基于噬菌体展示技术的内毒素检测方法

Detection of Endotoxin Based on Phage Display Technology

利用噬菌体展示技术,经过两轮非特异性筛选,一轮特异性筛选,获得内毒素高效特异结合十二肽。经生物分子相互作用系统表征,结果表明,筛选获得的多肽与内毒素具有良好的亲和性,解离平衡常数KD为3.52×10^(-9);将获得的多肽人工合成,采用EDC/NHS共价偶联法与乳胶微球偶联,建立了内毒素比浊快速检测方法,内毒素标准品在0.03～0.48EU/mL浓度范围内,增强免疫比浊法吸光度值与内毒素标准品浓度呈正线性关系,相关系数R2>0.99,最低检测限达到0.03EU/mL。与市售内毒素鲎试剂检测试剂盒对比,在检测血清样本时,检测结果一致性良好。该研究为临床体外诊断内毒素快速检测提供技术基础。

In this paper,phage display technology was used to obtain endotoxin-specific binding of dodecapeptide after two rounds of non-specific screening and one round of specific screening. The molecular interaction experiments show that the dodecapeptide has good affinity with endotoxin,the dissociation constant is 3.52·10-9,and the dodecapeptide is synthesized and coupled with latex microspheres and Immuno-turbidimetric is established to detect endotoxin. The linear range is 0.03~0.48 EU/mL,R2=0.9925,and the detection limit is 0.03 EU/mL. It has good consistency with commercial kit. A technical basis is provided for the clinical diagnosis of endotoxin in vitro rapid detection and diagnosis.