Development and optimization of a fluorescence polarization immunoassay for orbifloxacin in milk

Abstract

A novel monoclonal antibody for orbifloxacin (ORB) was produced for the first time and used to develop a homogeneous fluorescence polarization immunoassay (FPIA) for ORB determination in milk. Three coating antigens and six fluorescent tracers were prepared, and evaluated by ELISA and FPIA methods, respectively. Heterogeneous tracer, LOM-BDF, was selected to develop the FPIA, providing high sensitivity and stability. Moreover, a new optimization strategy for tracer and Ab concentration was proposed by integrating Z′ factor into checkerboard titration, and Z′ factor >0.8 was considered as a prerequisite to ensure the robustness of quantitative FPIA. The optimized FPIA provided a detection limit of 3.9 ng mL⁻¹ and IC₅₀ of 24.5 ng mL⁻¹ with an expected Z′ factor of 0.81. Saturated (NH₄)₂SO₄ precipitation was applied for milk sample pre-treatment due to high tolerance to elevated-ionic strength in the FPIA. Mean recoveries of fortified milk ranged from 74.3 to 112% at the adding levels of 10, 20 and 40 ng mL⁻¹. The results indicate that the developed FPIA is efficient and especially suitable for fast screening of ORB in milk.