ASSEMBLY OF ANTIBODIES IN LIPID-MEMBRANES FOR BIOSENSOR DEVELOPMENT

An investigation of the incorporation of antibody in lipid films of a composition that has been used for biosensor preparation is reported. IgG that is incorporated into lipid monolayers prepared from 7:3 mixtu res of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic ac id is edge-active, and enters and penetrates the fluid region of the m ixed-phase system when monolayers are held at low pressure (<20 mN/m). It was found that there is an ''exclusion pressure'' observed in pres sure-area (pi-A) curves that are collected for monolayers that contain antibody. This term refers to a specific threshold of lateral pressur e (which is reached by monolayer compression) that can cause explusion of antibody from the interior of a membrane. Microscopic images of mo nolayers containing the fluorescent phospholipid nitro-benzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE), or antibody labeled wit h tetramethylrhodamine isothiocyanate (TRITC), were used to determine the structure of membranes, and the location of effects on structure c aused by IgG. Ellipsometric measurements of lipid monolayers that were cast onto silicon wafers by the Langmuir-Blodgett method were used to study the thickness of monolayers and to investigate the structural c hanges that occurred at the ''exclusion pressure.'' Both the use of fl uorescent antigen and ellipsometry indicated that antibody binding act ivity was present and was dependent on compression pressure. The effec ts of pH and ionic strength of sub-phase, antibody concentration, incu bation time, and lateral pressure have been examined. The results may indicate the conditions that can be used to improve the incorporation of active IgG for preparation of biosensors that are based on lipid me mbranes.