Optimization of Immobilized Bacterial Disaccharides for Surface Plasmon Resonance Imaging Measurements of Antibody Binding

Abstract

The interactions between proteins and immobilized carbohydrates are crucial to biological events such as cell signaling and immune response. The modification of surfaces with carbohydrates to create sensing platforms provides a pathway to study these interactions in a laboratory setting. In this work, a family of structurally related Salmonella disaccharide epitopes is immobilized on thin gold films in an array format to probe antibody binding with surface plasmon resonance (SPR) imaging. The disaccharides are modified with an alkyl thiol linker for facile immobilization to gold. Small differences in the stereochemistry of the immobilized, modified disaccharides are shown to greatly influence the binding of a monoclonal antibody. Specifically, binding is only observed to an immobilized abequose dideoxyhexose relative to a tyvelose or a paratose analogue. However, both the amount and relative strength of bound antibody depends on the distribution of disaccharide moieties in a mixed monolayer of the epitope and a nonbinding diluent molecule. We thoroughly characterize the mixed monolayers with a variety of techniques to understand the optimal density and distribution of the disaccharide for antibody capture. This work reinforces the importance of controlling the density of ligands at the interface for optimized surface based bioassays.

Valency and density matter: Deciphering impacts of immunogen structures on immune responses against a tumor associated carbohydrate antigen using synthetic glycopolymers

“…These steric crowding effects have been observed in study of self-assembled monolayer of glycans where enzymes and lectins actually bound more weakly to surfaces with higher glycan densities. 44, 45 …”

Plasmonic imaging of protein interactions with single bacterial cells

“…(Fantner et al, 2010;Lower et al, 2011) The ex situ approaches include the study of reconstituted artificial membranes (Früh et al, 2011;Hirst et al, 2013), membrane protein embedded liposomes (Liu and Boyd, 2013), and extracted surface constituents (e.g., membrane proteins (Holden et al, 2006) and sugars (Grant et al, 2008) from bacteria. Given the complexity of the bacteria, in situ study of intact bacterial cells in their native environments are more attractive.…”

Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N2-dG adducts by capillary electrophoresis immunoassay

“…As B binds independently to P 1 and P 2 , the binding constants (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) can be obtained: …”

“…(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) shows that Eq. (3-17) is consistent with the definition of binding constant of B and P 1 , as shown in the Eq.…”

“…Qualitative and quantitative study of such binding is essential not only to the understanding of the molecular basis for immune functions, and also important to the method development of immunoassays. Various techniques have been developed for binding study, such as enzyme-linked immunosorbent assay (ELISA) [2][3][4], surface plasmon resonance (SPR) [5][6][7], gel electrophoresis mobility shift assay (EMSA) [8,9], and affinity chromatography [10,11]. Most immunoassays heavily rely on the adsorption of antigen or antibody on solid/liquid surface, e.g.…”