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Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder

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Abstract

A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9′-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL-1 and the IC50 (50% inhibition) value was 164.7 ng mL-1. The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography–mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure.

Figure Performance comparison of tree tracers and two antibodies. (a), antibody A; (b) antibody B

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Abbreviations

BSA:

Bovine serum albumin

CAAT:

2-chloro-4,6-diamino-1,3,5-triazine

CAC:

Codex Alimentarius Commission

CR:

Cross-reactivity

DCC:

N,N′-dicyclohexylcarbodiimide

DMF:

N,N-dimethylformamide

EDA:

Ethylenediamine

EDC:

1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride

EDF:

1-(2-aminoethyl)-3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl)thiourea

ELISA:

Enzyme-linked immunosorbent assay

FITC:

Fluorescein isothiocyanate isomer I

FPIA:

Fluorescence polarization immunoassay

HPLC:

High-performance liquid chromatography

LOD:

Limit of detection

MS:

Mass spectrometry

NHS:

N-hydroxysuccinimide

TLC:

Thin-layer chromatography

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Acknowledgements

This work was supported by the National Department Public Benefit Research Foundation (201003008-08) and China Guangdong Provincial Science and Technology Projects (cgzhzd1005, 2009B040500002).

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Authors and Affiliations

  1. The Institute of Food Quality and Safety, South China Agricultural University, Guangzhou, 510642, China

    Qiang Wang, Yuan-Ming Sun, Zhen-Feng Li, Hui Liu, Zhen-Lin Xu, Yu-Dong Shen & Hong-Tao Lei

  2. Institute of Agri-Food and Land Use, Queen’s University Belfast, Belfast, BT9 5AG, UK

    Simon A. Haughey

  3. Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119998, Russia

    Sergei A. Eremin

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  1. Qiang Wang
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Correspondence to Hong-Tao Lei.

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Wang, Q., Haughey, S.A., Sun, YM. et al. Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder. Anal Bioanal Chem 399, 2275–2284 (2011). https://doi.org/10.1007/s00216-010-4599-2

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