Development of a Generic Enzyme-Immunoassay for the Detection of Fluoro(quinolone)-Residues in Foodstuffs Based on a Highly Sensitive Monoclonal Antibody

An enzyme immunoassay (EIA) was developed based on a generic and highly sensitive monoclonal antibody (mAb) enabling simultaneous detection of all fluoroquinolone/quinolone antibiotics (FQs) approved for the treatment of food-producing animals. To generate the group-specific antibody, norfloxacin was conjugated to glucose oxidase (GlcOX) by one-step carbodiimide method and used as immunogen. For the establishment and optimization of a direct, competitive EIA, three different FQ-horseradish peroxidase (HRP) conjugates were generated. Best results were obtained by using a hapten-heterologous conjugate (clinafloxacin-HRP) prepared by applying a simple and highly efficient periodate method. Under optimized conditions, the assay showed a very high sensitivity, e.g., the detection limit for norfloxacin was 0.02 ng/mL. Furthermore, due to the high affinity of the employed mAb, all FQs approved within the EU for the treatment of food-producing animals can be detected well below their maximum residue levels (MRLs) with IC 50 values ranging from 0.16 ng/mL (marbofloxacin) to 3.82 ng/mL (sarafloxacin). Additionally, the established EIA showed cross-reactivity with 23 other FQs and IC 50 values ranged from 0.05 ng/mL (rufloxacin) to 73.2 ng/mL (pradofloxacin). Thus, the established EIA could be potentially applied both for the detection of approved and illegally used quinolones in food. To demonstrate the applicability of the assay, artificially contaminated milk, meat, fish, and shrimp samples were analyzed; mean recovery rates were 88.9%, 76.2%, 74.4%, and 77.9%, respectively. These data demonstrate that the developed EIA is suited for the rapid and sensitive monitoring of FQs residues in animal-derived food.