Development Of A Cytokine Chip

细胞因子芯片的开发

• 批准号: 6836997
• 负责人: Terry M. Phillips
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6836997
• 关键词: T lymphocyte; biocytin; bioengineering /biomedical engineering; biotin; cytokine; densitometry; glass; high throughput technology; immunologic assay /test; laser spectrometry; nanotechnology; neutralizing antibody; recombinant proteins; technology /technique development

Cytokines serve a variety of regulatory functions, one of the most important being the regulation of T-cell differentiation. Secreted cytokine profiles have been demonstrated to be predictive of different T-cell differentiation, especially cytokines regulating cell-mediated and humoral immunity. In order to measure different cytokine profiles simultaneously in individual samples, a basic cytokine chip has been developed based on solid-phase immunoaffinity extraction of the analytes of interest. Arrays of recombinant streptavidin spots were derivatized to the surfaces of silanized glass microscope slides via a carbonyldiimidazole bridge. Anti-cytokine antibodies were biotinylated via their carbohydrate moieties and bound to the steptavidin. Samples were labeled with a laser dye prior to analysis and incubated with the chip. Following removal of non-reactive materials by washing, the bound analytes were read in a scanning laser densitometer. Improvements in both the immobilization chemistry and techniques for keeping the antibody molecules active have increased the sensitivity of the chip to 1.0 - 1.8 pg/ml for all five cytokines studied. Further studies are underway to improve this sensitivity as well as optimize the chip reading mechanism. The chip now available exhibits very good selectivity and a high degree of specificity when using model mixtures of cytokine. However, further studies are required to determine the efficiency of this chip against human samples and to increase the chip selectivity to 50 cytokines, chemokines, and growth factors. This refined chip will have applications in the analysis of a number of important regulatory pathways including hematopoesis, inflammation neurogenic regulation, cellular regulation, and wound healing.

细胞因子具有多种调节功能，最重要的是T细胞分化的调节。已证明分泌的细胞因子谱图可以预测不同的T细胞分化，尤其是调节细胞介导的和体液免疫的细胞因子。为了同时测量各个样品中的不同细胞因子谱，基于感兴趣的分析物的固相免疫疗法提取，已经开发了基本的细胞因子芯片。重组链霉亲和素斑点的阵列通过羰基二咪唑桥衍生在硅烷化玻璃显微镜载玻片的表面上。抗周期因子抗体通过其碳水化合物部分进行生物素化，并与steptavidin结合。在分析之前，将样品用激光染料标记，并与芯片孵育。通过洗涤去除非反应性材料后，在扫描激光密度计中读取结合的分析物。对所有五种研究的细胞因子，固定化学和保持抗体分子的固定化学和技术的改进都提高了芯片的敏感性至1.0-1.8 pg/ml。正在进行进一步的研究以提高这种敏感性并优化芯片阅读机制。现在，使用细胞因子模型混合物时，可用的芯片具有很好的选择性和高度的特异性。但是，需要进一步的研究来确定该芯片对人类样品的效率，并提高对50个细胞因子，趋化因子和生长因子的芯片选择性。这种精致的芯片将在分析许多重要的调节途径中应用，包括造血，炎症神经源性调节，细胞调节和伤口愈合。