Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy
Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy
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DOI：
10.1021/la050162e
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发表时间：
2005-06-07
期刊：
影响因子：
3.9
通讯作者：
Dufrêne, YF
中科院分区：
文献类型：
--
作者：
Berquand, A;Xia, N;Dufrêne, YF
我们用原子力显微镜（AFM）来探讨抗溶菌酶抗体（Fv）的单个Fv片段的抗原结合力。为了检测单个分子识别事件，将基因工程组氨酸标记的Fv片段偶联到用氨三乙酸和三（乙二醇）封端的烷硫醇的混合自组装单层（SAM）修饰的AFM针尖上，而溶菌酶（Lyso）共价固定到羧基和羟基封端的烷硫醇的混合SAM上。使用X射线光电子能谱（表面化学组成）、AFM成像（水溶液中的表面形态）和表面等离子体共振（SPR，水溶液中的特异性结合）来验证官能化过程的质量。AFM力-距离曲线记录在加载速率为5000 pN/s之间的Fv-和溶血修饰的表面产生的非结合力的分布组成的整数倍的基本力量子的类似50 pN，我们归因于一个单一的抗体-抗原对的破裂。注射含有游离Lyso的溶液导致粘附概率显著降低，表明测得的50 pN解结合力是由于特异性抗体-抗原相互作用所致。为了研究相互作用的动力学，在各种加载速率下记录力-距离曲线。解束缚力与对数（加载速率）的曲线图显示了两个不同的线性制度与上升的斜率，表明多个障碍存在于能源景观。将低强度区的数据外推至零力，得到解离动力学解离速率常数（k（off）约为1 × 10（-3）s（-1）），在SPR估算的k（off）范围内。
We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of similar to 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximate to 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.