The intracellular antibody capture technology: towards the high-throughput selection of functional intracellular antibodies for target validation.
Several approaches have been developed over the past decade to study the complex interactions that occur in biological system. The ability to carry out a comprehensive genetic analysis of an organism becomes more limited and difficult as the complexity of the organism increases because complex organisms are likely to have not only more genes than simple organisms but also more elaborate networks of interactions among those genes. The development of technologies to systematically disrupt protein networks at the genomic scale would greatly accelerate the comprehensive understanding of the cell as molecular machinery. Intracellular antibodies (intrabodies) can be targeted to different intracellular compartments to specifically interfere with function of selected intracellular gene products in mammalian cells. This technique should prove important for studies of mammalian cells, where genetic approaches are more difficult. In the context of large-scale protein interaction mapping projects, intracellular antibodies (ICAbs) promise to be an important tool to knocking out protein function inside the cell. In this context, however, the need for speed and high throughput requires the development of simple and robust methods to derive antibodies which function within cells, without the need for optimization of each individual ICAb. The successful inhibition of biological processes by intrabodies has been demonstrated in a number of different cells. The performance of antibodies that are intracellularly expressed is, however, somewhat unpredictable, because the reducing environment of the cell cytoplasm in which they are forced to work prevents some antibodies, but not others, to fold properly. For this reason, we have developed an in vivo selection procedure named Intracellular Antibody Capture Technology (IACT) that allows the isolation of functional intrabodies. The IAC technology has been used for the rapid identification of antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by the selected intracellular antibodies. Several optimizations of the IAC technology for protein knock-out have been developed so far. This system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in different cell lines.
Visintin
Michela
M
Lay Line Genomics SpA, Via di Castel Romano 100, 00128 Rome, Italy.
Quondam
Michele
M
Cattaneo
Antonino
A
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016503:Drug Delivery Systems / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology*
D000906:Antibodies