Fig 1.
Phage lateral-flow assay detecting Norwalk VLPs.
Assay membrane is nitrocellulose (FF80HP, 5x40 mm), sample pad is Fusion 5 (5x20 mm), Absorbent pad is CF5 (10x30 mm). Control line consists of anti-M13 antibodies (0.25 μg/cm) and test line is anti-Norwalk monoclonal antibodies (1.0 μg/cm).
Fig 2.
Detection of Norwalk VLPs using a sandwich ELISA.
A) Antibody pair screening for the detection of Norwalk VLPs; values correspond to the absorbance for a sample for 109 VLPs offered; background absorbance for no VLP sample was subtracted (typical value ~0.1). Red color denotes maximum ΔOD450 observed in the ELISA, yellow lowest, and a smooth color gradient in between. Black box denotes the sandwich pair that was used in LFA. B) Sandwich ELISA detecting Norwalk VLPs where F2 was used as the capturing antibody. For the detection biotinylated F1 and streptavidin HRP (antibody sandwich, closed symbols), or the phage construct (Antibody-NeutrAvidin-AviTag phage) and anti-M13/ HRP conjugate (phage sandwich; open symbols) were used.
Fig 3.
Detection of Norwalk VLPs in lateral-flow assay (LFA).
Norwalk VLPs in 100 μL are detected using anti-Norwalk antibodies in the test line (T); gold nanoparticle (top row) and antibody-phage construct followed by HRP/anti-M13 conjugate (bottom row). Control line (C) consists of anti-mouse antibodies for the gold nanoparticle LFA and anti-M13 antibodies for the phage LFA. Nitrocellulose FF80HP was used as test membrane, Fusion 5 as sample pad and CF5 as absorbent pad. All images were equally gamma-corrected (gamma-correction factor = 0.45) to compensate for contrast lost in the overexposed, scanned images and better represent the naked-eye appearance of the raw strips.
Fig 4.
Evaluation of the Norwalk VLP (NVLP) phage LFA.
A) Intensity plots of representative lateral-flow assay strips detecting Norwalk VLPs in 100 μL PBS, using phage-antibody construct and HRP/anti-M13 antibody conjugate, made in ImageJ software. These intensity plots were analyzed using the gel analysis tool in ImageJ. First peak is for the control line (C) and the second peak is for the test line (T). B) Intensity vs. NVLP concentration in sample for phage LFA (n = 5 or 6, average ± 1 SD). C) Intensity vs. NVLP concentration in sample for gold nanoparticle LFA (n = 3; average ± 1 SD). For B) and C), the intensity of the test line divided by the sum of the intensities of the test and the control lines of each strip were calculated for each strip. The solid lines represent the average value for the no-target control and the dotted lines represent the no-target value plus three times the standard deviation of the background.