Immunosensor Based on Long-Period Fiber Gratings for Detection of Viruses Causing Gastroenteritis

Since the norovirus is the main cause of acute gastroenteritis all over the world, its fast detection is crucial in medical diagnostics. In this work, a rapid, sensitive, and selective optical fiber biosensor for the detection of norovirus virus-like particles (VLPs) is reported. The sensor is based on highly sensitive long-period fiber gratings (LPFGs) coated with antibodies against the main coat protein of the norovirus. Several modification methods were verified to obtain reliable immobilization of protein receptors on the LPFG surface. We were able to detect 1 ng/mL norovirus VLPs in a 40-min assay in a label-free manner. Thanks to the application of an optical fiber as the sensor, there is a possibility to increase the user’s safety by separating the measurement point from the signal processing setup. Moreover, our sensor is small and light, and the proposed assay is straightforward. The designed LPFG-based biosensor could be applied in both fast norovirus detection and in vaccine testing.

The GII.4 NoV 2012 variant (Hu/GII.4/Sydney/NSW0514/2012/AU) Vp1 DNA coding sequence (1637 bp) was synthesized by Gene Art Gene Synthesis (Thermo Fisher Scientific), and cloned into the baculovirus transfer vector pFastBac1 (Invitrogen, Carlsbad, CA) using EcoRI and NotI restriction sites. For generating a recombinant bacmid DNA, DH10Bac TM competent Escherichia coli cells were transformed with the pFastBac1-VP1 transfer vector. Recombinant baculovirus (rBV-VP1) was assembled in Spodoptera frugiperda (Sf9) insect cells transfected with bacmid DNA. Recombinant baculoviruses containing the vp1 gene were identified by the immunoperoxidase monolayer assay (IPMA) and by PCR reaction with specific primers based on the pUC/M13 forward and reverse primers.
For the production of NoV VLPs, Sf9 cells in suspension culture were infected with recombinant baculovirus at an MOI of 3 and harvested 60 h post-infection. Supernatant was collected after centrifugation in a microfuge at 8500 rpm for 10 min.
NoV VLPs were concentrated by ultracentrifugation (Beckman SW-28 rotor) at 82,000 × g for 1 h at 4 °C, and pellet was resuspended in NTA buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.8, 0.1% v/v Tween 20). One milliliter of concentrated NoV VLPs were loaded onto a 10%-60% discontinuous sucrose gradient and ultracentrifugated at 82,000 × g for 1.5 h at 4 °C (SW 41 Ti Beckman rotor) as described before [1,2]. Fractions (1 mL) were collected from the sucrose gradients with thin pipette tips to determine the density and composition of the NoV VLPs samples. Samples were analyzed by SDS/PAGE, Western blotting and ELISA. Purified Nov VLPs were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM).

SDS-PAGE
For SDS-PAGE, proteins were dissolved in the denaturing loading buffer and heated for 10 min in 70 °C. Aliquots of 35 µL of each sample were loaded on 10%-20 % precast WedgeWell Gel (Thermo Fisher Scientific) and run at a constant voltage of 165 V. Polyacrylamide gel was stained with Coomassie brilliant blue solution for 30 min in RT and destained o/n in RT.

Western blot analysis
After semi-dry immunotransfer, PVDF membranes were blocked for 1 h in a 5% semi-skimmed milk solution and incubated overnight with primary antibodies (rabbit anti-Norovirus antibodies (ab92976) to Norovirus GII.4 Abcam) 1:100 in 5% milk solution. The next day, membranes were washed and incubated with secondary goat-anti rabbit-HRP antibodies (1:3000, Jackson) or with secondary goatanti rabbit-AP antibodies (1:2000, Santa Cruze) solution. Reaction was developed with ECL Plus Chemiluminescent substrate for horseradish peroxidase (Thermo Scientific) or BCIP/NBT stock solution for alkaline phosphatase (Sigma Aldrich).

Dynamic light scattering
Particle sizing was performed using a Malvern Instrument Zeta Sizer NanoS dynamic light scattering instrument (Malvern, Worcestershire, UK). Measurements were taken in water at 25 °C. Sample solutions were passed through 0.45-μm filters and equilibrated at room temperature for 10 min prior to measuring; each measurement duration was 10 s. The results were calculated as the average of six consecutive measurements.

Transmission electron microscopy
The NoV VLPs purified from the supernatant of infected Sf9 cells were diluted in TM buffer (Tris and MgCl2 buffer). Particles were adsorbed onto carbon-coated grids, stained with 2% uranyl acetate, and examined immediately in a Philips CM 100 electron microscope.

ELISA
A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µL/well of 14 samples originated from sequential fractions of 0.5 mL after ultracentrifugation. Each sample was tentimes diluted in 1× PBS. The coated plate was incubated o/n at 4 °C. Protein samples were discarded, the plate was washed 4 × 5 min with 200 µL/well of washing buffer (PBS/0.05% Tween 20) and blocked for 1 h -37 °C/45 min -RT with 250 µL/well of blocking buffer. After 1.5 h of incubation, blocking buffer was discarded and the plate was washed as above. Then, 100 µL/well of primary antibody solution (rabbit anti-Norovirus antibodies (ab92976) to Norovirus GII.4, Abcam) diluted 1:1000 in 3% milk/PBST was added and incubated 1 h, 37 °C. Antibody solution was discarded and the plate was washed as previously. Then, 100 µL/well of secondary antibodies solution (Goat anti-mouse, Jackson ImmunoResearch) diluted 1:1200 in 3% milk/PBST was added and incubated 1 h, RT. Finally, after the last plate washing step (6 × 5 min with 200 µL/well), 100 µL/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), incubated in darkness until the desired blue color developed, and stopped by adding 50 µL of 0.5 M sulfuric acid to each well. Absorbance was measured at 450 nm.

RHDV VLP
The negative control Rabbit hemorrhagic disease virus (RHDV) VLPs were produced according to [1]. Briefly, the vp60 gene of polish strain SGM was cloned into pFast Bac1 transfer vector using Not1 and XhoI restriction sites. Recombinant baculovirus was obtained according to manufacturer's instructions (Invitrogen). In order to produce RHDV VLPs, Sf9 cells were infected with recombinant baculovirus at a MOI of 5 and harvested 60 h post-infection. Supernatant was collected after centrifugation in a microfuge at 8500 rpm for 10 min. VLPs from the supernatant were purified by sucrose gradient (60%-10%) centrifugation at 80,000 × g for 1.5 h at 4 °C (SW 41 Ti Beckman rotor).