Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly

Although nowadays many different libraries for the selection of antibodies are available, hybridoma cells and immunized animals are still an important source for the production and selection of specific binders. Therefore, this protocol describes the isolation of the coding mRNA, PCR amplification of VL and VH domains and the final assembly of these domains into the scFv format, fused to an M13 phage coat protein for enrichment by phage display and for subsequent expression as a soluble single-chain Fv (scFv) fragment. Compared to our previously published descriptions we designed an entirely new set of oligonucleotide primers for gene amplification, based on the complete mouse genome data available today, ensuring a best possible coverage of murine germline sequences on the DNA level with as few non-natural amino acid combinations introduced as possible. In the standard procedure detailed below, critical steps are emphasized and potential pitfalls and general remarks of this method highlighted at the end.