title: Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder.
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 IC(50) (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.
authors.affiliation: The Institute of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.forename: Simon A
authors.initials: SA
authors.lastname: Haughey
authors.forename: Yuan-Ming
authors.initials: YM
authors.lastname: Sun
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Eremin
authors.forename: Zhen-Feng
authors.initials: ZF
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Zhen-Lin
authors.initials: ZL
authors.lastname: Xu
authors.forename: Yu-Dong
authors.initials: YD
authors.lastname: Shen
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Lei
mesh_terms: D000818:Animals; D002417:Cattle; D003611:Dairy Products / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis*; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D008892:Milk / Q000737:chemistry; D011208:Powders / Q000032:analysis; D014227:Triazines / Q000032:analysis*
chemical_list: D011208:Powders; D014227:Triazines; C011907:melamine