Metathion hapten, complete antigen and antibody as well as preparation method and application of metathion hapten, complete antigen and antibody

Abstract

The invention discloses a meta-dephosphorized hapten, a complete antigen, an antibody and a preparation method and application thereof. The meta-dephosphorized hapten has the following structural formula. The metathion hapten is designed for metathion and can be used for preparing the metathion antibody, and the metathion antibody prepared through animal immunization and experimental verification can specifically recognize metathion. 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] This invention belongs to the field of immunoassay technology, specifically relating to a rapid phosphorus hapten, a complete antigen, an antibody, and their preparation methods and applications. Background Technology

[0002] Mevinphos (MVP) is a water-soluble contact and systemic organophosphate insecticide commonly used to control chewing and sucking insects and spider mites. It kills pests by inhibiting the activity of acetylcholinesterase, blocking nerve signal transmission. Mevinphos has high toxicity; experimentally measured acute toxicity in rats is 3 mg / kg (Rtecs Number - GQ5250000 - Chemical Toxicity Database). Its high water solubility allows it to accumulate easily in the environment, potentially causing adverse effects on humans. Mevinphos residue levels in fruits and vegetables are strictly controlled and are a necessary testing indicator for exported fruits and vegetables. Excessive mevinphos residues can not only lead to food safety issues but also trigger related international trade disputes. Its use has been completely banned by major agricultural countries and organizations worldwide (including the EU, China, the US, and Australia). Therefore, developing and applying effective detection technologies to ensure that methamidophos residues meet safety standards is of great significance. Establishing a rapid, simple, and reliable detection method is therefore crucial. Immunoassay, a rapid detection and analysis technique based on the principle of antigen-antibody specific binding, is widely used in various fields due to its advantages of speed, simplicity, high sensitivity, accuracy, high throughput, and portability. This invention does not currently report on the hapten prepared from the methamidophos structure. Summary of the Invention

[0003] This invention provides a rapid phosphorus-inactivating hapten with the following structural formula:

[0004]

[0005] This invention provides a method for preparing a rapid phosphorus hapten, comprising the following steps: using tert-butyl acetoacetate as the starting material, the mixture is sequentially subjected to bromination, phosphorylation, acid hydrolysis, coupling with tert-butyl 4-hydroxybutyrate, acid deprotection, coupling with tert-butyl p-hydroxybenzoate, and finally acid deprotection to obtain the rapid phosphorus hapten.

[0006] A rapid phosphorus hapten complete antigen is obtained by coupling the rapid phosphorus hapten with a carrier protein; the carrier protein is bovine lactoferrin or bovine serum albumin.

[0007] This invention also provides a method for preparing rapid phosphorus-inactivating complete antigen, comprising the following steps:

[0008] S1: Dissolve the rapid phosphorus hapten described in claim 1 in an organic solvent, add EDC and NHS for activation, and obtain solution A;

[0009] S2: Dissolve the carrier protein in carbonate buffer at pH 9.6 to obtain solution B;

[0010] S3: Add solution A dropwise to solution B while stirring, maintain pH = 9.6 for reaction, and purify after reaction to obtain the complete antigen.

[0011] Preferably, in S1, the molar ratio of the rapid phosphorus hapten, EDC and NHS is 1:1.5-2:1.5-2.

[0012] Preferably, in step S3, the molar ratio of the phosphoamic acid hapten in solution A to the carrier protein in solution B is 30-40:1.

[0013] The present invention also provides a phosmet antibody, which is prepared by immunizing animals with the phosmet complete antigen and can specifically bind to phosmet (see Table 1).

[0014] Table 1 Results of Cross-Reactivity Rate Measurement

[0015]

[0016] Preferably, the antibody is a polyclonal antibody, a monoclonal antibody, or a genetically engineered antibody.

[0017] Accordingly, the present invention also provides an immunoassay kit for detecting phosphorus residues in fruits, vegetables, water bodies or soil, comprising the phosphorus antibody described in the present invention.

[0018] The present invention also provides the use of a phosmet hapten or a phosmet complete antigen in the preparation of an immunoassay agent for the detection of phosmet.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This methamidophos hapten is designed specifically for methamidophos and can be used to prepare methamidophos antibodies. Animal immunization and experimental verification have shown that the prepared methamidophos antibodies can specifically recognize methamidophos. This invention also discloses the preparation method and application of the aforementioned methamidophos hapten.

[0021] The indirect competitive enzyme-linked immunosorbent assay (ELISA) method constructed in this invention has an IC50 sensitivity for rapid phosphorus inactivation. 50 The detection limit is up to 6.33 ng / mL, with a detection range of 1.80-22.23 ng / mL and a detection limit of IC50. 10 The concentration was 0.86 ng / mL. The hapten prepared in this invention and the immunoassay based on it have a wide linear range and high sensitivity, which can meet the needs of rapid screening. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the preparation process of a rapid phosphorus hapten in one embodiment.

[0023] Figure 2 This is a mass spectrometry analysis chromatogram of a rapid phosphorus hapten as an example.

[0024] Figure 3 This is an ultraviolet (UV) identification image of a rapid phosphorus-inactivated artificial antigen from one embodiment.

[0025] Figure 4 This is an ELISA competition standard curve of the rapid phosphorus-inactivating antibody obtained in Example 4. Detailed Implementation

[0026] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings:

[0027] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0028] Example 1: Synthesis of rapid phosphorus hapten; the preparation flowchart is as follows: Figure 1 As shown:

[0029] The preparation method of rapid phosphorus hapten is as follows:

[0030] Step A: Add 4.0 g of tert-butyl acetoacetate to a clean 100 mL single-necked flask and dissolve it in 60 mL of chloroform. Add 5.0 g of N-bromosuccinimide in portions at room temperature. After the addition is complete, react at room temperature for 4.5 hours. After the reaction is complete, evaporate the solvent under reduced pressure and purify directly by column chromatography to obtain 6.0 g of a colorless oil.

[0031]

[0032] Step B: Add 6.0 g of the product from the previous step to a clean 250 mL single-necked flask, dissolve in 80 mL of dichloroethane, and add 3.8 g of trimethyl phosphite dropwise at 60 °C. After the addition is complete, slowly raise the temperature to 80 °C and react for 4.0 hrs. After the reaction is complete, evaporate the solvent under reduced pressure and directly purify by column chromatography to obtain 5.0 g of a colorless oil.

[0033]

[0034] Step C: Add 5.0 g of the product from the previous step to a 100 mL single-necked flask and dissolve it in 30 mL of dichloromethane. Add 15 mL of LTA at room temperature with stirring, and react at room temperature for 2 hours until the starting material is almost completely reacted. Evaporate the solvent under reduced pressure and directly purify by column chromatography to obtain 3.9 g of the final product.

[0035]

[0036] Step D: In a 100 mL single-necked flask, dissolve 3.0 g of the product from the previous step in 30 mL of dichloromethane. While stirring at room temperature, add 1.2 eq of tert-butyl 4-hydroxybutyrate, 2 eq of EDC.HCl, and 0.5 g of DMAP. React overnight at room temperature until the reactants are almost completely reacted. Evaporate the solvent under reduced pressure and directly purify by column chromatography to obtain 3.2 g of the final product (MW: 352).

[0037]

[0038] Step E: Add 3.0 g of the product from the previous step to a 100 mL single-necked flask and dissolve it in 30 mL of dichloromethane. Add 15 mL of LTFA while stirring at room temperature, and react at room temperature for 2 h until the starting material is almost completely reacted. Evaporate the solvent under reduced pressure and directly purify by column chromatography to obtain 2.1 g of the final product (immunogen MVP-H1), MW: 296.

[0039] Step F: In a 100 mL single-necked flask, dissolve 1.5 g of the product from the previous step in 30 mL of dichloromethane. While stirring at room temperature, add 1.2 eq of tert-butyl p-hydroxybenzoate, 2 eq of EDC·HCl, and 0.1 g of DMAP. React overnight at room temperature until the starting material is almost completely reacted. Evaporate the solvent under reduced pressure and directly purify by column chromatography to obtain 1.2 g of the final product.

[0040]

[0041] Step G: Add 1.0 g of the product from the previous step to a 100 mL single-necked flask and dissolve it in 10 mL of dichloromethane. Add 5 mL of LTFA while stirring at room temperature and react at room temperature for 2 h until the starting material is basically completely reacted. Evaporate the solvent under reduced pressure and directly purify by column chromatography to obtain 0.6 g of the final product (coated with original MVP-H2), MW: 330.

[0042]

[0043] Mass spectrometry (ESI) analysis confirmed that the molecular weights were correct. The MVP-H1 positive spectrum with a Na peak at 319 and the MVP-H2 positive spectrum with a Na peak at 353, both indicating the target molecular weight. This demonstrates that the hapten synthesis was correct. The mass spectrometry results are as follows: Figure 2 show.

[0044] Advantages of designing phosmet hapten: To achieve coupling of the hapten molecule with the carrier protein, a spacer arm with an active carboxyl group at the end needs to be introduced through chemical modification. This 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 against phosmet.

[0045]

[0046] Example 2: Complete Synthesis of Rapid Phosphorus-Induced Antigen

[0047] Immunogen: 13.2 mg of methamidophos hapten, 11.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 6.9 mg of N-hydroxysuccinimide (NHS) were dissolved in 600 μL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature in the dark for 4 h to obtain the activated hapten, referred to as solution A. 50 mg of bovine lactoferrin was dissolved in 5 mL of pH 9.6 carbonate buffer, referred to as solution B. Solution A was added dropwise to solution B with stirring in an ice bath. After addition, the pH was adjusted to 9.6 with 3M NaOH. The reaction was carried out overnight in the dark, and the complete antigen was obtained after dialysis purification.

[0048]

[0049] Coating agent: 5.4 mg of methamidophos hapten, 5.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 3.2 mg of N-hydroxysuccinimide (NHS) were dissolved in 600 μL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature in the dark for 4 h to obtain the activated hapten, referred to as solution A. 100 mg of bovine serum albumin was dissolved in 20 mL of pH 9.6 carbonate buffer, referred to as solution B. Solution A was added dropwise to solution B with stirring in an ice bath. After addition, the pH was adjusted to 9.6 with 3M NaOH. The reaction was carried out overnight in the dark, and the complete antigen was obtained after dialysis purification.

[0050]

[0051] like Figure 3 As shown, all synthesized artificial antigens exhibit characteristic absorption peaks of both the hapten and the carrier protein, or their absorption peaks shift relative to the original carrier protein, indicating successful hapten conjugation.

[0052] Example 3: Preparation of anti-rapid phosphorus monoclonal antibody

[0053] The experiment was conducted as follows: Eight-week-old SPF Balb / c female mice (Ethics Certificate No.: D202411-4, Guangdong Provincial Experimental Animal Center) were used, and the routine immunization cycle was as follows: First immunization (immunogen + Freund's complete adjuvant, subcutaneous injection), second immunization (immunogen + Freund's incomplete adjuvant, subcutaneous injection), third immunization (immunogen + Freund's incomplete adjuvant, subcutaneous injection), fourth immunization (immunogen + Freund's incomplete adjuvant, subcutaneous injection), and fifth booster immunization (immunogen, intraperitoneal injection), every two weeks. Blood was collected from the tail tip of the animals on the 7th day after each immunization, every two weeks, with a blood volume of 150-200 μL each time, for measuring serum antibody levels. After the fifth booster immunization, the blood obtained was incubated at 37°C for half an hour, centrifuged at 12000 rpm for 10 min, and the obtained antiserum was used for detection.

[0054] Example 4: Establishment of a standard curve for the detection of rapid phosphorus inactivation by antiserum

[0055] The working concentrations of the coating agent and the rapid phosphorus antibody were determined using a matrix titration method. The working concentration of the coating agent was 250 ng / mL, and the working concentration of the rapid phosphorus antibody was 100 ng / mL. Three parallel experiments were conducted (n=3).

[0056] The steps for indirect competitive ELISA detection with antiserum are as follows:

[0057] Coating: Dilute the coating agent to an appropriate concentration with carbonate buffer, add 100 μL to each well of the microplate, and coat overnight in a 37°C water bath.

[0058] Washing: Tap off the liquid in the well, wash the plate twice with a plate washer, add 250μL of washing solution to each well, and spin dry the liquid in the well.

[0059] Sealing: Add 120μL of 1% skim milk powder to each well, seal at 37℃ for 2 hours, shake off the liquid in the well, and invert in a 37℃ oven for 1 hour for later use.

[0060] Sample addition and incubation: Dilute methamidophos into a series of gradient standard solutions, diluted to 250, 125, 62.5, 31.25, 15.62, 7.81, 3.91, 1.95, 0.98, and 0.49 ng / mL, respectively. Add 50 μL to each well, then add 50 μL of the diluted antiserum solution. Incubate in a 37°C water bath for 40 min, then wash the plate 5 times with a plate washer, adding 250 μL of washing buffer to each well, and then spin dry the liquid in the well.

[0061] Add secondary antibody: Add 100 μL of HRP-goat anti-mouse diluted 5000 times to each well, react in a 37℃ water bath for 30 min, and then wash the plate as in S4.

[0062] Color development: Mix equal volumes of TMB substrate solution and substrate buffer solution, add 100 μL of the mixture to each well, place in a 37°C water bath for 10 min for color development, and then add 50 μL of 10% H2SO4 stop solution to each well.

[0063] Measurement: Absorbance (OD) was read using an ELISA reader at a wavelength of 450 nm.

[0064] Calculation: Calculate the IC of the suppression curve using the four-parameter fitting module in Origin 8.5. 10 IC 20 IC 50 IC 80 Values, see standard curve. Figure 4 An indirect competitive ELISA immunoassay method for phosphate-degradable phosphorus was established. The standard curve obtained by this method has an IC50 value of [missing information]. 50 The value was 6.33 ng / mL, and the detection limit was (IC50). 10 The effective concentration was 0.86 ng / mL, and the linear detection range was 1.80-22.23 ng / mL.

[0065] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A rapid phosphorus-inactivating hapten, characterized in that, It has the following structural formula:

2. A method for preparing the rapid phosphorus hapten as described in claim 1, comprising the following steps: using tert-butyl acetoacetate as the starting material, sequentially undergoing bromination, phosphorylation, acid hydrolysis, coupling with tert-butyl 4-hydroxybutyrate, acid deprotection, coupling with tert-butyl p-hydroxybenzoate, and finally acid deprotection to obtain the rapid phosphorus hapten.

3. A rapid phosphorus-inactivating complete antigen, characterized in that, The fast-acting phosphorus hapten of claim 1 is obtained by coupling with a carrier protein; the carrier protein is bovine lactoferrin or bovine serum albumin.

4. A method for preparing the rapid phosphorus-inactivating complete antigen as described in claim 3, characterized in that, The process includes the following steps: S1: Dissolve the rapid phosphorus hapten described in claim 1 in an organic solvent, add EDC and NHS for activation, and obtain solution A; S2: Dissolve the carrier protein in carbonate buffer at pH 9.6 to obtain solution B; S3: Add solution A dropwise to solution B while stirring, maintain pH = 9.6 for reaction, and purify after reaction to obtain the complete antigen.

5. The method as described in claim 4, characterized in that, In S1, the molar ratio of the rapid phosphorus hapten, EDC and NHS is 1:1.5-2:1.5-2.

6. The method as described in claim 4, characterized in that, In S3, the molar ratio of the rapid phosphorus hapten in solution A to the carrier protein in solution B is 30-40:

1.

7. A rapid phosphorus-inactivating antibody, characterized in that, The antibody is prepared by immunizing animals with the complete antigen of methamidophos as described in claim 3, and is capable of specifically binding to methamidophos.

8. The rapid phosphorus-inactivating antibody as described in claim 7, characterized in that, The antibody is a polyclonal antibody, a monoclonal antibody, or a genetically engineered antibody.

9. An immunoassay kit for detecting fast-acting phosphorus residues in fruits, vegetables, water bodies, or soil, characterized in that, It contains the rapid phosphorus-inactivating antibody as described in claim 7 or 8.

10. The use of a phosmet hapten as described in claim 1 or a phosmet complete antigen as described in claim 3 in the preparation of an immunoassay agent for detecting phosmet.

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