| Abstract: | A convenient new bacteriophage display vector, pSD3, has been constructed and used to generate rabbit monoclonal anti-pesticide antibody fragments. Following amplification of immunoglobulin light chain, and heavy chain variable region gene libraries, restriction enzymes Sfi I and PflM I are used to assemble scFv libraries in pSD3. This allows the number of stages involving the polymerase chain reaction and restriction enzyme digestion to be minimized to optimize maintenance of the original diversity of the variable region genes in the libraries. The vector also incorporates an amber codon, a 6xHis tag and a c-myc epitope to facilitate soluble single-chain Fv production detection and purification. Using the pSD3 system two anti-atrazine single-chain Fvs were isolated from a library derived from the spleen cells of a rabbit immunized with bovine serum albumin-atrazine conjugate. Characterization of single-chain Fvs by competition and equilibrium ELISA indicated good specificity and affinity to atrazine. |
