Production of a functional single-chain Fv fragment from the pan-leukocyte antibody WM65 using splicing by asymmetric PCR.

A method of assembly of a single-chain Fv fragment is described, whereby asymmetric polymerase chain reactions (APCR) and primer extension were used to join immunoglobulin heavy and light chain variable region genes via a linker sequence. In this procedure heavy and light chain genes, together with a linker gene containing complementary sequences, were amplified by APCR to generate single-stranded products. The single stranded heavy or light chain genes were hybridized to the relevant single-stranded link product, and extended to produce double-stranded heavy-link and link-light genes. These genes then underwent another round of APCR, resulting in two single-stranded genes (heavy-link and link-light) containing extensive overlapping sequences. Hybridization and extension of these two single-stranded products allowed the formation of the complete heavy chain-link-light chain double-stranded product. Using this method, a functional single-chain Fv fragment based on the pan-leukocyte antibody WM65 was expressed and purified from E. coli. This method of immunoglobulin gene assembly by polymerase chain reaction (PCR) offers an alternative to the current methods of the genetic engineering of antibody fragments.