Fig. 1.

Construction of single-chain Fv fragment. mRNA was isolated from the murine hybridoma cells HB8696 producing anti-HER-2/neu mAb, followed by synthesis of first strand cDNA. PCR amplification was performed for both Ig heavy and light chains with the use of cDNA as a template. Sets of sense and antisense PCR primers were designed to amplify the V_H and V_L regions. After the first round of PCR, V_H and V_L genes were purified and on a second round PCR these were linked by a (Gly₄ Ser)₃ linker. The assembled scFv PCR products were ligated into the pHEN1 phagemid, which also contained the c-myc tag peptide as a recognition marker. E. Coli transformed with the pHEN1-scFv produced soluble scFv, which was isolated and purified from culture supernatants