A fully automated stand-alone flow injection immunoanalysis (FIIA) device for the determination of cephalexin in milk is developed with a main focus on the investigation of the influence of the sample matrix. The system is based on principles of flow-through immunoassays and on sequential addition of the assay components to an immunoreactor. Protein G is immobilised on the surface of the immunoreactor serving as affinity matrix for the polyclonal anti-cephalexin antibodies. A cephalexin–alkaline phosphatase conjugate is mixed with the analyte-containing sample and binds in a competitve manner to the corresponding antibodies in the immunoreactor. After substrate addition enzymatically generated p-aminophenol is detected at a carbon electrode at +150 mV vs. Ag/AgCl. One assay cycle takes 16 min including regeneration of the immunoreactor. The large excess of protein G allows for more than 150 regenerations without significant loss of signal height. Due to the high specificity of the anti-cephalexin antibodies, other -lactam antibiotics like penicillin, amoxicillin and cloxacillin do not interfere in the measurements, even when added at 10 mg l–1. To deactivate alkaline phosphatase present in milk, samples are heat-treated for 3 min prior to measurements. Cephalexin recoveries from two milk samples are 90 and 110%. The detection limit in milk is 1 µg l–1 (mean relative standard deviation of 3%), less than the maximum residue level of 4 µg per kg milk fixed for some -lactam antibiotics in the European Union. The device is suitable for fast quantitative data generation from consecutively measured samples and thus adds to analytical screening methods.
Meyer, U. J., Zhi, Z. L., Loomans, E., Spener, F., & Meusel, F. (1999). Automated stand-alone flow injection immunoanalysis system for the determination of cephalexin in milk. The Analyst, 124(11), 1605-1610. https://doi.org/10.1039/a907121k