Selection of chicken single-chain antibody fragments directed against recombinant VP7 of bluetongue virus

Abstract

Viral protein seven (VP7) is a major core protein and a group-reactive antigen that can be used for the diagnosis of bluetongue virus. VP7 gene of bluetongue virus serotype 4 was expressed in E. coli. Using phage display technology, anti-VP7st4 scFvs were selected from a chicken scFv library (Nkuku®) following different panning strategies. Polyclonal phage ELISA showed that VP7st4-specific scFvs were enriched after three rounds of panning. Six different scFvs (A1, H2, TA8, TC9, TD12 and SA12) were identified by sequence analysis. Stability of these scFvs was determined by incubation at different temperatures and after several freeze/thaw cycles. The scFvs were also tested in an inhibition ELISA. Inhibition with an anti-bluetongue virus guinea pig serum resulted in a 30% decrease in ELISA signal of A1. No inhibition was obtained with the rest of the scFvs when guinea pig and sheep serum were used. An anti-bluetongue virus chicken IgY inhibited the scFvs by 50% to 86%. A fragmented-gene library displaying peptides of VP7st4 was constructed. The library was subjected to three rounds of affinity selection against the anti-VP7st4 scFvs. Enrichment of clones specific to each scFv was observed. The clones were identified by sequence analyses. The regions on VP7st4 to which the scFvs bind could not be identified since no duplicate clones were selected.