Fluorescence Polarization Assay (FPA)
FPA was performed in duplicates using Brucella FPA test kit (Ellie LLC, USA) according to the manufacturer’s instruction. Reagents were allowed to come to room temperature. The test was performed by mixing 20 μL of serum, positive and negative controls with 1 mL of sample diluent and incubated at room temperature for 5 minutes. Blank readings of all samples and controls were taken using the FPA device (Brucella FPA®, Diachemix, LLC, USA), and the millipolarization (mP) units were recorded. This was followed by addition of 10 μL of the tracer to each tube mixing gently and incubating for 3 minutes, after which a second reading was obtained. For the samples, the change in mP was determined by deducting the negative control mP from each sample mP (based on the mean of the duplicates). The ΔmP values ≤ 10 were considered as negative, between 10 to 20 were considered as suspect, and values > 20 were considered as positive. A typical threshold level for the absolute mP is above 95 mP units.