Chicken monoclonal antibody isolated by a phage display system.

Abstract

A method for obtaining chicken mAb using a phage display system was established. Chickens were hyperimmunized with murine serum albumin, and polyadenylated RNA was extracted from their spleen cells. cDNA of V regions of heavy or lambda-chain genes were obtained by reverse transcription-PCR using a pair of newly designed primers. A phage display library of 1.4 x10(7) clones expressing the chicken Ab variable region as a single chain was constructed. The library was panned three times against the Ag, then screened by ELISA. Of a number of Ag-binding clones, five mAb were identified by DNA sequencing. The specificity of these mAb for serum albumin of various species was examined by ELISA. The results showed different degrees of cross-reactivity to rat serum albumin, but not to other albumin. This method provides a procedure for efficiently generating specific chicken mAb against murine proteins and against the proteins of other mammals, whose amino acid sequences are highly conserved in mammalian species. The nucleotide sequences of lambda-chains revealed components to develop diversification of chicken Ab molecules. Four to seven pseudogene sequences were found per lambda-chain, which are regarded as donors in gene conversion for preimmune repertoire formation. A total of 28 nucleotide substitutions, which may be somatic hypermutations in Ag-driven affinity maturation but could also be errors introduced during gene conversion or polymorphic residues, were found in five lambda-chains.