Simultaneous determination of four different antibiotic residues in honey by chemiluminescence multianalyte chip immunoassays

We are presenting the first method for identification and quantification of antibiotic derivatives in honey samples using regenerable antigen microarrays in combination with an automated flow injection system. The scheme is based on an indirect competitive immunoassay format using monoclonal antibodies bound to the surface of the microarray. The surface of glass slides was coated with epoxy-activated poly(ethylene glycol) and enables direct immobilization of the antibiotic derivatives. The antigen/antibody interaction on the surface of the chip can be detected by chemiluminescence (CL) read-out via CCD camera. The method allows for fast analysis of the four analytes simultaneously and without purification or extraction. An effective data evaluation method also was developed to warrant unambiguous identification of the spots and to establish grey levels of CL intensities. The software developed enables fast and automated processing of the CL images. Dose-response curves were obtained for the derivatives of enrofloxacin, sulfadiazine, sulfamethazine and streptomycin. Spiking experiments revealed adequate recoveries within the dynamic ranges of the calibration curves of enrofloxacin (92%+/- 6%), sulfamethazine (130%+/- 21%), sulfadiazine (89%+/- 20%) and streptomycin (93%+/- 4%).