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Hapten, artificial antigen and monoclonal antibody for phenothiazine medicaments, preparation methods of hapten and artificial antigen and application of monoclonal antibody

A monoclonal antibody and phenothiazine technology, applied in the field of food safety, can solve the problem that multiple phenothiazine drugs cannot be detected by multi-residue immunoassay, antibodies cannot recognize other phenothiazine drugs at the same time, and there is no cross-reactivity. And other issues

Inactive Publication Date: 2014-02-05
HEBEI AGRICULTURAL UNIV.
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1979, Midha et al. synthesized the hapten N-(2-carbonylethyl ester) desmethylchlorpromazine of chlorpromazine, and the prepared polyclonal antibody had a cross-reactivity rate of 100% with chlorpromazine, but with other phenothione Azines have no cross-reactivity
In 2010, Liu et al. synthesized a hapten based on acepromazine and prepared a monoclonal antibody. The cross-reactivity rate of this antibody with chlorpromazine was 100%, but the cross-reactivity rate with other phenothiazine drugs was extremely low (≤ 1%)
Therefore, the various antibodies currently available are haptens synthesized using chlorpromazine or acepromazine as a template, and the prepared antibodies cannot recognize other phenothiazine drugs at the same time, and of course they cannot be used for multi-residue immunoassay detection of various Phenothiazines

Method used

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  • Hapten, artificial antigen and monoclonal antibody for phenothiazine medicaments, preparation methods of hapten and artificial antigen and application of monoclonal antibody
  • Hapten, artificial antigen and monoclonal antibody for phenothiazine medicaments, preparation methods of hapten and artificial antigen and application of monoclonal antibody
  • Hapten, artificial antigen and monoclonal antibody for phenothiazine medicaments, preparation methods of hapten and artificial antigen and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the synthesis of the group-specific hapten of the phenothiazine drug of formula 1 structure

[0029] Experiment 1:

[0030] (a) Add 4mmol 2-chlorophenothiazine into the three-necked flask and dissolve it in 20 mL tetrahydrofuran, add 20mmol sodium hydride under stirring, and stir for 10 minutes;

[0031] (b) Add 4 mmol sodium bromoacetate to the above three-necked flask, heat to reflux, stop heating after 6 hours of reaction and lower to room temperature;

[0032] (c) Add 40 mmol of sodium hydride to the above-mentioned three-necked flask, stop the reaction after 8 hours of reflux reaction and cool down to room temperature;

[0033] (d) Add absolute ethanol to the above-mentioned three-neck flask to remove excess sodium hydride, filter under reduced pressure, and evaporate the obtained filtrate to dryness on a rotary evaporator to obtain a solid, which is acidified in ethanol and separated by column chromatography to obtain Compound shown in formula 1. ...

Embodiment 2

[0044] Example 2: Synthesis of family-specific artificial antigens of phenothiazines with the structure of Formula 2

[0045] Experiment 1:

[0046] (a) Dissolve 0.1 mmol of phenothiazine hapten in 3 mL of N,N-dimethylformamide, then add 20 μL of triethylamine and 0.1 mmol of isobutyl chloroformate, and stir at 4°C for 2 hours. get liquid A;

[0047] (b) Dissolve 0.02 mmol of bovine serum albumin in 2 mL of 1 mol / L sodium bicarbonate solution to obtain liquid B;

[0048] (c) Add liquid A to liquid B dropwise, and stir and react at 4°C for 12 hours to obtain a reaction liquid containing artificial antigens of phenothiazine drugs;

[0049] (d) Dialyze the above-mentioned reaction solution containing the artificial antigen of phenothiazine drugs with PBS at 4°C for 3 days, and filter the solution in the obtained dialysis bag with a filter membrane with a filter diameter of 0.2um under sterile conditions to obtain the following: The artificial antigen of the phenothiazine drugs...

Embodiment 3

[0060] Embodiment 3: the preparation of monoclonal antibody

[0061] (a) Using the artificial antigen of phenothiazine drugs prepared in the above example as the immunogen, immunize 5 Balb / C mice respectively, with an immunization dose of 100-300 μg / mouse, and the immunization method is as follows: mix the immunogen with an equal amount of Freund's complete adjuvant was fully emulsified and injected subcutaneously at multiple points on the back of the neck. At intervals of 2-3 weeks, after emulsifying the immunogen with an equal amount of Freund's incomplete adjuvant, booster immunization once, a total of 6 booster immunizations;

[0062] (b) Seven days after the last immunization, the mouse with the highest serum titer in each group in step (a) above was selected and sacrificed by cervical dislocation. Under sterile conditions, the spleen was removed, splenocytes were isolated, and fused with mouse myeloma SP2 / 0 cells at a ratio of 10:1. Positive hybridoma cells were scree...

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Abstract

The invention discloses a hapten, an artificial antigen and a monoclonal antibody for phenothiazine medicaments, preparation methods of the hapten and the artificial antigen and the application of the monoclonal antibody. The hapten contains a common structure of the phenothiazine medicaments and is connected with a carrier protein through introduced carboxyl in a preparation process of the artificial antigen for the phenothiazine medicaments, namely, the common structure is far away from the carrier protein and is completely exposed to an immunological recognition system of an animal. Therefore, a broad-spectrum specific antibody capable of recognizing a plurality of phenothiazine medicaments can be obtained when the artificial antigen is used for immunizing the animal as an immunogen, and is used for quickly realizing immunoassay and screening to improve the detection efficiency, shorten the detection time and lower the detection cost.

Description

technical field [0001] The invention relates to the field of food safety, in particular to a family-specific hapten, artificial antigen, and monoclonal antibody of phenothiazine drugs, and a preparation method and application thereof. Background technique [0002] As a class of sedatives, phenothiazines are mainly used to relieve abnormal agitation of animals, achieve sedation, chemical stabilization and cooperate with anesthesia. The common varieties of such drugs are chlorpromazine, promethazine, acepromazine, and perphenazine. , fluphenazine, thioridazine. In recent years, some unscrupulous feed merchants or breeders have randomly added phenothiazine drugs to the feed in pursuit of high feed conversion rate and high profit, so as to reduce the activity of animals and thus play a role in fattening. Used in the process of animal transportation, it can reduce the maintenance needs of animals and reduce the loss of weight and mortality on the way. However, these drugs are s...

Claims

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Application Information

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IPC IPC(8): C07D279/22C07K14/765C07K14/77C07K1/107C07K16/44G01N33/577
CPCC07D279/22C07K14/765C07K14/77C07K16/44G01N21/31G01N33/531G01N33/577
Inventor 王建平刘静张会彩高宝龙
Owner HEBEI AGRICULTURAL UNIV.
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