Flow cytometric immunoassay for sulfonamides in raw milk Original Research Article

Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this automated FCI, a previously developed biotinylated multi-sulfonamide mutant antibody (M.3.4) was applied in combination with fluorescent beads, directly coated with a sulfathiazole derivative, and streptavidin–phycoerythrin (SAPE) for the detection. With this FCI, at least 11 different sulfonamides could be detected (more than 50% inhibition at the 100 ng mL−1 level) and, after an incubation of 1 h, measurements were rapid (10 s per sample). For the application with raw milk, a 96-well microplate-based filtration step was included into the protocol to remove disturbing milk fat particles. Because of differences in sensitivity towards different sulfonamides, the FCI was considered and validated as a qualitative screening assay. For sulfadoxine, the most applied sulfonamide in Dutch dairy cattle, the detection capability (CCβ) was <50 μg L−1 and this level seems feasible for five other sulfonamides. For sulfadiazine, the CCβ was <200 μg L−1 and this level seems feasible for four other sulfonamides. A major advantage of the applied xMAP-technology, with its 100 different color-coded bead sets, is the possibility to develop multiplex immunoassays for the simultaneous detection of several antibiotics.