Oxafos hapten, complete antigen, antibody as well as preparation method and application of xafos hapten, complete antigen and antibody

Abstract

The invention discloses an imefos hapten, a complete antigen, an antibody as well as a preparation method and application of the imefos hapten. The imefos hapten has the following structural formula: # imgabs0 #. The efosfos hapten is designed for efosfos and can be used for preparing an efosfos antibody, and the efosfos antibody prepared through animal immunization and experimental verification can specifically recognize efosfos. The hapten prepared by the invention and an immunoassay method established on the basis of the hapten are wide in linear range and relatively high in sensitivity, and can meet the requirement of rapid screening.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to an ethoprophos hapten, a complete antigen, an antibody, and a preparation method and application thereof. Background Art

[0002] Ethoprophos, also known as chlorfenapyr, is a highly effective, broad-spectrum organophosphorus insecticide that has a contact killing effect on pests and can be used to control various insects. Organophosphorus insecticides have become the most widely used type of pesticides due to their low price and good insect repellent effect. However, the residues of such pesticides in agricultural products or the environment may cause health hazards to humans. Therefore, in order to protect human health and monitor pesticide exposure levels, it is urgent to establish a simple, convenient and rapid pesticide residue analysis method to detect chlorfenapyr in the environment and food. The detection method is of great significance.

[0003] Immunoassay is an analytical technique based on the principle of specific and reversible binding reaction between antigen and antibody. The immune reaction involves the comprehensive effects of highly complementary stereostructure, static electricity, hydrogen bonds and van der Waals forces between antigen and antibody molecules. It has the characteristics of strong specificity, low detection limit, low detection cost, simple operation, and suitability for on-site screening of large quantities of samples. It is evaluated as the most challenging and competitive rapid detection technology in the 21st century. There is no report on the hapten prepared by the present invention for the structure of chloranthiocarb. Summary of the invention

[0004] The object of the present invention is a hapten of chloranthiocarb, which has the following structural formula:

[0005]

[0006] Accordingly, the present invention also provides a method for preparing the ethosulf hapten, comprising the following steps:

[0007] Step A: Wash the petroleum ether to remove oil and dry the sodium hydride, add n-hexane to disperse it, cool it down, then add propyl mercaptan, turn off the refrigeration after adding, heat it up by itself and stir overnight to obtain the anthocyanin intermediate 1:

[0008]

[0009] Step B: dissolving phosphorus oxychloride in dichloromethane; adding dropwise the reaction system of the ethanophos intermediate 1; heating and stirring overnight; after the reaction is completed, filtering to remove insoluble matter, and drying the filtrate to obtain the ethanophos intermediate 2:

[0010]

[0011] Step C: weigh the ethanophos intermediate 2 and dissolve it in dichloromethane; then dissolve tert-butyl 4-hydroxybutyrate in dichloromethane and add it, and then add triethylamine; stir and react at room temperature; after the reaction is completed, spin dry and pass through a column to obtain the ethanophos intermediate 3: wherein the eluent used is ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20;

[0012]

[0013] Step D: dissolving the intermediate 3 of chloranthion in dichloromethane and then adding the mixture; after reacting at room temperature, removing the solvent by rotary evaporation, and passing through a column to obtain the hapten of chloranthion: wherein the eluent used is ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20;

[0014]

[0015] Correspondingly, the present invention also provides the use of the ethosulf hapten in the preparation of the ethosulf complete antigen.

[0016] Correspondingly, the present invention also provides a complete anthocyanin antigen, which is obtained by coupling the anthocyanin hapten provided by the present invention with a carrier protein; the carrier protein is bovine lactoferrin, bovine serum albumin or ovalbumin.

[0017] Accordingly, the present invention also provides a method for preparing a complete antigen of ethoprophos, comprising the following steps:

[0018] S1: dissolving the obtained hapten of chloranthion in DMF, then adding EDC and NHS under stirring, stirring at room temperature in the dark, to obtain an activated hapten, referred to as liquid A: wherein the molar ratio of the hapten of chloranthion, the EDC and the NHS is 1:1.5-2:1.5-2;

[0019] S2: dissolving the carrier protein in a carbonate buffer solution at pH = 9.6-9.8, wherein the carrier protein concentration is 5-10 mg / mL, which is called solution B;

[0020] S3: Add the above liquid A dropwise to liquid B under stirring in an ice bath. After the addition, adjust the pH value to 9.6-9.8 with a NaOH solution. Allow to react overnight in the dark and obtain a complete antigen with the following structure after purification by dialysis:

[0021]

[0022] Preferably, in S2, the molar ratio of the anthocyanidin hapten, the EDC and the NHS is 1:1.5-2:1.5-2;

[0023] Preferably, the molar ratio of the anthocyanidin hapten in the solution A to the carrier protein in the solution B is 30-40:1.

[0024] The present invention also provides an anthocyanin antibody, which is prepared by using the anthocyanin hapten described in the present invention or the anthocyanin complete antigen described in the present invention.

[0025] Preferably, the anthocyanidin antibody is a polyclonal antibody, a monoclonal antibody or a genetically engineered antibody.

[0026] The present invention also provides a preparation for detecting ethoprophos, comprising the ethoprophos hapten described in the present invention, the ethoprophos complete antigen described in the present invention or the ethoprophos antibody described in the present invention.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The hapten of ethoxyfen is designed for ethoxyfen and can be used to prepare ethoxyfen antibodies. The ethoxyfen antibodies prepared through animal immunization and experimental verification can specifically recognize ethoxyfen. The invention also discloses a preparation method and application of the hapten of ethoxyfen.

[0029] The indirect competitive enzyme-linked immunosorbent assay method constructed by the present invention is sensitive to chloranthate IC 50 The detection range is 2.94-20.86 ng / mL, and the detection limit is IC 10 The hapten prepared by the present invention and the immunoassay method established based on the hapten have a wide linear range and high sensitivity, and can meet the needs of rapid screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a flow chart of a method for preparing an ethosulf hapten according to an embodiment.

[0031] Figure 2 The figure is a mass spectrometry analysis spectrum of the anthocyanidin hapten in one embodiment.

[0032] Figure 3 This is an ultraviolet identification diagram of the anthracene artificial antigen according to an embodiment.

[0033] Figure 4 This is the ELISA competition standard curve of the anthocyanin antibody obtained in Example 4. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings:

[0035] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0036] Example 1 Synthesis of hapten of chloranthiocarb

[0037] A method for preparing an ethoprophos hapten, wherein the preparation method is as follows Figure 1 As shown, the following steps are included:

[0038] Step A: Weigh 2.5 g of sodium hydride washed with petroleum ether to remove oil and dried, add 50 mL of n-hexane to disperse, and cool to -78°C. Then weigh 7.7 g of propyl mercaptan and slowly add it. After adding, turn off the refrigeration and heat it up by itself and stir overnight to obtain a white turbid liquid (ethoxysulfuron intermediate 1).

[0039]

[0040] Step B: Take 7.8 g of phosphorus oxychloride and dissolve it in 125 mL of dichloromethane. Slowly add all the intermediate 1 to the reaction system at -75°C. After the addition, turn off the refrigeration and heat it up by itself and stir overnight. After the reaction is completed, filter to remove insoluble matter, and spin-dry the filtrate to obtain a light yellow liquid with acid mist (ethoprophos intermediate 2).

[0041]

[0042] Step C: Weigh 349 mg of intermediate 2 and dissolve it in 10 mL of dichloromethane. Weigh 160 mg of tert-butyl 4-hydroxybutyrate, dissolve it in 2 mL of dichloromethane and then add 121 mg of triethylamine. Stir the reaction at room temperature for 6 hours. After the reaction is completed, spin dry and pass through a column to obtain a colorless oil (ethoxysulfuron intermediate 3); the eluent used is ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20.

[0043]

[0044] Step D: Weigh the intermediate 3 (48.2 mg 0.135 mmol) and dissolve it in 4 mL of dichloromethane, then add 2 mL of TFA. React at room temperature for 1.5 h. After the reaction is completed, remove the solvent by rotary evaporation and pass through a column to obtain a white solid (ethofen hapten), MW: 300.06. The eluent used is ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20;

[0045] The mass spectrometry results are as follows Figure 2 The results showed that the mass-to-charge ratio (m / z) 301.0 ion peak in the positive ion mode ES I ionization full scan corresponded to the quasi-molecular ion peak [M+H]+, proving that the synthesis of the anthocyanidin hapten was successful.

[0046] Advantages of the design of the anthocyanidin hapten: In order to achieve the coupling of the hapten small molecule with the carrier protein, a spacer arm with an active carboxyl group at the end needs to be introduced through a chemical modification method. The hapten can be coupled with the carrier protein through the active ester method to synthesize an artificial antigen, which effectively improves the antibody's recognition activity for anthocyanidin.

[0047]

[0048] Example 2 Synthesis of complete antigen of ethoxysulf

[0049] Immunogen: Dissolve 5.6 mg of chloranil hapten, 5.4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 3.2 mg of N-hydroxysuccinimide (NHS) in 600 μL of N, N-dimethylformamide (DMF), stir for 4 hours at room temperature in the dark, and obtain the activated hapten, called liquid A. Dissolve 50 mg of bovine lactoferrin in 5 mL of pH = 9.6 carbonate buffer, called liquid B; add the above liquid A dropwise to liquid B under stirring in an ice bath, and after adding, adjust the pH to 9.6 with 3M NaOH. React overnight in the dark, and after purification by dialysis, obtain the following complete antigen:

[0050]

[0051] Coating source: Dissolve 13.6 mg of chloranthate hapten, 13.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 7.8 mg of N-hydroxysuccinimide (NHS) in 600 μL of N, N-dimethylformamide (DMF), stir for 4 hours at room temperature in the dark, and obtain the activated hapten, called solution A. Dissolve 100 mg of bovine serum albumin in 20 mL of pH = 9.6 carbonate buffer, called solution B; add the above solution A dropwise to solution B under stirring in an ice bath, and after adding, adjust the pH to 9.6 with 3M NaOH. React overnight in the dark, and after purification by dialysis, obtain the following complete antigen:

[0052]

[0053] like Figure 3 As shown, all the synthesized artificial antigens have characteristic absorption peaks of hapten and carrier protein or their absorption peaks are shifted relative to the original carrier protein, indicating that all the artificial antigens are successfully synthesized.

[0054] Example 3 Preparation of anti-methosulfan monoclonal antibody

[0055] The experiment is as follows: take 8-week-old SPF Balb / c female mice (Guangdong Provincial Experimental Animal Center), the routine immunization cycle is as follows: the first immunization (immunogen + Freund's complete adjuvant, subcutaneous injection), the second immunization (immunogen + Freund's incomplete adjuvant, subcutaneous injection), the third immunization (immunogen + Freund's incomplete adjuvant, subcutaneous injection), the fourth immunization (immunogen + Freund's incomplete adjuvant, subcutaneous injection), the fifth booster immunization (immunogen, intraperitoneal injection), once every 2 weeks. The tail tip of the animal is blooded on the 7th day after each immunization, once every 2 weeks, and the blood collection volume is 150-200μL each time, which is used to measure the serum antibody level. The blood obtained after the fifth booster immunization is incubated at 37°C for half an hour, centrifuged at 12000rpm for 10min, and the antiserum obtained is used for detection.

[0056] Example 4 Establishment of standard curve for antiserum detection of ethoprophos

[0057] The working concentrations of the coating source and the anthocyanin antibody were determined by the square array titration method, the working concentration of the coating source was 500 ng / mL, and the working concentration of the anthocyanin antibody was 100 ng / mL. Three parallel tests were performed (n=3).

[0058] The steps of antiserum indirect competition ELISA detection are as follows:

[0059] Coating: Dilute the coating source to an appropriate concentration with carbonate buffer, add 100 μL to each well of the ELISA plate, and coat overnight in a 37°C water bath.

[0060] Washing: Pat off the liquid in the wells, wash the plate twice with a plate washer, add 250 μL of washing solution to each well, and spin dry the liquid in the wells.

[0061] Blocking: Add 120 μL of 1% skimmed milk powder to each well, block at 37°C for 2 h, shake dry the liquid in the well, and place upside down in a 37°C oven for 1 h for later use.

[0062] Sample addition and incubation: dilute chloranthiocarb into a series of gradient standard solutions, dilute to 250, 125, 62.5, 31.25, 15.62, 7.81, 3.91, 1.95, 0.98, 0.49 ng / mL, add 50 μL to each well, then add 50 μL of the diluted antiserum diluent, react in a 37°C water bath for 40 minutes, wash the plate 5 times with a plate washer, add 250 μL of washing solution to each well, and spin dry the liquid in the well.

[0063] Add secondary antibody: add 100 μL of 5000-fold diluted HRP-goat anti-rabbit to each well, react in a 37°C water bath for 30 min, and wash the plate as in S4.

[0064] Color development: TMB substrate solution and substrate buffer were mixed in equal volumes, 100 μL of the mixture was added to each well, and the cells were placed in a 37°C water bath for 10 min of color development. Then, 50 μL of 10% H2SO4 stop solution was added to each well.

[0065] Determination: The absorbance (OD) was read using an enzyme-labeled instrument at a wavelength of 450 nm.

[0066] Calculation: The IC10, IC20, IC50, and IC80 values ​​of the inhibition curve were calculated using the four-parameter fitting module of Origi n8.5. The standard curve is shown in Figure 4 The obtained standard curve IC 50 The value was 7.83 ng / mL, and the detection limit was (IC 10 )1.65ng / mL, and the linear detection range was 2.94-20.86ng / mL.

[0067] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A hapten of ethoprophos, characterized in that It has the following structural formula:

2. A method for preparing the ethosulf hapten according to claim 1, characterized in that: The steps include: Step A: Wash the petroleum ether to remove oil and dry the sodium hydride, add n-hexane to disperse it, cool it down, then add propyl mercaptan, turn off the refrigeration after adding, heat it up by itself and stir overnight to obtain the anthocyanin intermediate 1: Step B: dissolving phosphorus oxychloride in dichloromethane; adding dropwise the reaction system of the ethanophos intermediate 1; heating and stirring overnight; after the reaction is completed, filtering to remove insoluble matter, and drying the filtrate to obtain the ethanophos intermediate 2: Step C: Weigh the ethanophos intermediate 2 and dissolve it in dichloromethane; weigh tert-butyl 4-hydroxybutyrate, dissolve it in dichloromethane and then add it, and then add triethylamine; stir at room temperature to react; after the reaction is completed, spin dry and pass through a column to obtain the ethanophos intermediate 3: Step D: Weigh the intermediate 3 of chloranthion and dissolve it in dichloromethane, then add it; after the reaction at room temperature, remove the solvent by rotary evaporation, and pass it through a column to obtain the hapten of chloranthion:

3. Use of the ethosulf hapten as claimed in claim 1 in the preparation of the ethosulf complete antigen.

4. A complete antigen of ethoprophos, characterized in that The method is obtained by coupling the ethoprophos hapten according to claim 1 with a carrier protein; the carrier protein is bovine lactoferrin, bovine serum albumin or ovalbumin.

5. A method for preparing the complete antigen of ethoprophos as claimed in claim 4, characterized in that: The steps include: S1: dissolving the anthocyanidin hapten obtained as claimed in claim 2 in DMF, then adding EDC and NHS under stirring, stirring at room temperature in the dark, to obtain an activated hapten, referred to as liquid A: wherein the molar ratio of the anthocyanidin hapten, the EDC and the NHS is 1:1.5:1.5; S2: dissolving the carrier protein in a carbonate buffer solution at pH = 9.6, with a carrier protein concentration of 5-10 mg / mL, referred to as solution B; S3: Add the above liquid A dropwise to liquid B under stirring in an ice bath. After the addition, adjust the pH value to 9.6 with a NaOH solution. Allow to react overnight in the dark and obtain a complete antigen with the following structure after purification by dialysis:

6. The method according to claim 5, characterized in that In the S2, the molar ratio of the anthocyanidin hapten, the EDC and the NHS is 1:1.5:1.

5.

7. The method according to claim 5, characterized in that The molar ratio of the anthocyanidin hapten in the solution A to the carrier protein in the solution B is 30:

1.

8. An antibody against ethoxychloride, characterized in that The anthocyanin antibody is prepared by using the anthocyanin hapten described in claim 1 or the anthocyanin complete antigen described in claim 3.

9. The ethoprophos antibody according to claim 8, characterized in that The anthocyanidin antibody is a polyclonal antibody, a monoclonal antibody or a genetically engineered antibody.

10. A preparation for detecting ethoprophos, characterized in that: The invention comprises the anthocyanidin hapten according to claim 1, the anthocyanidin complete antigen according to claims 4-5 or the anthocyanidin antibody according to any one of claims 7-8.

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