Li Ning, Chinthalapally Molangur, Holden Van K, Deepak Janaki, Dhilipkannah Pushpa, Fan Jonathan M, Todd Nevins W, Jiang Feng
Departments of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Environmental Science and Technology, College of Agriculture and Natural Resources, University of Maryland, College Park, MD 20742, USA.
J Clin Med. 2022 Nov 24;11(23):6923. doi: 10.3390/jcm11236923.
Cytokines play crucial roles in tumorigenesis and are potential biomarkers for cancer diagnosis. An Enzyme-linked Immunosorbent Assay (ELISA) is commonly used to measure cytokines but has a low sensitivity and can only detect a single target at a time. CRISPR-Associated Proteins (Cas) can ultra-sensitively and specifically detect nucleic acids and is revolutionizing molecular diagnostics. Here, we design a microplate-based CRISPR-ELISA assay to simultaneously profile multiple cytokines, in which antibodies are coupled with ssDNA to form antibody-ssDNA complexes that bridges CRISPR/Cas12a and ELISA reactions. The ssDNA triggers the Cas12a collateral cleavage activity and releases the fluorescent reporters to generate amplified fluorescent signals in the ELISA detection of cytokines. The CRISPR-ELISA assay can simultaneously measure multiple cytokines with a significantly higher sensitivity compared with conventional ELISA. Using the CRISPR-ELISA assay to profile plasma cytokines in 127 lung cancer patients and 125 cancer-free smokers, we develop a panel of plasma cytokine biomarkers (IL-6, IL-8, and IL-10) for early detection of the disease, with 80.6% sensitivity and 82.0% specificity. The CRISPR-ELISA assay may provide a new approach to the discovery of cytokine biomarkers for early lung cancer detection.
细胞因子在肿瘤发生过程中发挥着关键作用，并且是癌症诊断的潜在生物标志物。酶联免疫吸附测定法（ELISA）通常用于检测细胞因子，但灵敏度较低，且一次只能检测一个靶点。CRISPR相关蛋白（Cas）能够超灵敏且特异性地检测核酸，正在彻底改变分子诊断技术。在此，我们设计了一种基于微孔板的CRISPR-ELISA检测方法，用于同时分析多种细胞因子，其中抗体与单链DNA偶联形成抗体-单链DNA复合物，该复合物连接CRISPR/Cas12a和ELISA反应。单链DNA触发Cas12a的旁切活性并释放荧光报告分子，从而在细胞因子的ELISA检测中产生放大的荧光信号。与传统ELISA相比，CRISPR-ELISA检测方法能够同时检测多种细胞因子，且灵敏度显著更高。利用CRISPR-ELISA检测方法分析127例肺癌患者和125例无癌吸烟者的血浆细胞因子谱，我们开发了一组用于疾病早期检测的血浆细胞因子生物标志物（IL-6、IL-8和IL-10），灵敏度为80.6%，特异性为82.0%。CRISPR-ELISA检测方法可能为发现用于早期肺癌检测的细胞因子生物标志物提供一种新方法。
