期刊
PHARMACEUTICAL RESEARCH
卷 33, 期 10, 页码 2411-2420出版社
SPRINGER/PLENUM PUBLISHERS
DOI: 10.1007/s11095-016-1980-7
关键词
concentration; cyanovirin-N; HIV; magnetic nanoparticles; poly(N-isopropylacrylamide); sensitivity
资金
- University of Washington
- University of Washington CFAR Clinical Research and Retrovirology Core [P30-AI-027757]
- ACTG Laboratory Center [UM1-AI-106701]
- NIH [GM100558, CA174581]
- National Science Foundation
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, similar to 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 K-D of 0.6 nM to gp120. The temperature-responsive binary reagent system achieved a maximum viral capture of nearly 100% HIV, 1 x 10(5) virus copies in 100 mu l, 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 mu l (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.
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