Covalent immobilization of antibody fragments onto Langmuir-Schaefer binary monolayers chemisorbed on gold

Binary solid-supported monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycolipoate (DPPGL) and 1-palmitoyl-2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine (DSDPPC) have been studied by Langmuir isotherm measurements at the air-water interface and by scanning probe microscopy (SPM) of the solid-supported films. The pendant disulfide ring of the lipoic acid moiety of DPPGL was used as a linker to covalently bind pepsin-cleaved antibody Fab' fragments via a thiol-disulfide interchange reaction. The disulfide, group of the matrix lipid DSDPPC was used to covalently bind the binary monolayer onto an ultraflat gold substrate by Langmuir-Schaefer (LS) deposition. The aim is to combine high immunological sensitivity and specificity with improved adhesion properties and hence mechanical, thermal, and chemical stability of the film. SPM was used to demonstrate the binding of Fab' fragments of polyclonal anti-human IgG to two different lipid matrixes with DPPGL concentrations of 5 and 20 mol %. The subsequent antibody-antigen complex formation was further demonstrated by SPM. Fab' fragments bound to the matrix in a slightly tilted orientation, and also some aggregates were formed. Aggregate formation was more pronounced in the film with the higher linker concentration, increasing the unspecific binding of human IgG (hIgG).