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Preparation method of fluoroacetamide hapten and application of monoclonal antibody

A technology of fluoroacetamide and hapten, which is applied in the field of preparation of fluoroacetamide hapten and monoclonal antibody, can solve problems that hinder the application and promotion of immunological methods, difficult immunological detection methods, and lack of immunogenicity. Achieve the effect of good application prospect, high practical value and high sensitivity

Active Publication Date: 2019-04-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since fluoroacetamide is a small molecular substance with a relative molecular weight of only 76, it is not immunogenic. In addition, although fluoroacetamide has exposed amino groups, it is not easy to undergo chemical reactions due to the formation of amide bonds.
This brings great difficulties to the study of immunological detection methods of fluoroacetamide, and also hinders the application and promotion of immunological methods to a certain extent.

Method used

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  • Preparation method of fluoroacetamide hapten and application of monoclonal antibody
  • Preparation method of fluoroacetamide hapten and application of monoclonal antibody
  • Preparation method of fluoroacetamide hapten and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Fluoroacetamide hapten (fluoroacetamide phe) and its synthesis and identification

[0081] 1. Synthesis of fluoroacetamide phe (such as figure 1 shown)

[0082] 1) Synthesis of fluoroacetyl chloride

[0083] An aqueous solution (34 mL) of KOH (19 g, 0.339 mol) was slowly added dropwise to ethyl fluoroacetate (30 g, 0.283 mol) in ethanol (300 mL) at room temperature, forming a white precipitate. The mixture was stirred overnight at room temperature (30-35°C). The solvent was spin-dried, and the obtained sodium salt was redissolved in hydrochloric acid (3M, 200mL), and NaCl solid was added to make the solution saturated, then extracted with ether for 4 to 5 times, the combined organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was spun After drying, fluoroacetic acid (15 g) was obtained.

[0084] In the three-neck flask with a thermometer and a condenser tube inserted, add PCl 5 (44g, 0.211mol, 1.1eq), cooled in an ice bath to...

Embodiment 2

[0095] Example 2 Fluoroacetamide hapten (fluoroacetamide liner) and its synthesis and identification

[0096] 1. Synthesis of fluoroacetamide liner (such as figure 2 shown)

[0097] 10g (76mmol) of 6-aminocaproic acid was added to 100ml of anhydrous methanol, the solution was cloudy, 23g (227mml) of triethylamine was added, stirred at room temperature for 10min, 12g (113mmol) of ethyl fluoroacetate was added dropwise, the reaction was refluxed overnight, and the solution Become clear, cool to room temperature, add acetic acid to adjust the pH to 5-6, concentrate the reaction solution, drain the oil pump to obtain an oily liquid, add EtOAc to dissolve, wash EA twice with saturated saline, dry, spin dry, and drain the oil pump to obtain The product is 7g, and the yield is about 48%.

[0098] 2. Identification of fluoroacetamide liner

[0099] Its structure was determined by proton NMR spectroscopy:

[0100] 1 H NMR (400MHz, CDCl 3 )δ: 6.48(bs,1H,NH); 4.87(s,1H,CH 2 ); 4....

Embodiment 3

[0102] Example 3 Preparation of fluoroacetamide artificial antigen

[0103] 1. Preparation and identification of fluoroacetamide coating agent

[0104] 1.1 Preparation of fluoroacetamide coating agent

[0105] 50mg of fluoroacetamide hapten (formula II or III) dissolved in 1ml N,N-dimethylformamide (DMF), N,N'-dicyclohexylcarbodiimide (DCC) 80mg, N - 60 mg of hydroxysuccinimide (NHS) was added to the above fluoroacetamide hapten solution, stirred at room temperature for 2 hours; 160 mg of BSA was dissolved in 20 ml of PBS buffer (0.01mol / L PH=7.4), and stirred The activated drug was added dropwise to the protein solution and reacted overnight at 4°C; then the reaction solution was put into a dialysis bag, dialyzed with saline solution at 4°C for 48 hours, and the water was changed 4 times; save.

[0106] 1.2 Identification of fluoroacetamide coating agent

[0107] Fluoroacetamide artificial antigen is prepared into a solution in PBS for flight mass spectrometry analysis, a...

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Abstract

The invention relates to the technical field of biological chemistry, and particularly discloses a preparation method of fluoroacetamide hapten and application of a monoclonal antibody. The fluoroacetamide hapten is synthesized by taking ethyl fluoroacetate and p-aminophenylacetic acid as main raw materials; the fluoroacetamide hapten and carrier protein bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) are coupled through an active lipid method, and thus a conjugate is prepared. Through experimental verification, it shows that the prepared conjugate enables a living body to generate the antibody against fluoroacetamide, that is to say, the prepared conjugate is fluoroacetamide artificial antigen. The prepared fluoroacetamide artificial antigen is suitable for enzyme-linked immunological analysis of fluoroacetamide.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to a preparation method and application of a fluoroacetamide hapten and a monoclonal antibody. Background technique [0002] Fluoroacetamide (fluoroacetamide), also known as 2-fluoroacetamide, dimethadamide, Qiu's rodenticide. The pure product is tasteless, odorless white needle-like crystal or white powder, easily soluble in water (1:5), hydrolyzed into fluoroacetic acid in acidic and neutral aqueous solution, and hydrolyzed into fluoroacetic acid in alkaline aqueous solution Sodium (sodium fluoroacetate). The production process is simple, and it is obtained by fluorinating chloroacetamide. Fluoroacetamide can enter the body through the skin, respiratory tract and digestion, causing poisoning events. If the rescue is not timely, it will cause death, affect the generation of adenosine triphosphate and the metabolic process of intermediate products, and the generated fluoroace...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/795C07K1/02C07K16/44C12N5/20G01N33/53C07C233/47C07C233/54C07C231/02C07C231/12C07C53/18C07C51/09C07C53/40C07C51/60C07C229/42C07C227/18
CPCC07C51/09C07C51/60C07C227/18C07C231/02C07C231/12C07C233/47C07C233/54C07K14/765C07K14/795C07K16/44G01N33/53C07C53/18C07C53/40C07C229/42
Inventor 王战辉沈建忠温凯江海洋杨玲史为民张西亚张素霞
Owner CHINA AGRI UNIV
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