Highly efficient immobilisation of antibody fragments to functionalised lipid monolayers

Abstract

The covalent attachment of Fab' fragments of polyclonal anti-human IgG to a lipid with a terminal linker group was examined by means of quartz crystal microbalance and surface plasmon resonance measurements. The linker lipid was embedded in binary or ternary monolayers of dipalmitoylphosphatidylcholine (DPPC) and cholesterol. Atomic force microscopy images of the films deposited on silanised SiO(2) substrates showed that Fab' fragments take a standing position, thus giving site-directed immobilisation. Human IgG forms a network on interaction with the antibodies. Non-specific binding of bovine serum albumin was found to be very low when DPPC was used as the host matrix. At an optimal Fab' fragment concentration a binding capacity above 60% was obtained. However, if the surface concentration of the immobilised antibodies was too high, the binding capacity decreased due to steric hindrance. The results demonstrate that the covalent coupling of Fab' fragments to N-(epsilon-maleimidocaproyl)-dipalmitoylphosphatidylethanolamine (DPPE-EMC) embedded in a host monolayer matrix of DPPC is a promising approach to achieve a site-directed immobilisation of antibodies with high antigen-binding efficiency.

Investigating the properties of supported vesicular layers on titanium dioxide by quartz crystal microbalance with dissipation measurements

“…AFM, on the other hand, offers invaluable information concerning the organization of the surface-bound layers at nanometer length scales, and therefore provides information complementary to that provided by QCM-D. 21,22 In this study, it was used to examine the properties of SVLs formed on the surface of titanium dioxide as a function of vesicle size, bilayer composition, and vesicle concentration in solution. 21,22 In this study, it was used to examine the properties of SVLs formed on the surface of titanium dioxide as a function of vesicle size, bilayer composition, and vesicle concentration in solution.…”

Development of QCM biosensor to detect a marine derived pathogenic bacteria Edwardsiella tarda using a novel immobilisation method

“…Generally, conventional immobilisation methods based on adsorption or covalent binding via the amino group of the protein are not site-directed and produce a random orientation of the antibody, which results in a very low binding efficiency (Vikholm et al, 1999). The strategy chosen to create the sensing layer should enable to control both the amount and the orientation of the antibody on the transducer while preserving its bioactivity (Briand et al, 2007).…”

Direct Optical Detection in Bioanalytics

“…Lekkala and Sadowski [133] have used the Kretschmann configuration in conjunction with lock-in amplifiers for the highly sensitive detection of intensity changes at near-resonance conditions. The system was used for the optimization of antibody orientation on various lipid-modified surfaces [134].…”