School of Biotechnology, National Centre for Sensor Research and Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
School of Biotechnology, National Centre for Sensor Research and Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
Biosens Bioelectron. 2015 May 15;67:708-14. doi: 10.1016/j.bios.2014.10.039. Epub 2014 Oct 23.
Microcystins are a major group of cyanobacterial heptapeptide toxins found in freshwater and brackish environments. There is currently an urgent requirement for highly-sensitive, rapid and in-expensive detection methodologies for these toxins. A novel single chain fragment variable (scFv) fragment was generated and is the first known report of a recombinant anti-microcystin avian antibody. In a surface plasmon resonance-based immunoassay, the antibody fragment displayed cross-reactivity with seven microcystin congeners (microcystin-leucine-arginine (MC-LR) 100%, microcystin-tyrosine-arginine (MC-YR) 79.7%, microcystin-leucine-alanine (MC-LA) 74.8%, microcystin-leucine-phenylalanine (MC-LF) 67.5%, microcystin-leucine-tryptophan (MC-LW) 63.7%, microcystin-arginine-arginine (MC-RR) 60.1% and nodularin (Nod) 69.3%, % cross reactivity). Following directed molecular evolution of the parental clone the resultant affinity-enhanced antibody fragment was applied in an optimized fluorescence immunoassay on a planar waveguide detection system. This novel immuno-sensing format can detect free microcystin-LR with a functional limit of detection of 0.19 ng mL(-1)and a detection range of 0.21-5.9 ng mL(-1). The assay is highly reproducible (displaying percentage coefficients of variance below 8% for intra-day assays and below 11% for inter-day assays), utilizes an inexpensive cartridge system with low reagent volumes and can be completed in less than twenty minutes.
微囊藻毒素是在淡水和半咸水环境中发现的一种主要的蓝藻七肽毒素。目前迫切需要高度敏感、快速和廉价的这些毒素检测方法。生成了一种新型的单链片段可变 (scFv) 片段，这是首例报道的重组抗微囊藻毒素禽类抗体。在基于表面等离子体共振的免疫分析中，抗体片段与七种微囊藻同系物显示出交叉反应性 (微囊藻亮氨酸精氨酸 (MC-LR) 100%，微囊藻酪氨酸精氨酸 (MC-YR) 79.7%，微囊藻亮氨酸丙氨酸 (MC-LA) 74.8%，微囊藻亮氨酸苯丙氨酸 (MC-LF) 67.5%，微囊藻亮氨酸色氨酸 (MC-LW) 63.7%，微囊藻精氨酸精氨酸 (MC-RR) 60.1%和节球藻毒素 (Nod) 69.3%，%交叉反应性)。在亲本克隆的定向分子进化后，所得的亲和力增强的抗体片段被应用于在平面波导检测系统上优化的荧光免疫分析中。这种新型免疫传感格式可以检测游离的微囊藻毒素-LR，其功能检测限为 0.19 ng mL(-1)，检测范围为 0.21-5.9 ng mL(-1)。该测定法具有高度重现性（日内测定的变异系数低于 8%，日间测定的变异系数低于 11%），使用廉价的试剂盒系统，试剂体积低，可在不到二十分钟内完成。
