細胞因子檢測是藥物研發過程中不可或缺的環節，細胞因子以及可溶性受體的水平檢測，常被用在藥物早期研究階段候選藥物生物學功能評價、臨床前及臨床藥效學研究、免疫原性研究以及細胞因子釋放綜合征（CRS）風險的評估過程中。

在細胞因子觀察指標中，最為重要的兩大類是Th1和Th2細胞因子，包括但不限于IL-2、IL-4、IL-6、IL-10、IFN-γ、TNF-α等。Th1和Th2細胞是輔助T淋巴細胞的兩個亞群，Th1細胞主要分泌IL-2、IFN-γ、TNF-α等，主要介導細胞毒和局部炎癥有關的免疫應答，輔助抗體生成，參與細胞免疫及遲發型超敏性炎癥的發生，故稱為炎癥性T細胞；Th2細胞主要分泌IL-4、IL-6、IL-10等，與B細胞增殖、分化、成熟有關，能促進抗體生成，增強抗體介導的體液免疫應答。在正常情況下，機體中Th1和Th2處于相對平衡的狀態，當平衡被破壞時，可能會引起多種疾病，如腫瘤、自身免疫性疾病及變態反應等，即為“Th1/Th2平衡飄移”。通過研究Th1/Th2細胞分泌的細胞因子，明確平衡漂移狀態，選擇性的調節Th1或Th2細胞水平以恢復平衡，可達到治療疾病的目的。

以CRS風險評估為例，在2014年FDA發布的《Guidance for Industry Immunogenicity Assessment for Therapeutic Protein Products》和2021年CDE 發布的《藥物免疫原性研究技術指導原則》中均有提到針對免疫調節類藥物要嚴控細胞因子釋放綜合征發生的風險，除了在常規的動物體內毒性試驗中進行細胞因子相關檢測外，還應進行體外細胞因子釋放試驗。細胞因子的檢測項目應與受試藥物臨床安全性考慮密切相關，一般情況下，觀察指標包括但不限于 IL-2、IL-6、IL-10、IFN-γ 和 TNF-α。

ClinMax^TM多因子檢測原理

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.

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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