Abstract
BackgroundColistin (polymyxin E) is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention, treatment, and growth promotion. However, the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings, and its residue in animal-origin food may also pose serious health hazards to humans. Thus, it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food.ResultsA one-step enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic assay (LFIA) for colistin were developed based on a newly developed monoclonal antibody. The ELISA showed a 50% inhibition value (IC50) of 9.7 ng/mL with assay time less than 60 min, while the LFIA had a strip reader-based detection limit of 0.87 ng/mL in phosphate buffer with assay time less than 15 min. For reducing the non-specific adsorption of colistin onto sample vial, the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy. The spiked recovery experiment showed that the recoveries of colistin from feed, milk and meat samples were in the range of 77.83% to 113.38% with coefficient of variations less than 13% by ELISA analysis and less than 18% by LFIA analysis, respectively. Furthermore, actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis.ConclusionsThe developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.
Highlights
Colistin is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention, treatment, and growth promotion
Antibody preparation and characterization Colistin is a small molecule which cannot elicit animal body to produce a specific antibody, it was covalently linked with bovine serum albumin (BSA) by ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) coupling method to form an immunogen for antibody preparation
The low dosage group led to Half maximal inhibitory concentration (IC50) values in the range of 68 to 133 ng/mL, while the high dosage group resulted in IC50 values of 76 to 149 ng/mL (Additional file 1: Table S1), indicating that the two immunization dosages did not produce significant difference on the affinities of sera antibodies
Summary
A one-step enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic assay (LFIA) for colistin were developed based on a newly developed monoclonal antibody. The ELISA showed a 50% inhibition value (IC50) of 9.7 ng/mL with assay time less than 60 min, while the LFIA had a strip reader-based detection limit of 0.87 ng/mL in phosphate buffer with assay time less than 15 min. The spiked recovery experiment showed that the recoveries of colistin from feed, milk and meat samples were in the range of 77.83% to 113.38% with coefficient of variations less than 13% by ELISA analysis and less than 18% by LFIA analysis, respectively. Actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis
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