Simultaneous separate and group determination of tylosin and tilmicosin in foodstuffs using single antibody-based immunoassay

An approach to modify the specificity of competitive indirect ELISA for determination of veterinary macrolides tylosin (TYL) and tilmicosin (TMN) using different structural design of coating antigen is described. The homologous and heterologous assay formats using rabbit antiserum against BSA–TYL conjugate were developed. The first format allowed selective determination of TYL (cross-reactivity for TMN was negligible). Heterologous hapten desmycosin (DMN) based conjugate being immobilised on the plates changed the specificity to group recognition (cross-reactivity for TMN was 103.4%). Using two coating antigens but single antibody and the same TYL standard solutions the described tandem test was capable for simultaneous determination of the respective analytes with their possible differentiation. The detection limit of assay that was far below the current MRLs and achieved 0.07 ng mL−1 for TYL and 0.14 ng mL−1 for TMN allowed to minimise matrix effect by sample dilution. The ELISAs of antibiotics in milk, eggs, honey and chicken muscle were optimised and showed acceptable recovery rate. A range of foodstuff samples was analysed using the developed tandem test.