Preparation of Artificial Antigen and Development of IgY-Based Indirect Competitive ELISA for the Detection of Kanamycin Residues

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 IC 50 value of anti-Kana IgY was 4.48 ng/ mL and the regression curve equation was y =−15.62x+ 60.17 (R 2 =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.

Effect of dietary supplementation with hyperimmunized hen egg yolk powder on diarrhoea incidence and intestinal health of weaned pigs

“…The purity of IgY extract obtained by PEG-6000 method was confirmed by SDS-PAGE under reducing condition ( Figure S1). IgY has a molecular weight of 180 kDa and is composed of two subunits, a heavy chain (70 kDa) and a light chain (25 kDa), which was consistent with a previous study (He et al, 2016). The titres of anti-ETEC IgY were determined by indirect ELISA.…”

Detection of kanamycin and gentamicin residues in animal-derived food using IgY antibody based ic-ELISA and FPIA

“…In order to monitor IgY antibody titre, an indirect ELISA had been performed as previous described (He, Wang, & Zhang, 2015). Firstly, the concentrations of antibodies and coating antigens were optimized by checkerboard titration.…”

“…IgY is usually used to treat rotavirus (Vega, Bok, Saif, Fernandez, & Parreño, 2015), allergic asthma (Wei-xu, Qin, Zhu, Yuan-yi, Li-feng, Zhi-yong, et al, 2014), alimentary tract diseases (Rahman, Van Nguyen, Icatlo Jr, Umeda, & Kodama, 2013), etc. Though chicken IgY antibodies have been widely used in vitro immunological detection and diagnosis, very few reports are available on the applications for antibiotic residue detection (He, Hu, Thirumalai, Schade, Du, & Zhang, 2016;He, Wang, & Zhang, 2016;.…”

Chemiluminescence imaging immunoassay for multiple aminoglycoside antibiotics in cow milk

“…According to the regulation of European Union (European Commission 2010), the maximum residue limits (MRL) for kanamycin (KAN) and streptomycin/dihydrostreptomycin (STR/diSTR) in cow milk are 150 and 200 ng mL À1 , respectively. Besides conventional chromatography methods (Preu et al, 1998;Oertel et al, 2004;Kaufmann & Maden, 2005;Liu et al, 2017), immunoassays have been applied to screen the antibiotics in animal-origin food due to be sensitive, rapid and easy-operated (Verheijen et al, 2000;Abuknesha & Luk, 2005;He et al, 2016;. Besides detecting one target specifically by immunoassay (Gu et al, 2016;Kong et al, 2017), another demand to develop the multiplex assays for more targets is growing concern because of its benefits, such as enhancing sample throughput, shorting assay time and decreasing sample consumption and cost (Zhang et al, 2004).…”