Time-resolved fluoroimmunoassay for quantitative determination of tylosin and tilmicosin in edible animal tissues

Abstract

To quantitatively determine tylosin and tilmicosin in edible animal tissues, a time-resolved fluoroimmunoassay (TRFIA) has been developed and validated. For this purpose, desmycosin-O-carboxymethoxylamine-BSA was fixed onto microtiter plates, standards and samples were loaded and, finally, diluted europium-labeled anti-tylosin antibodies were added. Results show that the limit of detection for tylosin was 0.03 ng mL −1 and that for tilmicosin was 0.05 ng mL −1 . The recoveries were 73.6% to 120.5%, with coefficients of variation below 15.6% in various biological matrices spiked with tylosin and tilmicosin at concentrations of 50-200 ng g −1 . There was good correlation (R 2 >0.99) between the TRFIA, an enzyme-linked immunosorbent assay and high performance liquid chromatography data. In conclusion, the new TRFIA is applicable to the detection of tylosin and tilmicosin and is an effective and economical method that will enable high-throughput sample screening. The method is expected to be widely applicable.

Development of Time-Resolved Fluorescence Immunochromatographic Assays for Simultaneously Detecting Tylosin and Tilmicosin in Milk in Group-Screening Manner

“…Time-resolved fluoroimmunoassay (TRFIA) has higher sensitivity than most of the conventional methods, while they decrease the matrix interference in the assay [26,30]. An Eu 3+ fluorescent lanthanide-based TRFIA for detecting T&T in edible tissues is described, which offers cost and efficiency advantages, as well as simplified analysis steps [31]. Li et al developed an LFIA based on the antibody-labeled time-resolved fluorescent microspheres (TRFM) as tracers; the cutoff values for T&T in milk were 2 ng/mL and 4 ng/mL, respectively [32].…”

Design of Novel Haptens and Development of Monoclonal Antibody-Based Immunoassays for the Simultaneous Detection of Tylosin and Tilmicosin in Milk and Water Samples

“…Tylosin is a macrolide antibiotic obtained from the culture fluid of Streptomyces fradiae and has a wide antibacterial spectrum against gram-positive organisms, selected gram-negative organisms, as well as mycoplasma, Rickettsia, and Chlamydia [1,2]. Tilmicosin is the semisynthetic derivative of tylosin ( Figure 1), which has a similar antibacterial spectrum to tylosin [3]. Furthermore, tilmicosin has a better antibiosis effect than tylosin against gram-negative organisms, mycoplasma, pasteurella, and can be used to control mycoplasma avian disease and mastitis of lactating cows [4][5][6].…”

Simultaneous detection of tylosin and tilmicosin in honey using a novel immunoassay and immunochromatographic strip based on an innovative hapten

“…Several methods have been developed for monitoring TYL and TIM residues, such as fluorescence immunoassay (Bang-Ce, Songyang, Peng, & Xiao-Hong, 2008;Su et al,. 2011;Wei et al, 2013), microbiological assay (Litterio, Calvinho, Flores, Tarabla, & Boggio, 2007;Nouws et al, 1999;Pol-Hofstad, Driessen-Van Lankveld, Tomassen, De Jong, & Van Egmond, 2008), electrochemical enzyme-linked immunosorbent assay (ELISA) (Ammida et al, 2004;Draisci et al, 2001), thin-layer chromatography coupled to microbiological detection (Vincent Gizzi, Holst, Jong, & Michard, 2007), high-performance liquid chromatography (HPLC) (Clark, Dowling, & Boison, 2009;Prats, Francesch, Arboix, & Perez, 2002;Zheng et al, 2011), and HPLC coupled with mass spectrometry (HPLC-MS) (Juan, Moltó, Mañes, & Font, 2010;Nozal Nalda et al, 2006;Thompson, Noot, Calvert, & Pernal, 2003). However, most of these assays are time consuming and require expensive equipment, specialized technical personnel, and complicated sample pre-treatment and, thus, are unsuitable for extensive screening and field detection.…”