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.

Signal Enhancement in Oriented Immunosorbent Assays: A Balance between Accessibility of Antigen Binding Sites and Avidity

“…These results suggested that avidity, which measures the total strength of binding between an antibody and its antigen, was the leading force involved in the binding of multimeric analytes. These results agreed with the observation of Bush and Knotts [ 22 ]; indeed, the authors, thanks to the developed coarse-grain model, found that multiple binding sites stabilize the immunocomplexes in microarrays.…”

Enzyme immobilization studied through molecular dynamic simulations

“…In a recent work in this regard, the Knotts group tried to devise a reliable heuristics to identify optimal attachment locations in typical proteins ( Smith et al, 2021 ), a protocol which may be promising when applied to enzyme immobilization. Using coarse-grained MD simulations they initially predicted how external factors-like confinement, tethering configuration, surface hydrophobicity and binding site valency-may affect protein stability and folding pathways ( Rathore et al, 2006 ; Bush et al, 2015 ; Bush et al, 2017 ; Bush et al, 2018 ). Additionally, they performed tethering and PEGylation at a limited number of sites in the loop regions of lysozyme to prove the capability of predicting well-performing functionalization sites ( Wei et al, 2010 ; Wei et al, 2011 ; Wei et al, 2013 ).…”

Antibody-Antigen Binding Events: The Effects of Antibody Orientation and Antigen Properties on the Immunoassay Sensitivity

“…The multiple binding leads to the stabilization of the Ab-Ag complexes, suggesting that avidity is the leading force involved in the binding of multimeric Ags. This occurs in both oriented and non-oriented ELISAs [59,60]. A clue to the role of affinity and avidity on the analytical sensitivity of ELISAs was obtained by comparing a non-oriented and an oriented ELISA for ferritin, a multimeric protein.…”