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Salin mimic agent hapten, artificial antigen and antibody and preparation methods thereof

An artificial antigen and hapten technology, applied in the field of immune detection, can solve the problems of poor anti-environmental interference, limitation, and low detection sensitivity, and achieve good immune activity, low preparation cost, and high sensitivity.

Active Publication Date: 2021-06-01
中国人民解放军火箭军工程设计研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method for detecting DMMP is gas chromatography, which has high sensitivity and good selectivity, but there are disadvantages such as expensive and bulky equipment, cumbersome sample pre-treatment process, and limited laboratory conditions.
Portable detection methods for DMMP mostly use surface acoustic wave (SAW) sensors, quartz crystal microbalance (QCM) sensors, semiconductor sensors, etc., but they also face the problems of poor specificity, poor ability to resist environmental interference, and low detection sensitivity

Method used

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  • Salin mimic agent hapten, artificial antigen and antibody and preparation methods thereof
  • Salin mimic agent hapten, artificial antigen and antibody and preparation methods thereof
  • Salin mimic agent hapten, artificial antigen and antibody and preparation methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Synthetic route of hapten (I):

[0036]

[0037] Weigh 2.125g (12.8mmol) of p-hydroxyphenylpropionic acid and 1.8g of KOH (32mmol), dissolve them in 100mL of anhydrous methanol at 0°C, and then add 2.770g (19.2mmol) of chlorophosphoric acid di After the methyl ester was added dropwise, it was stirred and heated, and the temperature was slowly raised to 65°C, and the stirring reaction was continued for 12 hours.

[0038] After the reaction, the reaction solution was distilled under reduced pressure to remove methanol to obtain the initial product, which was dissolved in 15 mL of ethyl acetate, then 15 mL of 2 mol / L NaOH solution was added, and the pH value was adjusted to about 2.0 with concentrated hydrochloric acid.

[0039] Extract with ethyl acetate 3×20 mL, discard the water layer, combine the ethyl acetate layers, wash the ethyl acetate layer with 2×10 mL of distilled water, add anhydrous magnesium sulfate to dry overnight, and distill off the ethyl acetate ...

Embodiment 2

[0066] (1) Synthetic route of hapten (II)

[0067]

[0068] In the synthesis of hapten (II), except that the reactant is 1.680g (12.8mmol) 6-aminocaproic acid and the amount of alkali used is 0.9gKOH (16mmol), all the other reactants and synthesis steps are the same as the hapten (I) in Example 1 )Synthesis.

[0069] (2) Antigen H2 synthesis route

[0070]

[0071] The synthesis steps of the antigen H2-Protein are the same as the synthesis steps of the antigen H1-Protein in Example 1.

[0072] After calculation, the coupling ratio of hapten (Ⅱ) to BSA was 58:1, and that to OVA was 61:1.

[0073] (3) Preparation of DMMP polyclonal antibody

[0074] The DMMP polyclonal antibody was prepared by immunizing animals with the antigen H2-BSA solution synthesized above, and the synthesis steps and dialysis bag pretreatment steps were the same as in Example 1.

[0075] Calculated after the pretreatment of the dialysis bag, the coupling ratio of hapten H2 to BSA was 58:1, and t...

Embodiment 3

[0083] In the method for preparing DMMP polyclonal antibody by immunizing animals with antigen H1-BSA solution in Example 1, an indirect non-competitive ELISA method was used for square array titration to determine the binding titer of antiserum and coating agent.

[0084] The immunogen is a conjugate of a hapten and BSA, and the coating is a conjugate of the corresponding hapten and OVA. Coating concentrations were set to 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL and 0.625 μg / mL, and coating buffer (carbonate buffer with pH=9.6 solution) for dilution. The specific operation steps are as follows:

[0085] a) Antigen H1-Protein coating in Example 1: Coat the enzyme-linked reaction plate with serially diluted coating original, 100 μL per well, overnight at 4°C.

[0086] b) Washing: Take out the enzyme-linked reaction plate and return to room temperature, discard the coating solution, wash the reaction plate with PBST solution five times, each time for 3 minutes, 300 μ...

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Abstract

The invention relates to the field of immunodetection, in particular to a preparation method of a sarin mimetic agent dimethyl methylphosphonate hapten, an artificial antigen and an antibody. The dimethyl methylphosphonate hapten is simple in structure, easy to synthesize and environment-friendly. The dimethyl methylphosphonate artificial antigen takes bovine serum albumin and ovalbumin as carrier proteins, and the synthesized artificial antigen is large in coupling ratio and has good immunocompetence; and the artificial antigen retains a phosphonate group, so that the preparation of a high-quality antibody is facilitated. The synthetic artificial antigen is subjected to animal immunization to obtain the artificial dimethyl methylphosphonate polyclonal antibody, and the prepared antibody is high in titer and strong in specificity; and the dimethyl methylphosphonate artificial antigen can be used for developing a dimethyl methylphosphonate immune rapid analysis technology, method and product.

Description

technical field [0001] The invention relates to the field of immune detection, in particular to a preparation method of sarin simulant dimethyl methylphosphonate hapten, artificial antigen and antibody. Background technique [0002] In recent years, the use of enzyme-linked immunosorbent assay (ELISA) to detect small molecular weight substances has attracted much attention due to its advantages of simple operation, high sensitivity, strong specificity, fast and accurate, and its application in the detection of pesticide residues has developed rapidly. A large number of ELISA analysis methods for methamidophos, methyl parathion, pyrethroid pesticides, fipronil insecticides, diflubenzuron pesticides, etc. have been reported. With the rapid development of biotechnology and sensor technology, ELISA methods have been widely used in biocontrol bacteria monitoring, herbicide residue detection, pesticide residue (water and soil) detection, and environmental pathogen monitoring. [...

Claims

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Application Information

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IPC IPC(8): C07F9/12C07F9/24C07K1/107C07K14/765C07K14/77C07K16/44
CPCC07F9/12C07F9/2458C07K14/765C07K14/77C07K1/1077C07K16/44C07K2317/33C07K2317/76
Inventor 刘晓芳刘红芹赵海洋李晋峰刘朝辉
Owner 中国人民解放军火箭军工程设计研究院
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