Location: Food and Feed Safety ResearchTitle: Development and optimization of a fluorescence polarization immunoassay for orbifloxacin in milk Author
Submitted to: Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2014
Publication Date: 6/7/2014
Publication URL: http://handle.nal.usda.gov/10113/58816
Citation: Mi, T., Liang, X., Ding, L., Zhang, S., Eremin, S.A., Beier, R.C., Shen, J., Wang, Z. 2014. Development and optimization of a fluorescence polarization immunoassay for orbifloxacin in milk. Analytical Methods. 6:3849-3857. Interpretive Summary: A fluorescence polarization immunoassay (FPIA) for determination of orbifloxacin (ORB) in milk was developed and optimized. In an FPIA method, light is measured when a smaller molecule like ORB containing a fluorophore label combines with an antibody. As the fluorophore-labeled ORB rotates in solution, it gives off light, and when ORB binds to an antibody, the type of light drastically changes allowing one to make measurements of amounts of chemical in solution. ORB is in the quinolone family of synthetic broad-spectrum antibiotics. The majority of quinolones in clinical use belong to the subset called fluoroquinolones. They are called fluoroquinolones because they have a fluorine atom attached to the central ring system of the quinolone molecule. Fluoroquinolones have been widely used in humans as well as in food-producing animals to control disease and in animals to improve growth and feed efficiency. Immunoassays make use of antibodies and are used as an analytical tool to analyze for a specific compound. Antibodies are substances that are produced by the immune system in response to foreign substances which enter the body. Once the antibodies to a foreign substance are isolated, they can be used in a method to detect the presence of that foreign substance. A robust assay was developed and resulted in a detection limit of 4.7 ng/mL ORB. The developed FPIA was used to analyze for ORB in spiked milk samples, which required only simple sample pretreatment.
Technical Abstract: A homogeneous microplate-based fluorescence polarization immunoassay (FPIA) for determination of orbifloxacin (ORB) in milk was developed and optimized. A monoclonal antibody of ORB was prepared, and six fluorescent tracers were synthesized from ORB and lomefloxacin (LOM) using three derivatives of fluorescein isothiocyanate (FITC), and they were evaluated and compared by FPIA. The tracer, LOM-BDF, was selected and used to develop the FPIA for ORB because of its high sensitivity at short incubation times. A novel strategy for the optimization of tracer concentration was proposed based on the Z’ factor. A robust assay was developed using a 20 nM concentration of tracer, due to a >0.75 Z’ factor. The developed method resulted in a detection limit of 4.7 ng/mL and a detection range between 7.3 and 90.7 ng/mL with a 0.81 Z’ factor. No significant cross-reactivity was observed with the 16 fluoroquinolones tested except for sparfloxacin (78.3%) and LOM (12.4%). Satisfactory assay sensitivity was obtained using a buffer at pH 8.0, and elevated-ionic strength did not significantly affect the analytical performance. However, more than 10% methanol or 2.5% acetonitrile was not tolerated in the FPIA. The developed FPIA was used to analyze for ORB in spiked milk samples, which only required simple sample pretreatment, and the recoveries of three spiked concentrations at 10, 20, and 40 ng/mL for FPIA were 110, 82.0, and 75.8%, and the coefficients of variation were 27.7, 19.5, and 15.2%, respectively.