Preparation of Artificial Antigen and Development of IgY-Based Indirect Competitive ELISA for the Detection of Kanamycin
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Preparation of Artificial Antigen and Development of IgY-Based Indirect Competitive ELISA for the Detection of Kanamycin Residues Jinxin He 1 & Yuan Wang 1 & Xiaoying Zhang 1
Received: 13 March 2015 / Accepted: 29 June 2015 # Springer Science+Business Media New York 2015
Abstract The present study was aimed to evaluate the feasibility to use chicken egg yolk antibodies (IgY) for the detection of Kanamycin (Kana) residues in food stuffs. Bovine serum albumin (BSA) was conjugated with Kana by carbodiimide method, and the laying chickens were immunized with Kana-BSA conjugate. PEG-6000 method was employed to extract IgY. The peak titer of anti-Kana IgY was 1:256,000 after the fifth booster immunization. The optimal dilution for anti-Kana IgY was 1:25,000 to obtain optical density (OD) of 1.0 at 2 μg/mL of OVA-Kana coating concentration. The indirect competitive ELISA (ic-ELISA) showed that the IC50 value of anti-Kana IgY was 4.48 ng/ mL and the regression curve equation was y =−15.62x+ 60.17 (R2 =0.98, n=3). The ic-ELISA showed a lower crossreactivity (less than 0.01 %) with other drugs (except gentamicin—1.91 %). Recoveries from milk, pork, and chicken powder samples were in the range of 82.02 to 98.20 %, with relative standard deviation lower than 5.13 %. Our results indicate that generated anti-Kana IgY can be used in routine screening analysis of Kana residues in food samples.
Keywords Kanamycin . Egg yolk antibody (IgY) . ELISA . Drug residues
* Xiaoying Zhang [email protected] 1
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
Introduction Kanamycin (Kana) is an aminoglycoside antibiotic produced by Streptomyces kanamyceticus. Kana has been widely used in animal husbandry for disease management and growth promotion. However, the residual Kana present in animal-derived food causes health hazards including the neurotoxicity and renal toxicity, which could lead to vestibular and cochlear damage as well as kidney dysfunction (Luft et al. 1987). Thus, the Kana residue in animal sourced food products has become a potential threat for human health. In order to deal with this health-related complications, the strict regulations for antibiotic usage and permissible residual limits in food stuffs have been established in Europe (EMEA/MRL/886/03-FINAL 2003). In the past decades, various methods have been reported for the detection of Kana residues such as microbiological agar diffusion (M-agar) assay (Maitra et al. 1979), gas chromatography (GC) (Isoherranen and Soback 1999), and capillary electrophoresis (CE) (Long et al. 2003), high-performance liquid chromatography (HPLC) (Blanchaert et al. 2013), electrochemical immunosensor (Wei et al. 2012), electrochemical aptasensor (Zhou et al. 2015). These are often complex and require tedious sample pretreatment steps. The enzyme-linked immunosorbent assay (ELISA) has been proven for simple sample pretreatment with high sensitivity and selectivity (Chen et al. 2008). Antibody extraction from the eggs of immunized hens has been
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