Improving RT-PCR detection from frozen, unpreserved first-void clinical samples APPLICATION NOTE SKU 44XXX Download full application note here Key Advantages > Capture and concentrate Zika virus from urine to boost assay sensitivity and eliminate the need for invasive blood collection. > Overcome the sensitivity challenges in frozen storage conditions by minimizing the impact of sample degradation through virus enrichment. > Circumvent the low input sample volume restrictions of commercial extraction methods by capturing more virions from larger volumes upstream. > Easily integrate into existing workflows using sample collection devices, commercial RNA extraction methods and RT-PCR assays. Introduction Increasing geographic distribution of mosquito vectors and subsequent transmission by international travel has created a need for a technology that is capable of accurately and reliably detecting low-concentration viral pathogens, like Zika, in biological samples. Non-invasive collection methods, such as urine sampling are ideal, but they are limited by low molecular assay sensitivity in urine samples. This sensitivity issue is further exacerbated when collected samples need to be stored for longer periods of time or transported prior to testing. First-void urine improves the sensitivity of molecular assays and offers an alternative to serum sampling. However, testing of urine samples that are stored for extended periods of time (> 1 year) in the absence of a preservative can result in false-negative results. Nanotrap particles can capture, concentrate, and preserve viral pathogens from biological fluids upstream of a range of analytical methods, including immunoassays and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). In this study, Nanotrap particles were evaluated for their ability to significantly improve the RT-PCR detection of Zika virus in frozen, archived clinical first-void urine samples. This work was performed in collaboration with Escuela Superior Politecnica del Litoral (ESPOL), Ghent University, Eucagen LLC., and Novosanis.

Nanotrap® Virus Particles capture and concentrate coronavirus OC43 for improved RT-PCR detection APPLICATION NOTE SKU 44XXX Download full application note here Key Advantages > Nanotrap® processing is simple and efficient, creating quality input sample material for downstream detection. > Bring low-abundance samples into the assay dynamic range and eliminate false negatives. Introduction Coronaviruses are a large family of viruses that usually cause mild to moderate upper-respiratory tract illnesses in humans. Three times in the 21st century, coronavirus outbreaks have emerged from animal reservoirs to cause severe disease and global transmission concerns, including the most recent outbreak for COVID-19 disease, caused by SARS-CoV-2. Effective diagnostic tools are an essential element to the containment of any epidemic. A major challenge for public health authorities with the COVID-19 outbreak has been that it appears that asymptomatic patients are contagious for as long as 1-2 weeks. Additionally, the early nucleic acid tests that have been developed using reverse-transcription polymerase chain reaction (RT-PCR) are not sensitive enough to identify infections in many asymptomatic patients. According to the head of the Chinese Academy of Medical Sciences and an early study of samples collected in China in late 2019 and early 2020, 50% - 70% of infected patients were undetected due to false-negative test results. Nanotrap® Virus Particles have already been shown to effectively capture and concentrate multiple strains of influenza and RSV, as well as the coronavirus strain 229E from saliva, thus enhancing detection, even in co-infection scenarios. Here we demonstrate the capture and concentration of a second coronavirus strain (HCoV-OC43) from viral transport media (VTM) for improved RT-PCR detection, suggesting that Nanotrap® Virus Particles might be able to capture and concentrate additional strains of coronavirus, including SARS-CoV-2.

Poster presented at HEAL Investigators Meeting, January 16-17, 2020 Objective: To develop and demonstrate the utility of a universal Nanotrap® technology solution for improved detection of fentanyl and fentanyl derivatives in urine and oral fluid samples. Results: 1) Nanotrap® particles improved sensitivity of Neogen’s ELISA for 9 fentanyl analogs. 2) Nanotrap® particles enriched fentanyl analogs from synthetic urine and oral fluid samples. The protocol used in these experiments is compatible with Thermo DRI assays and LC-MS platforms. Next Steps: Further studies are planned to establish fentanyl and analog LLODs as well as integration strategies for other analytical platforms. Impact: Nanotrap® particles can provide universal pre-concentration of multiple fentanyl analogs from a single sample prior to analysis. This pre-concentration approach can expand the utility and performance of existing assays in POC and reference laboratory settings while helping to address the current technical bottlenecks.

PLOS ONE, January 7, 2020 Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.

Analysis of the Babesia microti proteome in infected red blood cells by a combination of nanotechnology and mass spectrometry International Journal of Parasitology, 2019 Proteomics of Babesia microti has lagged behind other apicomplexans despite recent genome and transcriptome studies. Here, we used a combination of nanotechnology and mass spectrometry to provide a proteomic profile of B. microti acute infection. We identified ∼500 parasite proteins in blood with functions such as transport, carbohydrate and energy metabolism, proteolysis, DNA and RNA metabolism, signaling, translation, lipid biosynthesis, and motility and invasion. We also identified surface antigens with roles in the immune response to the parasite. This first evaluation of the B. microti proteome in erythrocytes provides information for the study of intracellular survival and development of diagnostic tools using mass spectrometry.

AIDS: December 1, 2019 - Volume 33 Objective: The aim of this study was to measure the protein concentration and biological activity of HIV-1 Tat in cerebrospinal fluid (CSF) of individuals on suppressive antiretroviral therapy (ART). Design: CSF was collected from 68 HIV-positive individuals on ART with plasma viral load less than 40 copies/ml, and from 25 HIV-negative healthy controls. Duration of HIV infection ranged from 4 to more than 30 years. Methods: Tat levels in CSF were evaluated by an ELISA. Tat protein and viral RNA were quantified from exosomes isolated from CSF, followed by western blot or quantitative reverse transcription PCR, respectively. Functional activity of Tat was assessed using an LTR transactivation assay. Results: Tat protein was detected in 36.8% of CSF samples from HIV-positive patients. CSF Tat concentration increased in four out of five individuals after initiation of therapy, indicating that Tat was not inhibited by ART. Similarly, exosomes from 34.4% of CSF samples were strongly positive for Tat protein and/or TAR RNA. Exosomal Tat retained transactivation activity in a CEM-LTR reporter assay in 66.7% of samples assayed, which indicates that over half of the Tat present in CSF is functional. Presence of Tat in CSF was highly associated with previous abuse of psychostimulants (cocaine or amphetamines; P = 0.01) and worse performance in the psychomotor speed (P = 0.04) and information processing (P = 0.02) cognitive domains. Conclusion: Tat and TAR are produced in the central nervous system despite adequate ART and are packaged into CSF exosomes. Tat remains biologically active within this compartment. These studies suggest that Tat may be a quantifiable marker of the viral reservoir and highlight a need for new therapies that directly inhibit Tat.

POSTER High sensitivity detection of multiple tick borne infections in blood and urine using nanoparticle entrapment and mass spectrometry Presented at ASTMH 2018 Team detected parasite proteins in red blood cells, serum, and urine of B. microti-infected golden hamsters using a nanoparticle MS approach. Three biomarkers were characterized by WB, IFA, and ELISA. Method also detected previously unrecognized proteins from multiple tick borne pathogens in urine samples from patients with persistent Lyme disease. Attributes of the technology include: 1) Rapid harvest of analytes, protection from degradation, and exclusion of unwanted high abundance host proteins; 2) Presence of unique chemical baits immobilized in the nanoparticle core with high affinity and a very low off-rate; 3) Absolute specificity for the target amino acid sequence by using tandem MS and simultaneous investigation of peptides from different tick borne pathogens; and 4) Adaptation to full automation for high throughput testing. Further adaptation of nanotechnology into immunochromatographic tests can provide a platform for a one-step, rapid analysis of prospective carriers of multiple tick borne infections or for blood screening. Access full poster here

Journal of Visual Experiments (JoVE): Purification of High Yield Extracellular Vesicle Preparations Away from Virus This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research. Go to JoVE entry

Cells 2019 Access full publication here Human Immunodeficiency Virus-1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infecting nearly 37 million people worldwide. Currently, there is no definitive cure, mainly due to HIV-1's ability to enact latency. Our previous work has shown that exosomes, a small extracellular vesicle, from uninfected cells can activate HIV-1 in latent cells, leading to increased mostly short and some long HIV-1 RNA transcripts. This is consistent with the notion that none of the FDA-approved antiretroviral drugs used today in the clinic are transcription inhibitors. Furthermore, these HIV-1 transcripts can be packaged into exosomes and released from the infected cell. Here, we examined the differences in protein and nucleic acid content between exosomes from uninfected and HIV-1-infected cells. We found increased cyclin-dependent kinases, among other kinases, in exosomes from infected T-cells while other kinases were present in exosomes from infected monocytes. Additionally, we found a series of short antisense HIV-1 RNA from the 3' LTR that appears heavily mutated in exosomes from HIV-1-infected cells along with the presence of cellular noncoding RNAs and cellular miRNAs. Both physical and functional validations were performed on some of the key findings. Collectively, our data indicate distinct differences in protein and RNA content between exosomes from uninfected and HIV-1-infected cells, which can lead to different functional outcomes in recipient cells. KEYWORDS: HIV-1; RNA sequencing; extracellular vesicle; EV; proteomics

Viruses Abstract: Ebola virus (EBOV) disease can result in a range of symptoms anywhere from virtually asymptomatic to severe hemorrhagic fever during acute infection. Additionally, spans of asymptomatic persistence in recovering survivors is possible, during which transmission of the virus may occur. In acute infection, substantial cytokine storm and bystander lymphocyte apoptosis take place, resulting in uncontrolled, systemic inflammation in aected individuals. Recently, studies have demonstrated the presence of EBOV proteins VP40, glycoprotein (GP), and nucleoprotein (NP) packaged into extracellular vesicles (EVs) during infection. EVs containing EBOV proteins have been shown to induce apoptosis in recipient immune cells, as well as contain pro-inflammatory cytokines. In this manuscript, we review the current field of knowledge on EBOV EVs including the mechanisms of their biogenesis, their cargo and their eects in recipient cells. Furthermore, we discuss some of the eects that may be induced by EBOV EVs that have not yet been characterized and highlight the remaining questions and future directions. View full publication here >> Keywords: Ebola virus; exosome; extracellular vesicles; VP40; NP; GP; cytokine

POSTER Presented at ASM Clinical Virology Symposium 2019 Influenza viruses are responsible for seasonal epidemics each year in the United States that result in an average of 200,000 hospitalizations and tens of thousands of deaths. Accurate and early diagnosis of influenza viral infections is critical. Using simple and efficient pre-concentration workflows combined with existing molecular test formats can improve detection at earlier time points. Nanotrap® particles can improve flu virus detection by pre-concentrating flu virus from viral transport media samples for RT-PCR detection. Methods are amenable to automation. Nanotrap® particles preserve pathogens at elevated temperatures. Incorporating Nanotrap® particles in the transport media could further improve sample processing workflows and downstream assay. Access full poster here

POSTER Presented at ASM Clinical Virology Symposium 2019 Zika is spread mostly by the bite of an infected Aedes species mosquito. These mosquitoes bite during the day and night. Zika can be passed from a pregnant woman to her fetus. Infection during pregnancy can cause certain birth defects. There is no vaccine or medicine for Zika. Accurate and reliable Zika virus diagnostic tests are essential in limiting the spread of Zika virus infections. Urine and other non-invasive biopecimens are ideally suited for Zika screening and surveillance; but existing test methods are often limited due to poor assay sensitivity. Processing first-void urine with Nanotrap® particles significantly improves the detection of Zika virus by RT-PCR in those samples. These results have important implications for routine clinical diagnostics, as the combination of first-void urine and Nanotrap® particles can be used to enrich pathogens such as Zika virus. Incorporating Nanotrap® particles in the urine collection device along with a preservative could streamline sample processing and further improve downstream assay performance. Access full poster here