Selection of antibodies for intracellular function using a two-hybrid in vivo system

Research output: Contribution to journalArticle

Abstract

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.

Details

Authors
  • M Visintin
  • E Tse
  • H Axelson
  • T H Rabbitts
  • A Cattaneo
Organisations
Research areas and keywords

Keywords

  • Animals, Antibody Affinity, Antigen-Antibody Reactions, Blotting, Western, CHO Cells, Chloramphenicol O-Acetyltransferase, Cricetinae, Disulfides, Genes, Reporter, Immunoglobulin Fragments, Models, Immunological, Saccharomyces cerevisiae, Transfection, Transformation, Genetic, Two-Hybrid System Techniques
Original languageEnglish
Pages (from-to)11723-8
JournalProceedings of the National Academy of Sciences
Volume96
Issue number21
Publication statusPublished - 1999 Oct 12
Publication categoryResearch
Peer-reviewedYes