A new electrofusion method has been developed with high efficiency for hybridoma formation. This method induces more positive clones than conventional fusion methods do. This has led to the successful establishment of functional monoclonal antibodies that can modulate the function of antigens. Such antibodies are difficult to obtain using existing methods. Monoclonal antibodies that inhibit the luminescent activity of the Gaussia luciferase (Gluc) have been prepared using this method. The affinities of antibodies for their antigens were assessed qualitatively by Biacore measurements. Epitope mapping experiments showed that these antibodies recognize the conformation of Gluc antigens rather than their specific sequences. The amino acid sequence of an antigen-recognition region of an antibody was also determined. The mechanism of luminescence inhibition by the monoclonal antibodies is discussed.