Nanotrap particles can capture RVFV NP.

<p>A) Recombinant histidine-tagged NP (His-NP) at a starting concentration of 0.4 mg/ml and a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at ambient temperature. After 30 minutes, the (+)NT samples were centrifuged. Both bound (P) and 10uL of unbound (S) material was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then analyzed for presence of NP protein by western blot using antibodies directed against the histidine tag. No Nanotrap particle samples (-NT) at a 10 μl volume of 0.4 mg/ml His-NP were processed in parallel. B) Purified NP (obtained from BEI Resources) at 2 μg in a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at an ambient temperature. A control—NT sample (10 μl volume) was processed in parallel. Samples were processed as describe in panel A. After electrophoresis, NP was visualized by Commassie blue staining. C) His-NP (obtained from Immune Technology) at 1 μg/ml and a volume of one milliliter was incubated with 100 μl of NT45, NT53, and NT69. A control—NT sample (10 μl volume) was processed in parallel. The control sample is 100 μg/ml NP (volume of 10 μl). The samples were processed as in panel A and analyzed by western blotting for NP by using antibodies against NP. D) Viral supernatants at 1E+06 pfu/ml and a volume of 1 ml were incubated with 100 μl NT45, NT46, NT53, NT55, and NT69 for 30 minutes at an ambient temperature. Control—NT samples (10 μl volumes) at 1E+07pfu/ml and 1E+06 pfu/ml were processed in parallel. The samples were processed as in panel A and analyzed by western blot for NP.</p

Non-virion associated NP can be detected in viral supernatants.

<p>Vivaspin 20 centrifugal concentrators with a 300,000 Da MWCO were used to filter viral supernatants harvested from Vero cells infected at an MOI 1 with MP12. Five milliliters of viral supernatant was added to the concentrators and centrifuged at 1400 rpm for 7 minutes until approximately 500 μl remained on the top portion of the concentrator. A) The top (T) and bottom (B) fractions, as well as control sample (C) containing the original sample before processing were analyzed by western blot using antibodies against NP (EC22 antibody). C, T, and B lysates were undiluted (neat). Cytoplasmic extract (CE) control is 7.7 μg/ml of RVFV infected Vero cell lysates. B) Plaque assays were performed with both fractions and the control sample. C) Densitometry analysis was performed to determine the NP band densities in the T and B fractions. The percent of total NP was determined by dividing the top or bottom portion's band density by the total NP band density (top and bottom portions added together) and multiplying by 100. D) NP band density per pfu values were calculated by dividing the NP band densities by the total pfu values.</p

Nanotrap particles can capture and enrich NP from virally infected cells.

<p>A) One ml of cytoplasmic extract (CE) at 2.6 μg/ml obtained from RVFV infected Vero lysates were incubated with 100 μl of NT45, NT53, and NT69. No Nanotrap particle sample (-NT) was processed in parallel. After 30 minutes, the (+)NT samples were centrifuged. The unbound material (S) from the spin was saved and 10 μl was processed in parallel. The bound material (P) was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then loaded onto a NuPage 4–12% Bis-Tris gel. Samples were subsequently analyzed by western blot for NP. B) One ml of CE was serially diluted in 50mM Tris-HCl from 15 μg/ml to 0.75 μg/ml and incubated with 100 μl of NT45 for 30 minutes. No Nanotrap particle samples (-NT) were processed in parallel. The control sample is CE at 770 μg/ml (10 μl volume). The samples were processed as in panel A.</p

Schematic of antigen capture with Nanotrap particles.

<p>Nanotrap particles are incubated with samples for 30 minutes at room temperature, centrifuged, and unbound material is removed. The pellet is resuspended in lysis buffer and boiled for 10 minutes. The sample is then centrifuged and unbound supernatant is loaded onto an SDS PAGE gel.</p

Nanotrap particles can protect NP from degradation at increased temperatures and times.

<p>A and B) Cytoplasmic extracts from RVFV infected cells were diluted in 100% sheep serum for a final concentration of 7.7 μg/ml. C and D) Viral supernatant was diluted in 100% goat serum for a final titer concentration of 1E+06 pfu/ml. One milliliter of sample was incubated with NT45 for 24 to 120 hours at either ambient temperature (A and C) or at 37^°C (B and D). No Nanotrap particle (-NT) and control cytoplasmic extract (CE) samples at 7.7μg/ml or 0.77 μg/ml (B only) at 10 μl volumes were processed in parallel. Viral supernatants without Nanotrap particles containing samples were analyzed on a separate gel (E). After the incubation time, the (+)NT samples were centrifuged and washed once with 0.25M sodium thiocyanate and twice with diH₂O. Presence of NP was analyzed by western blot using antibodies against NP.</p

Capture of other viruses with NanoTrap particles.

<p>A) Six different types of NanoTrap particles were incubated with viral supernatants containing VEEV for 30 minutes at room temperature and washed 4 times with water. Viral RNA was extracted and quantitated by qRT-PCR assays. B) Seven different types of NanoTrap particles were incubated with 1 ml J1.1 supernatant for 30 minutes at room temperature and washed 4 times with water. The pellets were diluted in 100 ul water and RNA extracted. A cDNA synthesis using 150 ng of each sample was performed and followed by PCR using 10 ul cDNA. A DNA gel was run to determine viral capture. A sample with no reverse transcriptase added and a sample with just water were used as negative controls. The volume of each band was quantified and graphed. C) Bovine serum was spiked with RVFV (1.0E+6 pfu/ml) only or both RVFV (1.0E+6 pfu/ml) and HIV (100 µl of J.1. supernatants). Samples were incubated with NT53 for 30 minutes, viral RNA extracted and quantitated by qRT-PCR. Samples without NT53 were processed in parallel (black bars).</p

Description of the NanoTrap particles' bait and shell.

*<p>Vinyl Sulfonic Acid.</p

The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus

<div><p>Background</p><p>Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.</p><p>Results</p><p>Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. Importantly, NT53 capture of RVFV resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. NT53 was also capable of capturing and enhancing RVFV detection from serum samples. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Furthermore, both NP-40-lysed virus and purified RVFV RNA were bound by NT53. Importantly, NT53 protected viral RNA from RNase A degradation, which was not observed with other commercially available beads. Incubation of RVFV samples with NT53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. Finally, NanoTrap particles were capable of capturing VEEV and HIV, demonstrating the broad applicability of NanoTrap particles for viral diagnostics.</p><p>Conclusion</p><p>This study demonstrates NanoTrap particles are capable of capturing, enriching, and protecting RVFV virions. Furthermore, the use of NanoTrap particles can be extended to a variety of viruses, including VEEV and HIV.</p></div

RVFV capture by NanoTrap particles.

<p>A) Seven different types of NanoTrap particles were incubated with viral supernatants containing RVFV (1E+7 pfu/ml) for 30 minutes at room temperature and washed 4 times with water. Viral RNA was extracted from the particles with Ambion's MagMax Viral RNA extraction kit and quantitated by qRT-PCR assays. B) Percent detected virus was calculated compared to a sample processed without NanoTrap particle incubation. C) Viral supernatants were incubated with NT46, NT53, and NT69 for 30 minutes at room temperature and washed 4 times with water. Serial dilutions followed by plaque assays were performed to determine if full virus was bound by the particles.</p

Characterization of RVFV NanoTrap particle capture.

<p>A) Viral supernatants were serially diluted (2.5E+6 to 2.5E+1 pfu/ml) and incubated with NT53 for 30 minutes at room temperature. The pellets were washed 4 times with water and then particles were tested in plaque assays to determine if full virus was bound by the particles. B) Viral supernatants at 1.0E+6 and 1.0E+3 pfu/ml were incubated with NT53 for 30 minutes at room temperature. The sample was spun at 10,000 rpm for 5 minutes and the unbound viral supernatant was saved separately. NT53 was washed 4 times with water and then particles were tested in plaque assays to determine how much virus were bound verses unbound by the particles. The percentage of bound virus at 1.0E+6 and 1.0E+3 pfu/ml was graphed.</p