Phan Joseph C, Nehilla Barrett J, Srinivasan Selvi, Coombs Robert W, Woodrow Kim A, Lai James J
Department of Bioengineering, University of Washington, Box 355061, Seattle, Washington, 98195, USA.
Nexgenia, Inc., 4000 Mason Rd., Fluke Hall, Suite 312-1, Seattle, Washington, 98195, USA.
Pharm Res. 2016 Oct;33(10):2411-20. doi: 10.1007/s11095-016-1980-7. Epub 2016 Jul 11.
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.
为了提高现有HIV诊断检测方法的检测限，我们探索了使用温度响应性磁性纳米颗粒试剂系统结合氰病毒素-N来识别HIV，以便从约1毫升的较大样本体积中快速有效地浓缩病毒颗粒。
表达、生物素化氰病毒素-N（CVN）突变体Q62C，然后将其与热响应性聚合物-链霉亲和素共轭物复合。使用基质辅助激光解吸/电离（MALDI）和TZM-bl HIV抑制试验对蛋白质表达/活性进行确认。使用表面等离子体共振（SPR）对生物素化CVN突变体与gp120的识别进行表征。使用逆转录聚合酶链反应（RT-PCR）测量使用热响应性磁性纳米颗粒试剂系统进行的病毒分离和富集。
生物素化的Q62C对gp120的解离常数（KD）为0.6 nM。温度响应性二元试剂系统在30分钟内使用聚N-异丙基丙烯酰胺-Q62C在100μl中实现了近100% HIV（1×10⁵病毒拷贝）的最大病毒捕获。此外，通过处理1000μl的10倍大样本，相同的试剂系统实现了近9倍的富集（图3）。
这项工作展示了一种温度响应性试剂系统，该系统使用抗病毒凝集素CVN进行识别来富集HIV，这可能适用于即时检测环境。
