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Fast immunological detection method for directly detecting furaltadone metablolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

A technology of furaltadone and rapid immunization, which is applied in the direction of immunoglobulin, chemical instruments and methods, biological testing, etc., to achieve the effect of long cycle, strong professionalism and high cost

Active Publication Date: 2014-07-30
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 that the traditional AMOZ immunoassay technology needs to be derivatized, and provide an AMOZ hapten, artificial antigen and highly specific antibody

Method used

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  • Fast immunological detection method for directly detecting furaltadone metablolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)
  • Fast immunological detection method for directly detecting furaltadone metablolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)
  • Fast immunological detection method for directly detecting furaltadone metablolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of hapten HI:

[0033]Take 604mg (3mmol) of AMOZ and 266.5mg (3.6mmol) of glyoxylic acid and dissolve them in 2mL of absolute ethanol respectively, add the ethanol solution of glyoxylic acid to the ethanol solution of AMOZ dropwise during the stirring process, and stir at room temperature overnight. After the reaction was completed, vacuum filtration was performed, and the filter cake was washed twice with 2 mL of ethanol and diethyl ether successively, and the precipitate was collected to obtain 540.2 mg of the target product HI as a white solid, with a yield of 70%. ESI-MS analysis (positive) m / z 258.7 [M+H]+; 1H NMR (600MHz, d5-Pyridine, TMS): δ 7.56 (s, 1H), 4.99-4.97 (m, 1H), 4.16 (t , J=8.8Hz, 1H), 3.79 (dd, J1=6.7, J2=8.8, 1H), 3.68-3.64 (m, 4H), 2.66 (dd, J1=6.1, J2=13.5, 1H), 2.62 ( dd, J1=5.5, J2=13.4, 1H), 2.51-2.46 (m, 4H).

Embodiment 2

[0034] Example 2 Preparation method of hapten HII

[0035] Take 604mg (3mmol) of AMOZ and 353mg (3.6mmol) of maleic anhydride and dissolve them in 2mL of absolute ethanol respectively, add the ethanol solution of maleic anhydride to the ethanol solution of AMOZ dropwise during stirring, and stir the reaction at room temperature overnight. After the reaction was completed, vacuum filtration was performed, and the filter cake was washed twice with 2 mL of ethanol and ether, and the precipitate was collected to obtain 566 mg of the target substance HII as a white solid with a yield of 63%. ESI-MS analysis (negative) m / z 298.6 [M-H]-; 1H NMR (500MHz, d6-DMSO, TMS): δ 6.32 (d, J=2.9Hz, 2H), 4.86–4.78 (m, 1H), 3.74 (t, J = 8.1Hz, 2H), 3.58 (s, J = 4.6 Hz, 4H), 3.43 (t, J = 7.7 Hz, 1H ).

Embodiment 3

[0036] Example 3 Immunogen / coating preparation

[0037] The difference between the preparation of the immunogen and the coating source lies in the structure of the hapten and the type of carrier protein. The immunogen uses the hapten HI, and the carrier protein uses bovine serum albumin (BSA); the coating source uses the hapten HII. The carrier protein is ovalbumin (OVA).

[0038] Methods of Immunogen Preparation. Active ester method: Take 10 mg (0.04 mmol) of hapten HⅠ, dissolve in 500 μL dimethylformamide (DMF), stir and add 20.6 mg (0.1 mmol) 1,3-dicyclohexylcarbodiimide (DCC) and 11.5 mg (0.1 mmol) of N-hydroxysuccinimide (NHS), react overnight at 4°C with magnetic stirring, centrifuge and take the supernatant as liquid A. Weigh 20 mg of carrier protein BSA and dissolve in 2 mL of PBS solution (0.1 mol / L, pH 7.4), stir and dissolve to prepare solution B. Under magnetic stirring, absorb 384 μL of solution A and add it dropwise to solution B, and stir and reac...

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Abstract

The invention discloses a fast immunological detection method for directly detecting furaltadone metablolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The method comprises preparation and application of an AMOZ half antigen, an artificial antigen, an antibody and the like. The AMOZ half antigen has a molecular structure shown as a formula (I) or a formula (II), and the AMOZ artificial antigen has a molecular structure shown as a formula (III) or a formula (IV). An antibody prepared from the antigen can be used for specifically identifying AMOZ. The valence of the antibody is 1:64000, the lowest detection limit is 0.852ng / mL, and the half inhibiting concentration is 15.26ng / mL. The antibody can be directly applied to the detection of AMOZ. By adopting the method, the defect of derivatization of AMOZ in the conventional enzyme linked immunosorbent assay method is overcome; the method is easy, convenient, rapid and low in cost. The antigen and the antibody can be widely applied to residue detection of AMOZ in foods.

Description

technical field [0001] The invention belongs to the technical field of food safety immunoassay, and more specifically relates to a rapid immunoassay method for directly detecting furaltadone metabolite AMOZ. Background technique [0002] AMOZ is a metabolite of the nitrofuran antibiotic furaltadone, an indicator of the illegal use of nitrofuran antibiotics, and a class of potentially teratogenic, carcinogenic and mutagenic substances. The European Union banned the use of nitrofuran antibiotics in food animals in 1995, and listed all nitrofuran antibiotics as prohibited drugs in 1997. my country also promulgated a ban on the use of nitrofuran antibiotics in 2002. ban. After that, the European Union passed the 2003 / 181 / EC Commission Resolution in 2003, establishing the minimum required performance limit of 1 μg / kg for various detection methods of nitrofuran drug metabolites in poultry meat products and aquatic products. Now, in order to ensure food safety, regulatory agencies...

Claims

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Application Information

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IPC IPC(8): G01N33/53C07K14/795C07K14/77C07K14/765C07K16/44
CPCC07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/566
Inventor 孙远明刘凤银徐振林沈玉栋雷红涛王弘杨金易肖治理
Owner SOUTH CHINA AGRI UNIV
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