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Beta-agonist hapten, artificial antibody as well as preparation methods and applications of beta-agonist hapten and beta-agonist artificial antibody

An artificial antibody and agonist technology, applied in peptide preparation methods, chemical instruments and methods, animal/human proteins, etc., can solve problems such as the lack of antibodies that can recognize β-agonists at the same time, and achieve easy large-scale production and application , the preparation method is simple, the effect of broad application prospect

Active Publication Date: 2018-06-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is that there is no antibody capable of simultaneously recognizing multiple β-agonists in the prior art. In order to overcome the above-mentioned deficiencies in the prior art, a β-agonist hapten is provided, which is combined with the hapten The artificial antigen prepared by carrier protein coupling is the immunogen, and the broad-spectrum specific β-agonist artificial antibody prepared can detect multiple β-agonist drugs at the same time, and can be used for on-site rapid detection of food safety

Method used

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  • Beta-agonist hapten, artificial antibody as well as preparation methods and applications of beta-agonist hapten and beta-agonist artificial antibody
  • Beta-agonist hapten, artificial antibody as well as preparation methods and applications of beta-agonist hapten and beta-agonist artificial antibody
  • Beta-agonist hapten, artificial antibody as well as preparation methods and applications of beta-agonist hapten and beta-agonist artificial antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Synthesis and identification of β-agonist hapten

[0045] 1. The synthetic route is as follows: figure 1 As shown, weigh 220mg of R-(-)-salbutamol and dissolve it in 40mL of methanol, add 77mg of succinic anhydride, and mix well under nitrogen protection. The mixture was stirred at room temperature for 12 h, and a white suspension appeared. The reaction solution was placed in a 50mL centrifuge tube and centrifuged at 3000r / min for 15min, the supernatant was discarded, and the precipitate was washed 3 times with 100mL of absolute ethanol, and dried in vacuum for 24h to obtain a white solid.1 H-NMR hydrogen spectrum and mass spectrum identification.

[0046] 2. The results are as follows:

[0047] Proton spectrum results:

[0048] 1 H-NMR (DMSO, 400 MHz): δ (ppm) 1.12 (9H, s), 2.38-2.41 (2H, m), 2.53-2.56 (2H, m), 2.76-2.78 (2H, m), 4.61- 4.62 (1H, m), 5.05 (2H, d, J = 2.0 Hz), 6.77 (1H, d, J = 8.0 Hz ), 7.10 (1H, dd, J = 8.4, 2.0 Hz), 7.27 (1H, d, ...

Embodiment 2

[0051] Example 2 Synthesis and identification of β-agonist artificial antigen

[0052] 1. Synthesis of β-agonist artificial antigen

[0053] (1) Dissolve 7.5 μL of triethylamine in 0.15 mL of DMF, stir at 4°C for 15 min, then add 3.4 mg of the β-agonist hapten obtained in Example 1 and 1 μL of isobutyl chloroformate, and stir at 4°C React for 30 minutes to obtain the β-agonist hapten solution;

[0054] (2) Dissolve 8 mg of BSA in 1 mL of carbonate buffer to obtain a carrier protein solution;

[0055] (3) After stirring evenly, add the carrier protein solution obtained in step (2) dropwise into the β-agonist hapten solution obtained in step (1), and stir and react at 4°C for 12 hours;

[0056] (4) Put the reacted solution in a dialysis bag, dialyze with 0.01mol / L PBS solution (pH=7.4) at 4°C for 3 days, change the dialysate twice a day during the period, and obtain the β-agonist after the dialysis artificial antigen.

[0057] In addition, the carrier protein BSA was replace...

Embodiment 3

[0061] Example 3 Preparation of broad-spectrum specific β-agonist artificial antibody

[0062] 1. Immunity

[0063] Mix and emulsify the above-mentioned β-agonist artificial antigen solution with an equal amount of adjuvant, mix and emulsify with complete Freund's adjuvant for the initial immunization, and then immunize after emulsifying with the immunogen and incomplete Freund's adjuvant 4 weeks later. The booster immunization interval was 3 weeks, and the dose of each immunization was 6mL / bird. On the 7th day after the third immunization, blood was taken to detect the antiserum titer until the antiserum titer no longer increased. After the blood was collected, the antiserum was separated by natural coagulation at room temperature, placed overnight at 4°C, and kept at 4000r / min. After centrifugation for 30 min, the supernatant serum was taken.

[0064] 2. Purification (caprylic acid-ammonium sulfate method):

[0065] (1) Take 3 mL of antiserum and add 2 times the volume of...

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Abstract

The invention discloses a beta-agonist hapten. The molecular structure of the beta-agonist hapten is shown in the following formula (I) in the description. The beta-agonist hapten is successfully synthesized from R-(-)-salbutamol as a raw material by one-step chemical reaction. The hapten is coupled with carrier protein, an artificial antigen is prepared, and the broad-spectrum specific beta-agonist artificial antibody is obtained after immunization. The antibody can recognize 31 kinds of beta-agonist drugs and analogs of the beta-agonist drugs, the semi-inhibitory concentration for R-(-)-salbutamol is 0.5 ng / mL, the linear range is 0.11-40.86 ng / mL, and the minimum detection limit is 0.04 ng / mL. The antibody can detect multiple beta-agonist drugs simultaneously, can be used for on-site rapid detection of food safety and has broad application prospect.

Description

technical field [0001] The invention relates to the technical field of food safety detection, in particular to a β-agonist hapten and an artificial antibody as well as a preparation method and application thereof. Background technique [0002] β-agonist (β-agonist) is the abbreviation of β-adrenoceptor agonist (β-adrenoceptor agonist), which belongs to a kind of phenylethylamine drugs. Functional medicine, because it can change energy distribution, reduce fat production, promote fat decomposition, promote protein synthesis and reduce its degradation, in the 1980s, β-agonists were added to feed to improve the quality of cattle, sheep, pigs, etc. Reports on lean growth rates in livestock. When the cumulative intake dose of the human body exceeds the threshold or ingests high residual animal visceral tissue, β-agonists will produce obvious physiological toxicity, causing palpitations, trembling, dizziness, nausea and vomiting, vasodilation, nervousness, rapid heartbeat, etc. ...

Claims

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Application Information

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IPC IPC(8): G01N33/558C07K14/765C07K14/77C07K1/02C07K16/44
CPCC07K14/765C07K14/77C07K16/44G01N33/558
Inventor 雷红涛姜文梦王兰腾沈兴陈佳虹孙远明徐振林李向梅
Owner SOUTH CHINA AGRI UNIV
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