Description | Targets panned | DPIS

Direct Phage to Intrabody Screening (DPIS) is a technique that was developed in the Marasco laboratory that allows the rapid isolation of functionally active intrabodies. Single-chain antibodies (sFv) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use sFv-phage libraries as a direct source of intrabodies unless a preselection step was used to identify these rare sFvs from natural libraries. We developed DPIS to bypass this preselection step. Target specific sFvs that are isolated from the Mehta I and II human sFv-phage display libraries can be directly screened in pools as intrabodies without prior knowledge of their individual identity or purity. Antigen specific sFvs that are initially identified by phage ELISA screens are grouped into pools according to the OD reading of the antigen specific phage ELISA assays and then transferred as pools into eukaryotic expression vectors, expressed as intrabodies. Biological activities of the pooled intrabodies can then be studied directly in vivo in a biological screen that analyzes target protein function and good sFv intrabody candidates are identified. Using the pooling strategy, there is no loss of individual target specific sFvs in the transfer from prokaryotic to eukaryotic expression vectors. In addition, the initial assignments into sFv pools based on phage ELISA readings allows the segregation of individual sFvs into discrete or minimally overlapping intrabody pools. This DPIS strategy should allow investigators to bypass much of the in vitro sFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic sFv phage libraries already in existence to identify intrabodies that are active in vivo.

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