Sensitive detection of norovirus using phage nanoparticle reporters in lateral-flow assay

Abstract

Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair.

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.