Use of antibody fragments (Fv) in immunocytochemistry
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...
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Veröffentlicht in: | The journal of histochemistry and cytochemistry 1995-06, Vol.43 (6), p.607-614 |
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Zusammenfassung: | 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. |
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ISSN: | 0022-1554 1551-5044 |
DOI: | 10.1177/43.6.7769231 |