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Acrylamide hapten, artificial antigen, antibody and preparation method and application thereof

A technology of acrylamide and artificial antigen, which is applied in the field of food safety immunoassay, can solve the problems of poor repeatability and low titer of antibody preparation, and achieve the effects of short time, enhanced structural characteristics and high derivation efficiency

Active Publication Date: 2013-09-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to overcome the defects of low titer and poor repeatability of antibody preparation in traditional acrylamide detection technology, and provide an acrylamide hapten, artificial antigen and specific antibody

Method used

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  • Acrylamide hapten, artificial antigen, antibody and preparation method and application thereof
  • Acrylamide hapten, artificial antigen, antibody and preparation method and application thereof
  • Acrylamide hapten, artificial antigen, antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of hapten BI (n=1):

[0045] Take 0.212 g (2.9 mmoL) of acrylamide and dissolve it in 400 μL of carbonate buffer at pH 9.6, and take 0.1 g (0.5 mmoL) of p-mercaptophenylacetic acid and dissolve it in 800 μL of anhydrous methanol. The above-mentioned anhydrous methanol solution of p-mercaptophenylacetic acid was slowly added dropwise into the carbonate buffer of acrylamide, and stirred at 37 °C for 1 h in the dark. After the reaction was completed, use vacuum filtration method, add 2 mL of primary water to wash, take the precipitate, and dry it in vacuum at 37°C to obtain 256 mg of white solid 4-(3-amino-3-oxobutylthio)phenylacetic acid , and the yield was 82.05%. ESI-MS analysis (negative) m / z 239 [M-H]-; 1H NMR (CD3OD, 400 MHz) d 7.206-7.221 (d, J = 6.0 Hz, 2H), 6.670-6.685 (d, J = 6.0 Hz, 2H), 2.942-2.966 (m, 2H), 2.406-2.431 (m, 2H).

Embodiment 2

[0046] Example 2 Preparation method of hapten BII (n=0)

[0047] Take 0.284 g (3.99 mmoL) of acrylamide and dissolve it in 400 μL of 0.1 mol / L carbonate buffer solution with a pH of 9.6, and take 0.1 g (0.79 mmoL) of p-aminothiophenol and dissolve it in 800 μL of absolute ethanol. The above anhydrous methanol solution of p-aminothiophenol was slowly added dropwise into the acrylamide carbonate buffer, and stirred at 37 °C for 1 h in the dark. After the reaction, the mixed solution was adjusted to pH 3 with 4 M hydrochloric acid, extracted with ethyl acetate, the aqueous layer was adjusted to pH 9.6 with 4 M sodium carbonate, extracted with ethyl acetate, the mixed solution was poured into saturated sodium chloride, and brown-red Oily liquid 4-(3-amino-3-oxopropylthio)aniline 297 mg, yield 77.34%. ESI-MS analysis (negative) m / z 196 [M-H]-; 1 H NMR (CD 3 OD, 400 MHz) d 7.335-7.349 (d, J = 5.6 Hz, 2H), 7.233-7.247 (d, J = 5.6 Hz, 2H), 3.579 (s, 2H), 3.144-3.168 (m, 2H),...

Embodiment 3

[0048] Example 3 Immunogen / coating preparation

[0049] The difference between the preparation of the immunogen and the coating source lies in the carrier protein, the immunogen carrier protein uses keyhole limpet hemocyanin (KLH), and the coating source carrier protein uses ovalbumin (OVA). The preparation method of the immunogen is used as an example in the following.

[0050] Active ester method: Dissolve 0.0239 g (0.1 mmol) of hapten BⅠ (n=1) in 0.5 mL of dimethylformamide (DMF), stir and add 0.0512 g (0.2 mmol) of 1,3-dicyclohexylcarbodi Imine (DCC) and 0.023 g (0.2 mmoL) NHS were reacted overnight at 4 °C with magnetic stirring, and the supernatant was liquid A after centrifugation. Weigh 0.02 g of carrier protein KLH or OVA and dissolve it in 2 mL of PBS (pH8.0) solution with a concentration of 0.1 mol / L, stir and dissolve to prepare solution B. Under magnetic stirring, liquid A was gradually dropped into liquid B, and reacted at 4 °C for 12 h. After centrifugation...

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PUM

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Abstract

The invention discloses an acrylamide hapten, artificial antigen, antibody and a preparation method and application thereof. The acrylamide hapten has the molecular structure shown by the formula (I) or formula (II), wherein n is 0-6. The acrylamide artificial antigen has a molecular structure shown by the formula (III) or formula (IV), wherein n is 0-6. The titer of the antiserum prepared from the antigen disclosed by the invention can reach 1:1,024,000, the minimum detection limit is 1-3ng / mL, and the half inhibition concentration is 8.7ng / mL; the generated antibody has high specificity, high sensitivity and high accuracy; and the antigen and antibody disclosed by the invention have wide application prospects in the residue detection of acrylamide in food.

Description

technical field [0001] The invention belongs to the technical field of food safety immunoassay, and more specifically relates to an acrylamide hapten, an artificial antigen, an antibody and a preparation method and application thereof. Background technique [0002] Acrylamide is a highly water-soluble unsaturated amide small molecular compound, which has certain toxicity to the human body. Long-term exposure to acrylamide will poison the central or other peripheral nerves of humans and animals, resulting in damage to cellular DNA and reproductive toxicity. In 1994, the International Agency for Cancer (IARC) classified acrylamide as a Class 2A carcinogen; in April 2002, Swedish scholars reported for the first time that acrylamide widely exists in heat-processed foods. In 2010, the European Chemicals Agency (ECHA) added acrylamide to the list of substances of very high concern. WHO stipulates that the limit of acrylamide in drinking water is 1 μg / L, that of the European Union...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C323/62C07C323/60C07C319/18C07K14/765C07K14/77C07K14/795C07K1/107C07K16/44G01N33/577G01N33/531
Inventor 孙远明徐振林吴璟沈玉栋雷红涛肖治理王弘杨金易
Owner SOUTH CHINA AGRI UNIV
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