Objective: To clone anti-human death receptor 5 (DR5) single-chain variable fragments (scFvs) from a large natural phage antibody library and identify the antigen-binding activity.
Methods: Phage antibodies against human DR5 were screened from a large natural phage antibody library constructed in our laboratory by four rounds of panning process using eukaryotically expressed DR5-Fc protein. The antigen-binding activity of the obtained scFvs was determined by immunocytochemistry and ELISA, and then the scFVs were analyzed by DNA fingerprinting and sequence analysis.
Results: After 4 rounds of panning, we obtained 26 positive clones with specific binding ability to DR5 protein. DNA fingerprinting and sequence analysis showed that there were four different variable fragments that were derived from different gene families. Soluble scFv fragments were prepared and then confirmed to bind specifically to DR5 protein in SW480 cells by immunocytochemistry.
Conclusion: Specific anti-human DR5 scFvs has been obtained from a large phage antibody library.