Selection of a ScFv against a sheep parasite antigen using phage-display

Abstract

Trichostrongylus colubriformis is an important parasite in sheep and causes millions of dollars damage annually. Current control methods rely almost exclusively on anthelmintic drugs to which strains of T. colubriformis are rapidly becoming resistant. A recombinant protein antigen, T-17, from the fourth larval stage of T. colubriformis was obtained. Single-chain Fv (ScFv) antibody libraries were made from two hybridoma cell-lines each expressing a single anti-T-17 antibody. The two ScFv libraries were displayed on filamentous bacteriophage then panned against the T-17 antigen but failed to produce a functional anti-T-17 ScFv. A more diverse ScFv library was then made from the spleen of a mouse immunised with the T-17 antigen. Several phage-display panning methods were used and compared, one method using a two-step acid elution of the bound phage succeeded in selecting a clone producing ScFvs binding to the T -17 antigen which runs at 19 kDa under reducing conditions. The ScFvs from this clone and all of the other clones which gave positive T-17-binding signals in colony lift-assays and ELISA were binding to proteins in the T -17 preparation which ran at 37.5, 31, and 16.5 kDa. Evidence suggesting that these three proteins are also T-17 is discussed.