Pharmaceutical Research

, Volume 33, Issue 10, pp 2411–2420 | Cite as

Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System

  • Joseph C. Phan
  • Barrett J. Nehilla
  • Selvi Srinivasan
  • Robert W. Coombs
  • Kim A. Woodrow
  • James J. Lai
Research Paper
  • 350 Downloads

Abstract

Purpose

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.

Methods

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.

Results

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).

Conclusion

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 WORDS

concentration cyanovirin-N HIV magnetic nanoparticles poly(N-isopropylacrylamide) sensitivity 

ABBREVIATIONS

CVN

Cyanovirin-N

HIV

Human immunodeficiency virus

LCST

Lower critical solution temperature

LFA

Lateral flow assay

MALDI

Matrix-assisted laser desorption ionization

mNP

Magnetic nanoparticles

pNIPAAm

Poly(N-isopropylacrylamide)

SA

Streptavidin

SPR

Surface plasmon resonance

Notes

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.

Supplementary material

11095_2016_1980_MOESM1_ESM.pdf (378 kb)
ESM 1 (PDF 377 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Joseph C. Phan
    • 1
  • Barrett J. Nehilla
    • 3
  • Selvi Srinivasan
    • 1
  • Robert W. Coombs
    • 2
  • Kim A. Woodrow
    • 1
  • James J. Lai
    • 1
  1. 1.Department of BioengineeringUniversity of WashingtonSeattleUSA
  2. 2.Division of Allergy and Infectious DiseasesUniversity of WashingtonSeattleUSA
  3. 3.Nexgenia, Inc.SeattleUSA

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