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

Analytical Methods Pub Date: 2014-03-10 DOI: 10.1039/C3AY42034E

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.

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