Use of antibody fragments (Fv) in immunocytochemistry.

Abstract

We developed a novel antibody fragment (Fv) technique for localization and determination of the surface topology of membrane protein complexes by immunogold electron microscopy. Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) raised against bacterial membrane proteins were established. The cDNAs coding for the variable domains of the MAbs were cloned and expressed in Escherichia coli. The engineered Fv fragments served as trifunctional adapter molecules. The Fv fragment binds to the epitope of the membrane protein. The Strep tag fused to the VH chain was used for one-step affinity purification of the Fv fragments. Immunological detection of the membrane protein-bound Fv fragments in electron microscopy was accomplished either via the Strep tag with colloidal gold-labeled streptavidin or via the c-myc tag, which was fused to the VL chain, in combination with the c-myc tag-specific antibody 9E10 and a colloidal gold-labeled secondary antibody. We examined four Fv fragments directed against the cytochrome c oxidase or the ubiquinol-cytochrome c oxidoreductase of Paracoccus denitrificans and bacteriorhodopsin of Halobacterium halobium to show that this method is generally applicable. In all cases the Fv fragments showed the same results as their corresponding parent antibodies in electron microscopic immunostaining and other applications.

Projection Structure of yidC: A Conserved Mediator of Membrane Protein Assembly

“…yidC membranes were either adsorbed to Formvar-coated grids or to poly-L-lysin-coated slides and labelled. 31 Myc epitopes genetically fused to the C-terminus of yidC were detected by a monoclonal mouse antibody (9E10, Abcam) and visualised with a gold-coupled polyclonal goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, distributed by Dianova). Specimens were stained with uranyl acetate, osmic vapour and lead citrate.…”

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

“…Martinsried. PMs, spheroplasts of Paracoccus denitrt'jicans, and hybridoma cell lines were prepared as described (12). RIBRIOUX, KLEYMANN, HAASE, HEITMA".…”

“…These include use as immunotoxins in tumor therapy (9), in the immune complex chromatographic purification of biomolecules (lo), and in the co-crystallization of membrane proteins (11). They have also been employed in immunoelectron microscopy (EM) (12). although until now the use of such fragments required labeled secondary conjugates for visualization.…”

Cell-Free Expression and Assembly of ATP Synthase

“…46 Proteoliposomes/liposomes were adsorbed to nickel grids that were Formvar and carbon coated, glow discharged, and treated with 0.01% (w/v) poly-L-lysin. The N-terminal His-tag at the β-subunit of the TA2-F 1 F o was detected by a monoclonal mouse anti-His antibody (Novagen, Darmstadt, Germany) and visualized with an 18-nm gold-coupled secondary antibody goat anti-mouse (Dianova, Hamburg, Germany).…”