Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging
Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging
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DOI：
10.1021/ac025922u
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发表时间：
2002-10-15
影响因子：
7.4
通讯作者：
Corn, RM
中科院分区：
文献类型：
--
作者：
Wegner, GJ;Lee, HJ;Corn, RM
描述了利用表面等离子体共振（SPR）成像技术研究肽-抗体相互作用而设计的金表面肽阵列的表征。多肽阵列的制备分为两步：(i)利用一组平行微通道将化学试剂通过硫醇-二硫交换反应将多肽探针共价附着在表面；（ii）第二个微通道采用环绕式设计，用作小体积流动池（5 μ l），将抗体溶液引入肽表面。作为证明，利用肽阵列SPR成像监测FLAG表位标签和单克隆抗FLAG M2的相互作用。研究这种肽抗体对是因为它作为纯化融合蛋白的重要手段。通过在含有胺端表面和惰性烷硫醇的烷硫醇混合单层上创建肽阵列，可以精确控制FLAG肽的表面覆盖。肽表位的摩尔分数也通过含有FLAG肽和不相互作用的HA或半胱氨酸的反应溶液来控制。通过研究基于FLAG结合基序的变异，利用SPR成像可以区分由单个氨基酸取代而不同的肽。此外，利用肽阵列对信号进行定量分析，同时确定四种肽的抗体-肽相互作用的结合常数。测量了FLAG肽的结合常数K-ads，发现其结合常数为1.5 x 10(8) M-1，而基于结合基序的残基由丙氨酸取代而产生的变体的结合常数为2.8 x 10(7), 5.0 x 10(6)和2.0 x 10(6) M-1。
The characterization of peptide arrays on gold surfaces designed for the study of peptide-antibody interactions using surface plasmon resonance (SPR) imaging is described. A two-step process was used to prepare the peptide arrays: (i) a set of parallel microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol-disulfide exchange reaction; (ii) a second microchannel with a wraparound design was used as a small-volume flow cell (5 muL) to introduce antibody solutions to the peptide surface. As a demonstration, the interactions of the FLAG epitope tag and monoclonal anti-FLAG M2 were monitored by SPR imaging using a peptide array. This peptide-antibody pair was studied because of its importance as a means to purify fusion proteins. The surface coverage of the FLAG peptide was precisely controlled by creating the peptide arrays on mixed monolayers of alkanethiols containing an amine-terminated surface and an inert alkanethiol. The mole fraction of peptide epitopes was also controlled by reacting solutions containing FLAG peptide and the non-interacting peptide HA or cysteine. By studying variants based on the FLAG binding motif, it was possible to distinguish peptides differing by a single amino acid substitution using SPR imaging. In addition, quantitative analysis of the signal was accomplished using the peptide array to simultaneously determine the binding constants of the antibody-peptide interactions for four peptides. The binding constant, K-ads, for the FLAG peptide was measured and found to be 1.5 x 10(8) M-1 while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 x 10(7), 5.0 x 10(6), and 2.0 x 10(6) M-1.