title: Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System.
abstract: 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 × 10(5) 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.
authors.affiliation: Department of Bioengineering, University of Washington, Box 355061, Seattle, Washington, 98195, USA.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Phan
authors.affiliation: Nexgenia, Inc., 4000 Mason Rd., Fluke Hall, Suite 312-1, Seattle, Washington, 98195, USA.
authors.forename: Barrett J
authors.initials: BJ
authors.lastname: Nehilla
authors.affiliation: Department of Bioengineering, University of Washington, Box 355061, Seattle, Washington, 98195, USA.
authors.forename: Selvi
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: Division of Allergy and Infectious Diseases, University of Washington, Seattle, Washington, 98104, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Coombs
authors.affiliation: Department of Bioengineering, University of Washington, Box 355061, Seattle, Washington, 98195, USA. woodrow@uw.edu.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Woodrow
authors.affiliation: Department of Bioengineering, University of Washington, Box 355061, Seattle, Washington, 98195, USA. jilai@uw.edu.
authors.forename: James J
authors.initials: JJ
authors.lastname: Lai
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D015497:HIV-1 / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D020349:Surface Plasmon Resonance / Q000379:methods; D013696:Temperature
chemical_list: D000998:Antiviral Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D037102:Lectins; C107733:cyanovirin N
keywords: HIV; concentration; cyanovirin-N; magnetic nanoparticles; poly(N-isopropylacrylamide); sensitivity