Immunosensor with a controlled orientation of antibodies by using NeutrAvidin–protein A complex at immunoaffinity layer

The orientation of antibody was controlled by using NeutrAvidin–protein A complex on the gold surface of SPR biosensor. The surface density of receptor antibody (anti-hIgG) was compared by treatment of receptor antibody to the layer of avidin, NeutrAvidin, protein A, NeutrAvidin–protein A complex and bare gold surface of SPR biosensor. The ligand antibody (hIgG) was injected to each IA layer and the binding ratio of ligand antibody per unit receptor was estimated as a parameter of orientation control. The NeutrAvidin–protein A complex on gold surface of SPR biosensor showed the highest surface density of receptor antibody as well as the binding ratio of ligand antibody per receptor antibody. The NeutrAvidin–protein A complex was also prepared on biotin-labelled SAM, and the binding ratio of ligand per receptor was found to be significantly improved in comparison to the IA layer prepared by chemical coupling of receptor antibody to the SAM layer. The NeutrAvidin–protein A complex which showed the highest efficiency for the binding of ligand antibodies, was applied for the detection of a cancer marker called CEA. By using NeutrAvidin–protein A complex and sandwich assay for signal amplification, sensitivity was improved to be 1.5-fold higher than bare gold surface and the detection of CEA with the detection limit of 30 ng/ml was achieved.