Influence of surfactants and antibody immobilization strategy on reducing nonspecific protein interactions for molecular recognition force microscopy
Influence of surfactants and antibody immobilization strategy on reducing nonspecific protein interactions for molecular recognition force microscopy
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DOI：
10.1021/la048437y
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发表时间：
2004-10-26
期刊：
影响因子：
3.9
通讯作者：
Schoenfisch, MH
中科院分区：
文献类型：
--
作者：
Brogan, KL;Shin, JH;Schoenfisch, MH
用原子力显微镜(AFM)监测抗体修饰的探针与底物固定蛋白之间的特异性和非特异性相互作用。用定向或随机固定的抗卵清蛋白抗体修饰探针。利用蛋白A实现了完整的免疫球蛋白的定向固定化，并用戊二醛进行了随机固定化。非特异性相互作用可能会导致抗体-抗原结合事件的错误检测，即使抗原结合部位通过定向固定策略被正确定位。因此，评估了非离子和两性离子表面活性剂，包括吐温20、吐温80、Triton X-100和CHAPS，以确定是否可以在不影响所需的特异性抗体-抗原结合的情况下减少非特异性结合事件。采用酶联免疫吸附试验和表面等离子共振试验研究了抗体与抗原的结合与固定化策略和表面活性剂浓度的关系。这些研究的数据表明，蛋白A可以用来将完整的免疫球蛋白固定在原子力显微镜探针上进行测力实验，表面活性剂有助于提高这种测量的选择性。
Specific and nonspecific interactions between antibody-modified probes and substrate-immobilized proteins were monitored by atomic force microscopy (AFM). Probes were modified with anti-ovalbumin IgG antibodies immobilized in either an oriented or a random manner. The oriented immobilization of whole IgG was accomplished through the use of Protein A, and random immobilization was carried out with glutaraldehyde. Nonspecific interactions may lead to false detection of antibody-antigen binding events even when the antigen binding sites are properly positioned by an oriented immobilization strategy. Thus, nonionic and zwitterionic surfactants, including Tween 20, Tween 80, Triton X-100, and CHAPS, were evaluated to determine if nonspecific binding events could be reduced without compromising the desired specific antibody-antigen binding. Enzyme-linked immunosorbent assay and surface plasmon resonance assays were also employed to study antibody-antigen binding as a function of immobilization strategy and surfactant concentration. The data from these studies indicate that Protein A can be used to immobilize whole IgG onto AFM probes for force measurement experiments and that a surfactant is useful for improving the selectivity for such measurements.