2.6. Fluorescence Polarization Assay

We performed the FPA in 171 serum (93 control and 78 field samples) and 119 milk (41 control and 78 field samples) samples. We used a commercial kit and an FP reader, following manufacturer’s instructions. For serum samples, 20 μL of each sample and control were added to 10 mm × 75 mm borosilicate tubes containing one milliliter of FPA buffer and incubated for 15 min to 30 min at room temperature, after which basal fluorescence was measured. Next, 10 μL of FITC-labeled antigen were added to the tubes and incubated for 5 min at room temperature to obtain millipolarization (mP) values of samples and controls. We followed this procedure for milk samples, using 40 μL of untreated or citric acid-treated samples as explained above.