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.