基于表面等离子体共振的外泌体PD-L1蛋白检测技术

Exosomal PD-L1 Detection Methods Based on Surface Plasmon Resonance

血清中可溶性程序性死亡-配体1(programmed cell death 1 ligand 1,PD-L1)蛋白的存在形式包括外泌体表面、微囊泡表面以及分泌的游离形式等。前期研究表明,血清中外泌体表面PD-L1含量与多种癌症的预后评估显著相关,然而目前的常规检测方法通常将血清中所有可溶性PD-L1蛋白作为一个整体进行检测,无法有效分辨外泌体PD-L1与其他形式的PD-L1。本研究基于表面等离子体共振技术,建立了一种外泌体PD-L1的特异性检测方法。该方法首先通过抗体识别PD-L1蛋白,并将其捕获至检测芯片表面;再利用α-溶血素在外泌体膜上形成多个寡聚体,从而特异性检测外泌体PD-L1。该方法通过α-溶血素结合过程的信号变化对外泌体PD-L1含量进行检测,可以有效降低检测背景噪音并放大信号。使用α-溶血素放大信号前的线性范围为0.035~2.208 pg/mL,信号放大后的线性范围为0.004-0.552 pg/mL。方法学验证实验表明,该方法特异性、灵敏度和精密度良好,具有一定的临床应用前景。

Soluble programmed cell death 1 ligand 1(PD-L1)in the serum includes exosomal,microvesical and secreted forms of PD-L1.Previous studies have shown that the level of exosomal PD-L1 in the serum significantly correlated with the prognosis of various cancers.However,current analysis detects all forms of PD-L1 in the serum as a whole,without distinguishing exosomal PD-L1 from other forms.In this study,a specific detection method for exosomal PD-L1 was established based on surface plasmon resonance.This method first captures PD-L1 by antibody recognition and immobilizes it on the surface of the detection chip.Then,α-hemolysin was recruited to form multiple oligomers on the exosomal membrane.This method quantifies the content of exosomal PD-L1 by monitoring the signal change during the binding process ofα-hemolysin,effectively reducing background noises and amplifying the signal.The linear range before signal amplification withα-hemolysin was 0.035-2.208 pg/mL,and after signal amplification,it was 0.004-0.552 pg/mL.Methodological validation showed that this method has good specificity,sensitivity,and repeatability,and has certain clinical application prospects.