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

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 un known function are predicted from genomic sequences. A major hindrance is t he paucity of antibodies that function in eukaryotic cells, presumably beca use the antibodies fold incorrectly in the cytoplasm, To overcome this prob lem, we have developed an in vivo assay for functional intracellular antibo dies 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 act ivate a reporter gene. We find that several characterized antibodies can bi nd 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 w hich a single scFv was isolated from a mixture of half a million clones, in dicating 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 t hose made from spleen RNA after immunization with antigen, for intracellula r analysis of protein function based only on genomic or cDNA sequences.