Calculation of ELISA cut-off value. The optical density (OD) of three negative control sera was determined at serum dilutions of 1:100 to 1:512,000. E.g., for the dilution of 1:100: negative control 1 = 0.011, negative control 2 = 0.008, negative control 3 = 0.044. The mean (x) and standard deviation (SD) of these three negative sera were calculated, viz. x= 0.021, SD = 0.02. The cut-off value was taken as x+ 3SD, in this example 0.083. Thus, an OD value above this value was considered positive. The antibody titres for test samples 1 and 2 were determined to be 1:100 and 1:800, respectively (i.e. where the line interpolated between the dots crosses the red cut-off threshold), but the titre for sample 3 was zero. Thus, test sample 1 and 2 are considered positive.