【新品速递】流式多因子检测技术，助力临床前及临床样本快速检测

在细胞因子观察指标中，最为重要的两大类是Th1和Th2细胞因子，包括但不限于IL-2、IL-4、IL-6、IL-10、IFN-γ、TNF-α等。Th1和Th2细胞是辅助T淋巴细胞的两个亚群，Th1细胞主要分泌IL-2、IFN-γ、TNF-α等，主要介导细胞毒和局部炎症有关的免疫应答，辅助抗体生成，参与细胞免疫及迟发型超敏性炎症的发生，故称为炎症性T细胞；Th2细胞主要分泌IL-4、IL-6、IL-10等，与B细胞增殖、分化、成熟有关，能促进抗体生成，增强抗体介导的体液免疫应答。在正常情况下，机体中Th1和Th2处于相对平衡的状态，当平衡被破坏时，可能会引起多种疾病，如肿瘤、自身免疫性疾病及变态反应等，即为“Th1/Th2平衡飘移”。通过研究Th1/Th2细胞分泌的细胞因子，明确平衡漂移状态，选择性的调节Th1或Th2细胞水平以恢复平衡，可达到治疗疾病的目的。

以CRS风险评估为例，在2014年FDA发布的《Guidance for Industry Immunogenicity Assessment for Therapeutic Protein Products》和2021年CDE 发布的《药物免疫原性研究技术指导原则》中均有提到针对免疫调节类药物要严控细胞因子释放综合征发生的风险，除了在常规的动物体内毒性试验中进行细胞因子相关检测外，还应进行体外细胞因子释放试验。细胞因子的检测项目应与受试药物临床安全性考虑密切相关，一般情况下，观察指标包括但不限于 IL-2、IL-6、IL-10、IFN-γ 和 TNF-α。

ClinMax^TM多因子检测原理

Fig.1 A representative datapoint showing single bead population in P1 gate, the antibody encapsulated 6-plex beads classified by APC channel, and fluorescence signal was measured for each cytokine in PE channel. Data was generated on BD FACSLyric^TM Flow Cytometer, equipped with blue laser (488 nm) and red laser (640 nm).

Fig.2 A double logarithmic fitting curve was plotted on the human Th1/Th2 Cytokines: IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ 6-plex panel. Gradient diluted caliborator protein (IL-2/4/6/10, TNF-α, IFN-γ), capture antibody encapsulated 6-plex beads and biotinylated detection antibody formed a sandwiches complex, SA-PE was added in and reacted with detection antibody. The PE fluorescence intensity was measured in proportion to the recombinant protein. The present curve of cytokines 6-plex panel was ranging from 9.7 pg/mL to 10000 pg/mL, respectively.

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Fig. 3 Recombinant cytokines were spiked into 8 human serum samples, and then analyzed. 75-120% of cytokines were recovered from serum samples.

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