The effects of antigen size, binding site valency, and flexibility on fab-antigen binding near solid surfaces.

Abstract

Next generation antibody microarray devices have the potential to outperform current molecular detection methods and realize new applications in medicine, scientific research, and national defense. However, antibody microarrays, or arrays of antibody fragments ("fabs"), continue to evade mainstream use in part due to persistent reliability problems despite improvements to substrate design and protein immobilization strategies. Other factors could be disrupting microarray performance, including effects resulting from antigen characteristics. Target molecules embody a wide range of sizes, shapes, number of epitopes, epitope accessibility, and other physical and chemical properties. As a result, it may not be ideal for microarray designs to utilize the same substrate or immobilization strategy for all of the capture molecules. This study investigates how three antigen properties, such as size, binding site valency, and molecular flexibility, affect fab binding. The work uses an advanced, experimentally validated, coarse-grain model and umbrella sampling to calculate the free energy of ligand binding and how this energy landscape is different on the surface compared to in the bulk. The results confirm that large antigens interact differently with immobilized fabs compared to smaller antigens. Analysis of the results shows that despite these differences, tethering fabs in an upright orientation on hydrophilic surfaces is the best configuration for antibody microarrays.