Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal.
Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal.
复制标题
DOI：
10.1371/journal.pone.0152148
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Napier RM
中科院分区：
文献类型：
--
作者：
Badescu GO;Marsh A;Smith TR;Thompson AJ;Napier RM
特异性针对植物激素脱落酸（阿坝）的单链Fv片段抗体（scFv）已经在细菌大肠杆菌中作为融合蛋白表达。阿坝结合的动力学已经使用表面等离子体共振光谱法（BIAcore 2000）使用表面和溶液测定法测量。注意在部分质量传递限制条件下使用初始速率测量来计算每个样品中活性蛋白的浓度。融合产物、亲本单克隆抗体和游离单链抗体均具有低纳摩尔亲和力常数，但亲本单克隆抗体的解离速率常数较低，导致亲和力增加三倍。测试抗肿瘤特异性并测量结构-活性结合偏好。生物活性的（+）-阿坝对映体以比无活性的（-）-阿坝高三个数量级的亲和力被识别。阿坝的代谢产物包括菜豆酸、双菜豆酸和脱氧ABA，其亲和力比（+）-阿坝低100倍以上。scFv的这些性质使其适合作为生物报告物中的传感器结构域，其对天然存在形式的阿坝具有特异性。
A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA.