When testing clinical samples on the benchtop, test and control line conjugates were hand spotted prior to sample application. Four Ab pair conditions, of various levels of performance with rNP, were used for clinical sample validation. The test line conjugate was diluted to a final concentration of 0.10% and control line chicken IgY conjugate to 0.15% in 50 mM borate, pH 8.5. First, 1 µL of conjugate mixture was pipetted onto the conjugate pad and allowed to dry at ambient temperature for 10 minutes prior to application of the sample. All samples were diluted 1:25 in sample buffer containing 2.5% BSA and 1% IGEPAL CA-630 in 1x PBS. Samples were incubated on ice for 30 minutes prior to use. Second, 75 µL of each sample diluted in sample buffer was added to the conjugate pad and run at ambient conditions inside a biosafety cabinet for 20 minutes prior to being read in an LFA reader (Axxin, Fairfield, Australia).