Infectious Disease Research Institute, 1124 Columbia Street, Suite 400, Seattle, WA 98104, USA.
J Immunol Methods. 2011 Oct 28;373(1-2):54-62. doi: 10.1016/j.jim.2011.07.021. Epub 2011 Aug 3.
Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development. Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens.
Toll 样受体 (TLR) 激动剂目前正在被作为疫苗佐剂进行研究，目前有几个主要候选药物已经进入许可产品或晚期临床开发阶段。豚鼠被广泛用于药物和疫苗的临床前测试；然而，由于免疫工具和试剂的有限可用性，TLR 激动剂在该模型中的评估受到阻碍。在这项研究中，我们验证了使用一种称为 QuantiGene Plex 2.0 试剂系统的分支 DNA (bDNA) 测定法来测量豚鼠细胞 TLR 刺激后先天细胞因子和趋化因子 mRNA 水平。在豚鼠脾细胞和全血中用 TLR-4 或 TLR-7/8 激动剂进行体外刺激后，Th1 极化细胞因子 (TNF-α、IL-1β、IL-12) 和趋化因子 (CXCL1、CCL2) 的基因表达上调。这些数据证实了 QuantiGene 系统作为测量转录水平的 RT-PCR 替代方法以及用于剖析豚鼠 TLR 刺激免疫反应的高通量筛选工具的实用性。总的来说，QuantiGene 平台可靠、可重复且敏感。这些激动剂有可能被用作针对各种病原体的疫苗的佐剂成分。
