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Development of indirect competitive enzyme-linked immunoassay of colistin for milk and egg analysis

ABSTRACT Polyclonal antibodies were obtained by immunizing rabbits with polymyxin B (PMB) conjugated to bovine serum albumin. Based on the heterologous coating conjugate of PMB with periodate-oxidized gelatin and the resulting antibodies, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect residual colistin (COL), a structural analogue of PMB in livestock products. The developed analysis was characterized by close specificity towards PMB (100%) and COL (88%), sensitivity (IC50 = 5.7 ng/mL), dynamic range (IC20-IC80 = 1.0–80 ng/mL) and the detection limit of COL (IC10 = 0.4 ng/mL). It was possible to quantify the antibiotic in milk and eggs without special and labour-intensive sample pretreatment using a simple 10- and 50-fold dilution of the corresponding samples. The developed analysis system made it possible to record the maximum residue limit of COL in dairy products (50 μg/kg) and eggs (300 μg/kg) with 89–104% recovery of analyte.