The covalent coupling of antibody fragments to linkers embedded in a monolayer matrix of phosphatidylcholine and cholesterol was examined at the air-water interface by the means of a quartz crystal microbalance, QCM. Two linkers that bind the free thiols of the Fab′ fragment were investigated. The nonspecific binding of bovine serum albumin and the specific binding of antigen were also monitored with the QCM. Standardized radioimmunoassay was used to confirm the immunoreaction and determine binding parameters. The monolayer formation of the linker lipids in the ternary system of phosphatidylcholine and cholesterol was, moreover, demonstrated by film balance studies. The results demonstrate that the covalent coupling of Fab′ fragments to linking groups embedded in a phospholipid monolayer matrix is a promising approach to achieve a defined immobilization of antibodies at the sensor surface with high antigen binding effeciency.