Microfluidic Chip-Based Immunoassay for Reliable Detection of Cloxacillin in Poultry

By combining a chemiluminescence immunoassay with microfluidic chip technology, a simple assay was established for the sensitive detection of cloxacillin in poultry samples. The chip used in this approach was composed of an upper microchannel layer and a lower flat base layer. Each of the formed microchannels has a volume of 18 mu L, thus enabling a considerable reduction of reagent consumption as well as sample size needed for analysis by the indirect competitive immunoassay. To obtain labeled antigens for coating, the active ester method was employed to couple cloxacillin to glucose oxidase (GlcOx). The immobilized GlcOx-cloxacillin conjugate on the base layer competed with residues of cloxacillin in samples for binding sites of the monoclonal antibodies (mAb) against cloxacillin. Horseradish peroxidase-labeled anti-mouse IgG antibodies and chemiluminescent substrate were used for signal generation. Under optimized conditions, the IC50 and the limit of detection (LOD) of the assay were at 96.5 +/- 9.37 and 0.92 +/- 0.07 ng/mL, respectively, and thus well below the maximum residue level of 300 ng/mL set by the European Commission. Recoveries for spiked chicken and duck samples were in the range from 94 to 118 % with relative standard deviations lower than 11 %. The results demonstrate that the microfluidic chip-based immunoassay can be used as a rapid and reliable platform for determination of cloxacillin in poultry.