Adapted from the Proceedings of the National Academy of Sciences: As reported in HMS Beagle 10/15/99

This technology holds promise for treating HD. One idea is that if the right antibodies are present in a cell, then they can lock on to and inactivate the mutant huntingtin. Another idea is to find out what mutant huntingtin does in a cell and fix it by some other method.

The problem is that antibodies often fold incorrectly inside the cell and lose their binding capacity. A new technique allows researchers to quickly screen a very large number of clones to find functional antibodies inside cells.

This clever method uses hybrid antibody proteins and reporter genes that are activated when the antibody binds properly.

This should aid in the development of therapeutic and function-identification antibodies. --Jerry 10/15/99
 

 
Biochemistry, Vol. 96, Issue 21, 11723-11728, October 12, 1999
Michela Visintin, et al.

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.
 

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