Application of polarization fluorescence immunoassay for the determination of flunixin in milk

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The technique of polarization fluorescence immunoassay for the detection of residual amounts of nonsteroidal anti-inflammatory drug flunixin used for prophylaxis and treatment in veterinary medicine and animal husbandry was developed. The optimal analysis time was 15 min taking into account sample preparation, the linear range was 20-5000 ng/mL, and the limit of detection was 2 ng/mL. High selectivity of immunoreagents with respect to the antigen under study was shown; cross-reactivity coefficients with preparations having structural similarity to flunixin did not exceed 0.01 %. The method of flunixin determination in milk was tested by the injected-found method, it was shown that the application of the developed method of sample preparation allows effective detection of the investigated antigen within the linear range. These results indicate the possibility of using the developed technique with the use of polyclonal antibodies for express and sensitive determination of flunixin in milk.

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作者简介

D. Arutyunyan

Lomonosov Moscow State University

Email: eremin_sergei@hotmail.com

Faculty of Chemistry

俄罗斯联邦, Leninskie gory, 1, building 3, Moscow 119991

L. Mukhametova

Lomonosov Moscow State University

Email: eremin_sergei@hotmail.com

Faculty of Chemistry

俄罗斯联邦, Leninskie gory, 1, building 3, Moscow 119991

I. Shanin

Lomonosov Moscow State University

Email: eremin_sergei@hotmail.com

Faculty of Chemistry

俄罗斯联邦, Leninskie gory, 1, building 3, Moscow 119991

S. Kondakov

Lomonosov Moscow State University

Email: eremin_sergei@hotmail.com

Faculty of Chemistry

俄罗斯联邦, Leninskie gory, 1, building 3, Moscow 119991

S. Eremin

Lomonosov Moscow State University

编辑信件的主要联系方式.
Email: eremin_sergei@hotmail.com

Faculty of Chemistry

俄罗斯联邦, Leninskie gory, 1, building 3, Moscow 119991

参考

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2. Fig. 1. Scheme of synthesis of ethylenediaminethiocarbamyl fluorescein (EDF) and its conjugate with flunixin (Flu-EDF).

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3. Fig. 2. (a) Change in fluorescence polarization signal upon binding of Flu-EDF, Flu-BDF and Flu-GDF conjugates (final concentration 2.5 nM) with antiserum antiFlu-1 (dilution 800 times). (b) Dependence of fluorescence polarization on dilution of antisera antiFlu-1 (●), antiFlu-2 (○), antiFlu-3 (▼) and non-immune rabbit serum 4 (♦) upon binding to the Flu-EDF conjugate. n = 3, 25°C, pH 8.5.

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4. Fig. 3. Kinetics of binding of the Flu-EDF conjugate (final concentration 2.5 nM) and polyclonal antibodies to flunixin antiFlu-1 (dilution 1 : 800). n = 3.

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5. Fig. 4. Optimization of the conditions of the FPIA method for determining flunixin. Normalized calibration dependences, (a) obtained at different concentrations of Flu-EDF 2.5 (1) and 1.25 (2) nM, dilution of antibodies antiFlu 400 times; (b) for antisera with a dilution of 500 times antiFlu-1 (1), antiFlu-2 (2) and antiFlu-3 (3). n = 3.

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6. Fig. 5. (a) Calibration dependence of fluorescence polarization signal on flunixin concentration, (b) detection region at Flu-EDF concentration of 1.25 nM and 800-fold dilution of anti-Flu-1 antibodies. n = 3.

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7. Fig. 6. Structural formulas of cross-reactants: (a) – fluoxetine, (b) – indoxacarb, (c) – nicotinic acid, (d) – nimesulide, (d) – celecoxib, (e) – diclofenac.

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8. Fig. 7. Determination of matrix effect in milk supernatant by polarization fluorescence immunoassay. Flu-EDF (2.5 nM), anti-Flu-1 (800-fold dilution), pH 8.5, 25°C.

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