SELECTION OF SINGLE-CHAIN VARIABLE FRAGMENT ANTIBODIES TO BLACK-CURRANT REVERSION ASSOCIATED VIRUS FROM A SYNTHETIC PHAGE DISPLAY LIBRARY

Single-chain variable fragment (scFv) antibodies that bind to black cu rrant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immuni zations. Several different BRAV-specific phage scFv were obtained quic kly, after only three rounds of selection against immobilized virus an tigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) a bsorbance values that were greater than seven times the control health y plant extracts. In contrast, comparative tests using a rabbit antise rum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the p DAP2 vector for the rapid and efficient production of scFv-alkaline ph osphatase fusion proteins. Functional fusion proteins were obtained af ter expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative w hen traditional antisera failed to detect BRAV, a weak immunogen. Furt hermore, the genetic fusion of antibody fragments to alkaline phosphat ase obviates the need for further chemical coupling procedures, and th e fusion proteins can be obtained cheaply.