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Development of an indirect competitive immunoassay for determination of L-hydroxyproline in milk

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L-hydroxyproline can be used as a marker for hydrolysed leather protein. This is the first study reporting an indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of L-hydroxyproline in milk. A hapten for L-hydroxyproline was synthesised and used to produce a monoclonal antibody. The antibody was specific to the hapten and showed low crossreactivity to parent L-hydroxyproline. After evaluation of different coating antigen/antibody combinations, a heterologous ELISA was developed to determine L-hydroxyproline in milk. Milk samples were hydrolysed with concentrated sulfuric acid in microwave oven to release free L-hydroxyproline that was derivatised with p-hydroxybenzaldehyde. The p-hydroxyphenyl L-hydroxyproline was determined by the ELISA, and the limit of detection calculated as L-hydroxyproline was 0.04 µg/mL. The recoveries of L-hydroxyproline from fortified milk ranged from 88.6% to 102.5%, with coefficients of variation ranging from 4.1% to 9.7%. Therefore, ELISA could be used as a rapid method to monitor the presence of L-hydroxyproline in milk.
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Keywords: ELISA; L-hydroxyproline; milk; monoclonal antibody; p-hydroxybenzaldehyde

Document Type: Research Article

Affiliations: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, China

Publication date: April 3, 2014

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