Highly efficient immobilisation of antibody fragments to functionalised lipid monolayers.
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.
Vikholm
I
I
VTT Chemical Technology, P.O. Box 14021, FIN-33101, Tampere, Finland.
Viitala
T
T
Albers
W M
WM
Peltonen
J
J
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D008055:Lipids / Q000737:chemistry / Q000276:immunology*; D018625:Microscopy, Atomic Force; D015394:Molecular Structure; D010714:Phosphatidylethanolamines / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D008055:Lipids; D010714:Phosphatidylethanolamines; D015060:1,2-Dipalmitoylphosphatidylcholine; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine