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.

UV-induced reaction kinetics of dilinoleoylphosphatidylethanolamine monolayers.

The UV-induced reactivity of dilinoleoylphosphatidylethanolamine (DLiPE) Langmuir and Langmuir-Blodgett films has been studied by in situ measurements of the changes in the mean molecular area, UV-vis and Fourier transform infrared spectroscopy, and atomic force microscopy (AFM). Optimum orientation and packing density of the DLiPE molecules in the monolayer were achieved by adding uranyl acetate to the subphase. A first-order reaction kinetic model was successfully fitted to the experimental reaction kinetics data obtained at a surface pressure of 30 mN/m. Topographical studies of LB films by AFM were performed on bilayer structures as a function of subphase composition and UV irradiation time. The orientational effect of the uranyl ions on the monolayer molecules was observed as an enhanced homogeneity of the freshly prepared monomeric LB films. However, the long-term stability of these films proved to be bad; clear reorganization and loss of a true monolayer structure were evidenced by the AFM images. This instability was inhibited for the UV-irradiated films, indicating that the UV irradiation gave rise to a cross-linked structure.