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Monoclonal antibody and enzyme linked immunosorbent assay and kit for detecting promethazine drugs

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, which is applied in the field of drug detection and analysis and immunology, can solve the problems of insufficient identification types, large variability and poor stability of polyclonal antibodies, and achieve high accuracy of the method and easy operation. Simple and precise effect

Active Publication Date: 2015-04-22
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN103554056A discloses a phenothiazine drug hapten, which is prepared by reacting 2-chlorophenothiazine with sodium bromoacetate, and the obtained monoclonal antibody can simultaneously recognize chlorpromazine, perphenazine, and promethazine , fluphenazine, acepromazine, thioridazine 6 phenothiazines, the identified types are still not enough
Lowry P et al. (Lowry P., Benchikh M.E., McConnell R.I., Tohill A., Fitzgerald S.P. Development of a highly sensitive, generic polyclonal antibody for the detection of phenothizines [J]. Life sciences. 2010.) used acepromazine and cattle The immunogen is obtained by coupling thyroid protein, and the prepared polyclonal antibody can recognize chlorpromazine, promazine and acepromazine, but the polyclonal antibody has large variability and poor stability

Method used

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  • Monoclonal antibody and enzyme linked immunosorbent assay and kit for detecting promethazine drugs
  • Monoclonal antibody and enzyme linked immunosorbent assay and kit for detecting promethazine drugs
  • Monoclonal antibody and enzyme linked immunosorbent assay and kit for detecting promethazine drugs

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Experimental program
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Effect test

Embodiment 1

[0031] The preparation of embodiment 1 immunogen and coating former

[0032] Immunogen / coating preparation

[0033] Weigh 40 mg of perphenazine and 20 mg of succinic anhydride, dissolve in 10 mL of pyridine, and heat for 4 hours. After adding 20 mL of pre-cooled distilled water, use 2 mol / L HCl to adjust the pH to acidic, add ethyl acetate to extract, repeat 3 times, collect the reaction liquid and evaporate to dryness to obtain the hapten.

[0034] Reconstitute the product from the previous step with 2 mL of N,N-dimethylformamide, add 30 mg of N,N'-dicyclohexylcarbodiimide (DCC) and 20 mg of N-hydroxysuccinimide (NHS), and activate for 24 hours.

[0035] Weigh 140mg of HSA and 200mg of OVA and dissolve them in 15mL of PBS respectively. Under the condition of ice bath, slowly drop the reaction solution from the previous step into the protein solution, and react overnight at 4°C to obtain the immunogen and the coating original, respectively. The reaction process is as follows...

Embodiment 2

[0037] The preparation of embodiment 2 monoclonal antibody

[0038] Preparation of hybridoma cells: refer to "Veterinary Immunology" by Du Nianxing.

[0039] Balb / C mice (purchased from Experimental Animal Center of Hubei Academy of Medical Sciences) were immunized with the immunogen prepared in Example 1. The immunization procedure is as follows: for basic immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant is injected subcutaneously at the back of the neck of the mouse, and then every 15 days, protein emulsion containing 100 μg of immunogen emulsified with Freund’s incomplete adjuvant is injected subcutaneously. Protein emulsion of immunogen for booster immunization. From the third immunization, the tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was detected by indirect ELISA method. Immunization qualified mice (high titer, good ...

Embodiment 3

[0046] The establishment of embodiment 3 indirect competition ELISA detection method

[0047] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0048] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 .12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0049] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0050] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 .12H 2 O 2.9g, KCl 0.2g, Tween 200.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0051] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0052] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0053] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxide 6.4mL...

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Abstract

The invention discloses a specific monoclonal antibody for detecting various promethazine drugs such as anti-chlorpromazine, anti-promazine, anti-perphenazine drugs and an enzyme linked immunosorbent assay and kit for detecting the promethazine drugs. The monoclonal antibody disclosed by the invention is secreted from a hybridoma 3A5 with the preservation number of CCTCC NO: 201354. Compared with the prior art, the monoclonal antibody disclosed by the invention can be used for identifying various promethazine drugs simultaneously. The enzyme linked immunosorbent assay and kit disclosed by the invention have the advantages of being high in detection efficiency and sensitivity, good in precision and accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of drug detection analysis and immunology, and specifically relates to a monoclonal antibody against phenothiazine drugs, and also relates to an ELISA method and a kit for detecting phenothiazine drugs. Background technique [0002] Phenothiazines are a class of drugs that act on the central nervous system and have the mother nucleus structure of thiazepine, which can block dopaminergic neurons D 2 It has the functions of sedative and hypnotic, lowering body temperature, and fattening. Typically used to reduce stress during transport of food producing animals to slaughter. In addition, such drugs are often used as feed additives, which can increase the feed-to-meat ratio of animals; secondly, they also have the effects of lowering body temperature and suppressing emesis. Therefore, many unscrupulous traders add such prohibited drugs at will or do not abide by the withdrawal period, resulting in drug residu...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/543
Inventor 袁宗辉彭大鹏王涓潘源虎王玉莲冯亮刘振利
Owner HUAZHONG AGRI UNIV
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