A highly sensitive and specific polyclonal antibody-based enzyme-linked immunosorbent assay for detection of antibiotic olaquindox in animal feed samples

Abstract

The use of olaquindox (OLA) as an additive in animal feedstuffs has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of OLA in animal feed samples was developed. OLA was activated by N'N-carbonyldiimidazole and coupled with bovine serum albumin (BSA) and ovalbumin (OVA). It was found that the sensitivity and specificity of the two antisera were very similar, with the IC(50) values of 16 ng mL(-1) and 19 ng mL(-1), respectively. Cross-reactivity was less than 35% for four structurally related compounds and no recognition of five other antibiotics was observed. The better antiserum I was selected for further experiments, for example testing stability, solvent effect, accuracy, and precision. The IC(50) value for eight standard curves was in the range 12-18 ng mL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.31 +/- 0.11 ng mL(-1). The ELISA tolerated 5% methanol without significant influence on IC(50) value. The recoveries of spiked OLA in five different animal feed types including auxin, pig complex feed, fish complex feed, broiler concentrated feed, and pig premix feed were in the range 88.3-119.0% and the intra-assay relative standard deviation (RSD) was within 4.7-33.5% (n = 3). The ELISA for unspiked feed samples was confirmed by high-performance liquid chromatography (HPLC), with a high correlation coefficient of 0.9862 (n = 5). The proposed ELISA could be a feasible quantitative/screening method for OLA analysis in feed samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput, and low expense.

Multiresidue determination of antibiotics in feed and fish samples for food safety evaluation. Comparison of immunoassay vs LC-MS-MS

“…In this sense, the European Union has established maximum residue limits (MRLs) (Official Journal of the European Communities, 1990) in order to guarantee the safety of aquaculture produced marine fish; for total sulfonamide and tetracycline residues 100 ng g À1 was the set level. Moreover, additives authorisation and inclusions in feed materials are also regulated (Official Journal of European Communities, 1970;Zhao, He, Pu, & Deng, 2008). Therefore, a good analytical method is required for quality control of antibiotics in feeds.…”

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for monitoring quinoxaline-2-carboxylic acid in the edible tissues of animals

“…Synthesis of antigens QCA, CBQCA and CPQCA were conjugated to BSA with reference to the previous method (Duan and Yuan 2001;Situ and Elliott 2005;Zhao et al 2008) to prepare the immunising conjugates and were conjugated to OVA to prepare the coating conjugates.…”

Development and Validation of an Lc-Dad Method for the Routine Analysis of Residual Quinolones in Fish Edible Tissue and Fish Feed. Application to Farmed Gilthead Sea Bream Following Dietary Administration

“…Animal feed is a very complex matrix, with different composition for each species and even different starting materials for each production batch, yielding feed samples with different characteristics. Athough there are methods reported for sulfonamides and oxytetracycline, [10] for penicillin V and amoxicillin, [26] for tetracycline, [27] for sulfadiazine and trimethoprim, [28] for oxytetracycline, [29] for zinc bacitracin, polymyxin B, oxytetracycline, and sulfacetamide, [30] for oxytetracycline, tetracycline, and chloramphenicol, [31] and for olaquindox, [32] only a limited number of papers can be found on the determination of quinolones in fish feed. Sarafloxacin was mainly investigated in premix and fish feed; however, the described technique is applicable to several other quinolones: difloxacin, temafloxacin, norfloxacin, and ciprofloxacin.…”