(620bk) Screening and Characterization of Rabbit ScFv Antibodies Recognizing Cell Surface Antigen from ScFv-Displayed Phage Library

In cancer therapeutics, diagnostics in early stage of sickness such as canser is important and demand for methods to detect the certain antigen is considerably increased. In the present circumstances, cancer screening used is the focus of the organization of cancer was collected. These diagnostic methods are painful, costly, and risky to perform the analysis and diagnosis for patients, and need to conversion to technique of simple cancer screening such as blood test and urine analysis.

 Cell surface proteins such as CD antigens and/or receptors sometimes are specifically expressed depending on cell species and morphology. Therefore, antibodies targeting these proteins result in valuable diagnostic agents and/or therapeutic drugs.

 Here, we investigated screening and characterization of single-chain Fv antibodies specific to a certain membrane protein. The 2 kinds of oligo-peptides both of which was corresponding to a part of the target antigen, were designed and chemically-synthesized. These peptides were conjugated with carrier protein KLH, and were injected to rabbit. After extraction of total RNA from spleen, scFv-displayed phage library was constructed. After 4 rounds of biopanning procedure, antigen-binding activities of scFvs isolated were evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, dissociation rate constant levels were also evaluated by SPR sensor equipped with peptide-immobilized sensor chip.

 All of scFvs possessing the antigen-binding activities were ranked and top 5 clones were produced as soluble form by recombinant E. coli cells.

 All the isolated scFvs reacted with the cells displaying target protein. These results suggested that scFv library prepared from rabbit has huge diversity and suitable for development of affinity molecules against target membrane proteins. The characteristics of scFv antibodies were also analyzed.