Abstract:
Phage libraries are a versatile source of recombinant antibody fragments directed against a wide variety of antigens. Recombinant antibodies have the advantage that they can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage library was panned against the 16 kDa antigen of Mycobacterium tuberculosis. Three phage displayed antibodies were obtained which bound specifically to the antigen. In soluble scFv format, however, they produced low ELISA signals. For this reason they were able to be used as models for antibody engineering. Three mutant sub-libraries were created by random mutagenesis. High stringency panning of the mutant sub-libraries against the target antigen yielded stronger binders which produced ELISA signals of up to eleven times higher than the parent scFvs. An increase in affinity was confirmed by surface plasmon resonance. One mutant scFv with a single amino acid exchange also showed an increase in the yield of scFvs it produced. Upon shortening the linker sequence between the heavy and light chains, size exclusion chromatography showed that multimerisation had occurred. Dimers, trimers and tetramers were formed thus increasing the avidity of the scFvs. Tetramers derived from the unmutated scFv showed the greatest improvement in ELISA binding. To improve expression and purification, an alternate bacterial expression vector with a histidine tag was investigated. For this series of experiments the coding region for a chicken scFv directed against VP7 of bluetongue virus was transferred from the pHEN1 display vector to the pSANG 14-3F vector, which fuses the scFv gene to a bacterial alkaline phosphatase gene. A bi-functional chicken scFv-alkaline phosphatase fusion protein that exhibits both alkaline phosphatase activity and specific antigen binding was expressed in Escherichia coli and purified in a single step via metal affinity chromatography. Furthermore the scFv-AP fusion protein was directly detected in ELISA without the use of secondary detection reagents. This study confirms that the strategies used can efficiently enhance the characteristics of chicken scFvs.
