Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System
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In order to improve the detection limit of existing HIV diagnostic assays, we explored the use of a temperature-responsive magnetic nanoparticle reagent system in conjunction with cyanovirin-N for HIV recognition to rapidly and efficiently concentrate viral particles from larger sample volumes, ~ 1 ml.
Cyanovirin-N (CVN) mutant, Q62C, was expressed, biotinylated, and then complexed with a thermally responsive polymer-streptavidin conjugate. Confirmation of protein expression/activity was performed using matrix assisted laser desorption/ionization (MALDI) and a TZM-bl HIV inhibition assay. Biotinylated CVN mutant recognition with gp120 was characterized using surface plasmon resonance (SPR). Virus separation and enrichment using a thermoresponsive magnetic nanoparticle reagent system were measured using RT-PCR.
Biotinylated Q62C exhibited a KD of 0.6 nM to gp120. The temperature-responsive binary reagent system achieved a maximum viral capture of nearly 100% HIV, 1 × 105 virus copies in 100 μl, using pNIPAAm-Q62C within 30 minutes. Additionally, the same reagent system achieved nearly 9-fold enrichment by processing a 10-times larger sample of 1000 μl (Fig. 3).
This work demonstrated a temperature-responsive reagent system that provides enrichment of HIV using antiviral lectin CVN for recognition, which is potentially amenable for use in point-of-care settings.
KEY WORDSconcentration cyanovirin-N HIV magnetic nanoparticles poly(N-isopropylacrylamide) sensitivity
Human immunodeficiency virus
Lower critical solution temperature
Lateral flow assay
Matrix-assisted laser desorption ionization
Surface plasmon resonance
ACKNOWLEDGMENTS AND DISCLOSURES
We thank Reggie Gausman and Jose Ortega for technical assistance with the RT-PCR work. Funding support was provided by the University of Washington, the University of Washington CFAR Clinical Research and Retrovirology Core (P30-AI-027757) and the ACTG Laboratory Center (UM1-AI-106701), NIH GM100558 and CA174581, and the National Science Foundation Graduate Research Fellowship Program. BJN is an employee of Nexgenia, a company that is commercializing stimuli-responsive reagents for life science applications.
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