Methamidophos haptens, complete antigens, antibodies, and methods of making and using the same
By preparing a phosphonium hapten and coupling it with a carrier protein, an immunoassay method was established, which solved the problem of detecting phosphonium residues and achieved detection results with high sensitivity and a wide linear range.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
- Filing Date
- 2025-01-08
- Publication Date
- 2026-06-02
AI Technical Summary
There is a lack of simple and rapid methods in the existing technology to detect phosphonium residues in the environment and food, and the preparation of phosphonium hapten has not been reported.
A phosphonium hapten was prepared and a terminal active group was introduced through chemical modification for coupling with a carrier protein to prepare a complete phosphonium hapten. A detection method was then established by combining this with an immunoassay.
It achieves highly sensitive detection of phosmet, with a detection limit of 1.65 ng/mL and a linear range of 2.94-20.86 ng/mL, making it suitable for rapid screening.
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Figure CN119930678B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of immunoassay technology, specifically relating to a phosphophosphorus hapten, a complete antigen, an antibody, and their preparation methods and applications. Background Technology
[0002] Ethylphosphonium, also known as dichlorvos, is a highly effective, broad-spectrum organophosphorus insecticide with contact toxicity, used to control various insects. Due to its low cost and good insecticidal effect, organophosphorus insecticides have become one of the most widely used pesticides. However, pesticide residues in agricultural products or the environment may pose health hazards to humans. Therefore, to protect human health and monitor pesticide exposure levels, it is of great importance to establish a simple, convenient, and rapid method for analyzing pesticide residues to detect ethylphosphonium in the environment and food.
[0003] Immunoassay is an analytical technique based on the principle of specific and reversible binding reactions between antigens and antibodies. The immune reaction involves the combined effects of highly complementary stereostructures, electrostatics, hydrogen bonds, and van der Waals forces between antigen and antibody molecules. It is characterized by high specificity, low detection limits, low cost, simple operation, and suitability for on-site screening of large batches of samples, and is considered one of the most challenging and competitive rapid detection technologies of the 21st century. This invention does not currently report on haptens prepared from phosphine-free structures. Summary of the Invention
[0004] The purpose of this invention is to provide a phospholipase hapten with the following structural formula:
[0005] .
[0006] Accordingly, the present invention also provides a method for preparing the aforementioned phosphodiesterase hapten, comprising the following steps:
[0007] Step A: Wash and dry sodium hydride with petroleum ether, then disperse it in n-hexane and cool it down; then add propanethiol, turn off the refrigeration and allow it to heat up and stir overnight to obtain phosphine quenching intermediate 1:
[0008] ;
[0009] Step B: Dissolve phosphorus oxychloride in dichloromethane; add the reaction system of phosphonium oxychloride intermediate 1 dropwise; heat and stir overnight; after the reaction is complete, filter to remove insoluble matter, and evaporate the filtrate to obtain phosphonium oxychloride intermediate 2:
[0010] ;
[0011] Step C: Weigh out phosphonium ether intermediate 2 and dissolve it in dichloromethane; then dissolve tert-butyl 4-hydroxybutyrate in dichloromethane and add it, followed by triethylamine; stir the reaction at room temperature; after the reaction is complete, evaporate to dryness and pass through a column to obtain phosphonium ether intermediate 3: the eluent used is ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20;
[0012] ;
[0013] Step D: Dissolve the phosphine intermediate 3 in dichloromethane and add TFA; react at room temperature and remove the solvent by rotary evaporation, then pass through a column to obtain the phosphine hapten: the eluent used is ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20.
[0014] .
[0015] Accordingly, the present invention also provides the use of phosphine hapten in the preparation of phosphine complete antigen.
[0016] Accordingly, the present invention also provides a complete phosphonium hapten, which is obtained by conjugating the phosphonium 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 phosphine-free complete antigen, comprising the following steps:
[0018] S1: Dissolve the obtained phosphine hapten in DMF, then add EDC and NHS while stirring, and stir at room temperature in the dark to obtain the activated hapten, which is called solution A: wherein the molar ratio of the phosphine hapten, the EDC and the NHS is 1:1.5-2:1.5-2.
[0019] S2: Dissolve the carrier protein in a carbonate buffer solution with a pH of 9.6-9.8, with a carrier protein concentration of 5-10 mg / mL, and call it solution B;
[0020] S3: Add the above liquid A dropwise to liquid B with stirring in an ice bath. After addition, adjust the pH to 9.6-9.8 with NaOH solution. React overnight in the dark, and obtain the following structurally complete antigen after dialysis purification: ;
[0021] Preferably, in step S2, the molar ratio of the phosphophosphorus hapten, the EDC, and the NHS is 1:1.5-2:1.5-2;
[0022] Preferably, the molar ratio of the phosphodiesterase hapten in solution A to the carrier protein in solution B is 30-40:1.
[0023] The present invention also provides a phosphonium-free antibody, which is prepared using the phosphonium-free hapten or the phosphonium-free complete antigen described in the present invention.
[0024] Preferably, the phosphobromine antibody is a polyclonal antibody, a monoclonal antibody, or a genetically engineered antibody.
[0025] The present invention also provides a formulation for detecting phosphatidylcholine, comprising the phosphatidylcholine hapten, the phosphatidylcholine complete antigen, or the phosphatidylcholine antibody described in the present invention.
[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0027] This nephrine hapten is designed specifically for nephrine and can be used to prepare nephrine antibodies. Animal immunization and experimental verification have shown that the prepared nephrine antibodies can specifically recognize nephrine. This invention also discloses the preparation method and application of the aforementioned nephrine hapten.
[0028] The indirect competitive enzyme-linked immunosorbent assay (ELISA) method constructed in this invention has an IC50 sensitivity for chlorinated paraffin. 50 The detection limit is up to 7.83 ng / mL, with a detection range of 2.94-20.86 ng / mL and a detection limit of IC50. 10 The concentration was 1.65 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
[0029] Figure 1 This is a flowchart illustrating a method for preparing a phosphophosphorus hapten according to one embodiment.
[0030] Figure 2 This is a mass spectrometry analysis chromatogram of a phosphophosphorus hapten from one embodiment.
[0031] Figure 3 This is an ultraviolet (UV) identification image of a phosphonium-based artificial antigen as an example.
[0032] Figure 4 This is an ELISA competition standard curve of the phosphochloride antibody obtained in Example 4. Detailed Implementation
[0033] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings:
[0034] 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.
[0035] The preparation method of phosphophosphorus hapten is as follows: Figure 1 As shown, it includes the following steps:
[0036] Step A: Weigh 2.5 g of sodium hydride after washing with petroleum ether to remove oil and drying, add 50 mL of n-hexane to disperse, and cool to -78℃. Then weigh 7.7 g of propanethiol and slowly add it. After the addition is complete, turn off the refrigeration and allow the temperature to rise and stir overnight to obtain a white turbid liquid (phosphine intermediate 1).
[0037]
[0038] Step B: Dissolve 7.8 g of phosphorus oxychloride in 125 mL of dichloromethane. Slowly add the entire intermediate 1 reaction system dropwise at -75℃. After the addition is complete, turn off the refrigeration and allow the temperature to rise automatically while stirring overnight. After the reaction is complete, filter to remove insoluble matter, and evaporate the filtrate to obtain a pale yellow liquid with acid mist (phosphorus oxychloride intermediate 2).
[0039]
[0040] Step C: Weigh 2,349 mg of intermediate and dissolve it in 10 mL of dichloromethane. Then weigh 160 mg of tert-butyl 4-hydroxybutyrate, dissolve it in 2 mL of dichloromethane, and add it to the solution, followed by 121 mg of triethylamine. Stir the reaction at room temperature for 6 h. After the reaction is complete, evaporate to dryness and pass through a column to obtain a colorless oil (phosphine intermediate 3); the eluent used was ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20.
[0041]
[0042] Step D: Weigh 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 complete, remove the solvent by rotary evaporation, and pass the solution through a column to obtain a white solid (phosphophosphorus hapten), MW: 300.06. The eluent used was ethyl acetate and petroleum ether in a volume ratio of 1:10 to 1:20.
[0043] Mass spectrometry results as follows Figure 2 The results show that the ion peak with a mass-to-charge ratio (m / z) of 301.0 in the positive ion mode ESI ionization full scan corresponds to the quasi-molecular ion peak [M+H]+, proving that the synthesis of the mitochondrial hapten was successful.
[0044] Advantages of designing phosphine haptens: To achieve the coupling of small hapten molecules with carrier proteins, 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 artificial antigens, which effectively improves the antibody's recognition activity against phosphine.
[0045] Immunogen: 5.6 mg of phosphosulfuron-methyl hapten was dissolved in 600 μL of N,N-dimethylformamide (DMF) with 5.4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 3.2 mg of N-hydroxysuccinimide (NHS). 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.
[0046]
[0047] Coating agent: 13.6 mg of phosphosulfuron-methyl hapten was dissolved in 600 μL of N,N-dimethylformamide (DMF) with 13.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 7.8 mg of N-hydroxysuccinimide (NHS). 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 carbonate buffer at pH 9.6, 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] like Figure 3 As shown, all synthesized artificial antigens exhibit characteristic absorption peaks of haptens and carrier proteins, or their absorption peaks shift relative to the original carrier protein, indicating that all artificial antigen synthesis was successful.
[0050] The experiment was conducted as follows: Eight-week-old SPF Bal b / c female mice (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 ℃ for half an hour, centrifuged at 12000 rpm for 10 min, and the obtained antiserum was used for detection.
[0051] The working concentrations of the coating agent and the phosphochloride antibody were determined using a matrix titration method. The working concentration of the coating agent was 500 ng / mL, and the working concentration of the phosphochloride antibody was 100 ng / mL. Three parallel experiments were conducted (n=3).
[0052] The steps for indirect competitive ELISA detection with antiserum are as follows:
[0053] 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.
[0054] Washing: Tap off the liquid in the well, wash the plate twice with a plate washer, add 250 μL of washing solution to each well, and spin dry the liquid in the well.
[0055] Sealing: Add 120 μL of 1% skim milk powder to each well, seal at 37 ℃ for 2 h, shake off the liquid in the well, and invert in a 37 ℃ oven for 1 h for later use.
[0056] Sample addition and incubation: Dilute phosphine 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℃ water bath for 40 min. 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.
[0057] Add secondary antibody: Add 100 μL of HRP-goat anti-rabbit diluted 5000 times to each well, react in a 37 ℃ water bath for 30 min, and wash the plate as in S4.
[0058] Color development: Mix equal volumes of TMB substrate solution and substrate buffer, add 100 μL of the mixture to each well, place in a 37 ℃ water bath for 10 min for color development, and then add 50 μL of 10% H2SO4 stop solution to each well.
[0059] Measurement: Absorbance (OD) was read using an ELISA reader at a wavelength of 450 nm.
[0060] Calculation: Use the four-parameter fitting module in Origin 8.5 to calculate the IC10, IC20, IC50, and IC80 values of the suppression curve. The standard curve is shown below. Figure 4 The obtained standard curve IC 50 The value was 7.83 ng / mL, and the limit of detection was (IC50). 10 The effective concentration was 1.65 ng / mL, and the linear detection range was 2.94-20.86 ng / mL.
[0061] 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 phosphophosphorus hapten, characterized in that, It has the following structural formula: 。 2. A method for preparing the phosphophosphorus hapten as described in claim 1, characterized in that, It includes the following steps: Step A: Wash and dry sodium hydride with petroleum ether, then disperse it in n-hexane and cool it down; then add propanethiol, turn off the refrigeration and allow it to heat up and stir overnight to obtain phosphine quenching intermediate 1: ； Step B: Dissolve phosphorus oxychloride in dichloromethane; add the reaction system of phosphonium oxychloride intermediate 1 dropwise; heat and stir overnight; after the reaction is complete, filter to remove insoluble matter, and evaporate the filtrate to obtain phosphonium oxychloride intermediate 2: ； Step C: Weigh out intermediate 2 of phosphatidylcholine and dissolve it in dichloromethane; then weigh out tert-butyl 4-hydroxybutyrate, dissolve it in dichloromethane and add it, followed by triethylamine; stir the reaction at room temperature; after the reaction is complete, evaporate to dryness and pass through a column to obtain intermediate 3 of phosphatidylcholine. ； Step D: Weigh out phosphonium ether intermediate 3, dissolve it in dichloromethane, then add TFA; react at room temperature, remove the solvent by rotary evaporation, and pass the solution through a column to obtain the phosphonium ether hapten. 。 3. The use of the phosphine hapten as described in claim 1 in the preparation of the phosphine complete antigen.
4. A complete phosphine-free antigen, characterized in that, It is obtained by conjugating the phosphochloride hapten of claim 1 with a carrier protein; the carrier protein is bovine lactoferrin, bovine serum albumin or ovalbumin.
5. A method for preparing the complete phosphobromine antigen as described in claim 4, characterized in that, Includes the following steps: S1: Dissolve the phosphine hapten obtained as described in claim 2 in DMF, then add EDC and NHS under stirring, and stir at room temperature in the dark to obtain the activated hapten, referred to as solution A: wherein the molar ratio of the phosphine hapten, the EDC and the NHS is 1:1.5:1.5; S2: Dissolve the carrier protein in a carbonate buffer solution at pH 9.6, with a carrier protein concentration of 5-10 mg / mL, and call it solution B; S3: Add the above liquid A dropwise to liquid B with stirring in an ice bath. After addition, adjust the pH to 9.6 with NaOH solution. React overnight in the dark, and obtain the following structurally complete antigen after dialysis purification: 。 6. The method as described in claim 5, characterized in that, The molar ratio of the phosphophosphorus hapten in solution A to the carrier protein in solution B is 30:
1.
7. A formulation for detecting phosmet, characterized in that, Includes the phosphine hapten of claim 1 or the phosphine complete antigen of any one of claims 4-5.