Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites

A single-chain antibody library against Eimeria tenella sporozoites was constructed by phage display. Antibody-displaying phage was selected in five panning rounds against cryopreserved E. tenella sporozoites. A 1000-fold increase in phage output and a 3000-fold enrichment were obtained after three rounds of panning, as the binding clones became the dominant population in the library. Ten clones were randomly selected from the last selection round, and their nucleotide sequences were aligned and compared to chicken germ-line sequences. Analysis of the light chain variable regions revealed possible donor pseudogenes which act as donors in gene conversion events, and contribute to the diversification of the VL immune repertoire. Possible somatic hypermutation events, a consequence of affinity maturation, were also identified. Soluble antibody was produced in a non-suppressor E. coli strain, purified by nickel affinity chromatography, and characterized by immunoblotting. In an immunofluorescence assay, this recombinant antibody showed specific binding to E. tenella sporozoites.