraz ELISA kit and a prochloraz 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 prochloraz hapten comprises the following steps:

1) dissolving 2.69g of 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid in 60mL of 1, 2-dichloroethane, adding 0.57g of KOH, reacting at 60 ℃ for 6h, stopping the reaction, adding water for washing, and evaporating to dryness to obtain an intermediate 1;

2) adding 40mL of n-propylamine, 0.31g of anhydrous sodium iodide and 1.38g of potassium carbonate into the intermediate 1, heating and refluxing for 4h, stopping the reaction, performing rotary evaporation, removing the n-propylamine, adding water, shaking, extracting with ethyl acetate, collecting an organic phase, and recrystallizing the organic phase with n-hexane-dichloromethane to obtain an intermediate 2;

3) dissolving 21.2 g of the intermediate in 80mL of dichloromethane, adding 1.1g of bis (trichloromethyl) carbonate and 0.3mL of triethylamine, stirring at room temperature for 3h, adding 0.27g of imidazole, continuing stirring for 7h, stopping the reaction, adding water, extracting, washing, evaporating to dryness to obtain red oily matter, loading on a silica gel column, eluting with petroleum ether-ethyl acetate, separating and purifying to obtain the prochloraz hapten.

Example 2

A preparation method of prochloraz artificial antigen comprises the following steps:

dissolving 17mg of the prochloraz hapten prepared in the example 1 in 1mL of DMF, dissolving and clarifying, adding 15.3mg of carbodiimide and 11.2mg of N-hydroxysuccinimide, fully and uniformly mixing, and reacting for 4h to obtain solution A; dissolving 50mg of Bovine Serum Albumin (BSA) in 0.05mol/L PB buffer solution to obtain solution B; dripping the A solution into the B solution, reacting at 4 deg.C for 24h, dialyzing and purifying with 0.02mol/L PBS for 3 days, changing solution 3 times per day to obtain artificial antigen of prochloraz coupled with bovine serum albumin, packaging, and storing at-20 deg.C.

Example 3

dissolving 11mg of the prochloraz hapten prepared in the example 1 in 1mL of DMF, dissolving and clarifying, adding 9.7mg of carbodiimide and 7.45mg of N-hydroxysuccinimide, fully and uniformly mixing, and reacting for 4h to obtain solution A; dissolving Ovalbumin (OVA)50mg in PB buffer solution of 0.05mol/L to obtain solution B; dripping the A solution into the B solution, reacting at 4 deg.C for 24h, dialyzing and purifying with 0.02mol/L PBS for 3 days, changing solution 3 times per day to obtain artificial antigen of prochloraz coupled with ovalbumin, packaging, and storing at-20 deg.C.

Example 4

The preparation method of the prochloraz antibody comprises 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 at subcutaneous multiple points on the back of the neck, wherein the immunization dose of each mouse is 200 mu g of prochloraz 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 prochloraz artificial antigen coupled with ovalbumin by 0.05mol/L of carbonate buffer solution with pH of 9.6 at a ratio of 1:1000, coating an enzyme label plate by 100 mu L of each hole, incubating for 2h at 37 ℃, 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 mu L/well of the suspension dropwise onto a culture plate, and placing the suspension 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 screening

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, prochloraz is selected as a standard substance, and the inhibition effect of positive cells is measured by indirect competitive ELISA. And selecting a well with better inhibition on the prochloraz standard, carrying out 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 prochloraz monoclonal antibody.

4. Preparation of ascites

Liquid paraffin was injected into Balb/c mice for 6-8 weeks at 500. mu.L/mouse. 10 days later, hybridoma cells in logarithmic growth phase were collected in RPMI-1640 basic medium, counted in a hemocytometer at a cell concentration of 1.0X 10 and a microscope⁶～1.5×10⁶In 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 potency of the prochloraz 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 prochloraz monoclonal antibody to prochloraz and other bactericides is as follows: the prochloraz accounts for 100 percent, and the carbendazim, thiophanate-methyl, thiabendazole, iprodione, imazalil, benomyl, procymidone, chlorothalonil and pyrimethanil are all less than 1 percent. Therefore, the prepared antibody has better specificity.

The above embodiments only express the embodiments of the present invention, and the description is specific and detailed, but it should not be 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

1. A prochloraz hapten, which has the following structural formula:

2. the method for preparing prochloraz hapten as claimed in claim 1, comprising the following steps:

3. The method for preparing prochloraz hapten according to claim 2, wherein the step 1) comprises the following steps: dissolving 2.69g of 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid in 60mL of 1, 2-dichloroethane, adding 0.57g of KOH, reacting at 60 ℃ for 6h, stopping the reaction, adding water for washing, and evaporating to dryness to obtain an intermediate 1.

4. The method for preparing prochloraz hapten according to claim 2, wherein the step 2) comprises the following steps: adding 40mL of n-propylamine, 0.31g of anhydrous sodium iodide and 1.38g of potassium carbonate into the intermediate 1, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the n-propylamine, adding water, shaking, extracting with ethyl acetate, collecting an organic phase, and recrystallizing the organic phase with n-hexane-dichloromethane to obtain an intermediate 2.

5. The method for preparing prochloraz hapten according to claim 2, wherein the step 3) comprises the following steps: and (3) taking 21.2 g of the intermediate, adding 80mL of dichloromethane to dissolve the intermediate, adding 1.1g of bis (trichloromethyl) carbonate and 0.3mL of triethylamine, stirring at room temperature for 3 hours, adding 0.27g of imidazole, continuing stirring for 7 hours, stopping the reaction, adding water, extracting and washing, drying by distillation to obtain a red oily substance, loading the red oily substance on a silica gel column, and eluting with petroleum ether-ethyl acetate for separation and purification to obtain the prochloraz hapten.

6. A prochloraz artificial antigen which is a conjugate obtained by coupling a carrier protein and the prochloraz hapten as claimed in claim 1.

7. The prochloraz artificial antigen of claim 6, wherein said carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

8. The method for preparing prochloraz artificial antigen as claimed in claim 6, comprising the steps of:

9. A prochloraz antibody obtained by immunizing an animal with the prochloraz artificial antigen of claim 6, wherein the prochloraz antibody is specifically immunoreactive with prochloraz.

10. Use of the prochloraz antibody of claim 9 for detecting prochloraz residues.