Generation and characterization of recombinant scFv antibodies detecting Eimeria acervulina surface antigen

Abstract

In our previous attempt to generate monoclonal antibodies (MAbs) against coccidia parasites that more accurately reflect the natural avian humoral immune response, we produced two chicken B-cell hybridomas, 5D11 and 2-1. While both cell lines secreted antibodies reactive with sporozoites of Eimeria acervulina, they were produced in yields too low to conduct meaningful in vivo studies. To circumvent this problem, we produced four single chain variable fragment (scFv) antibodies from the VH and VL genes of hybridomas 5D11 and 2-1. The concentration of these recombinant antibodies expressed in E. coli and purified to homogeneity was 5–6 mg/L. Three of the antibodies exhibited antigen binding specificity to Eimeria surface antigens equivalent to that of the native MAbs. Nucleotide sequence analysis of the VL genes from hybridomas 5D11 and 2- 1 and genomic DNA revealed vestiges of gene conversion with Vl pseudogenes. These recombinant scFv antibodies will prove useful for further characterization of natural Eimeria surface antigens as potential vaccine candidates.