Use of nanogold and fluorescent-labeled antibody Fv fragments in immunocytochemistry

Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified by one-step Strep tag affinity chromatography, chemically labeled with the marker, and employed in microscopy to localize epitopes on the membrane protein bacteriorhodopsin in purple membranes of Halobacterium halobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases, methods involving directly labeled antibody fragments show results identical to those in which antibodies or Iv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Iv fragments, however, offers more all-around applications in immunocytochemistry, The directly labeled Iv fragments, half the size of an Fab fragment, are at the molecular level the smallest antibody fragments yet described for visualization of biomolecules in microscopy.