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

Abstract

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 Fv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Fv fragments, however, offers more all-around applications in immunocytochemistry. The directly labeled Fv 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.

Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains

“…Monomaleimido-Nanogold (MMI-NG) (15 kDa) (Nanoprobes, Yaphank, NY; 1.4-nm diameter core consisting of 67 gold atoms within a 3.4-nm organic shell) was mixed with the buffer containing antibodies. The product was purified by gel filtration and ion-exchange chromatography (17). Horseradish peroxidase (HRP) (40 kDa) (Sigma) was activated through the sulfosuccinimidyl-4-(N-maleimidomethyl)-1-carboxylate (SMCC) hetero-bifunctional linker (Pierce) in sodium borate.…”

“…However, features of these reporters were not satisfactory for simultaneous localization of multiple molecules within the same sample at molecular resolution. In most cases, the reporters were big enough to create steric hindrance problems (17). Often, the core of a small reporter had to be covered with stabilizing and functional group shells (17,18,23), which significantly increased its final size.…”

“…In most cases, the reporters were big enough to create steric hindrance problems (17). Often, the core of a small reporter had to be covered with stabilizing and functional group shells (17,18,23), which significantly increased its final size. Electrostatic charges were generating repulsion forces (16).…”

“…To achieve reporting functions, quantum dots (17,18), metalligands (19,20), colloidal gold beads (21), metal clusters (22,23), metal-binding domains (MBDs) (24), carborane clusters (25), photoconverted fluorochromes (26), and enzyme molecules (8,9) have been used. However, features of these reporters were not satisfactory for simultaneous localization of multiple molecules within the same sample at molecular resolution.…”

Glassy Carbon Electrode Modified with Gold Nanoparticles and DNA for the Simultaneous Determination of Uric Acid and Norepinephrine under Coexistence of Ascorbic Acid

“…Nanosized materials have found promising technological applications in many different areas, such as microelectronic devices, 2 photocatalysis, 3 electrocatalysis, 4 biomedical applications 5 and chemical processes. 6 Nanoparticle architectures on electrode supports attract substantial research efforts directed to the development of bioelectrochemistry.…”