Generation and characterization of recombinant ScFv antibodies detecting Eimeria acervulina surface antigens

In our previous attempt to generate monoclonal antibodies (MAbs) against co ccidia parasites that more accurately reflect the natural avian humoral imm une response, we produced two chicken B-cell hybridomas, 5D11 and 2-1. Whil e both cell Lines secreted antibodies reactive with sporozoites of Eimeria acervulina, they were produced in yields too low to conduct meaningful in v ivo studies, To circumvent this problem, we produced four single chain vari able fragment (scFv) antibodies from the VH and VL genes of hybridomas 5D11 and 2-1. The concentration of these recombinant antibodies expressed in E. coil and purified to homogeneity was 5-6 mg/L. Three of the antibodies exh ibited antigen binding specificity to Eimeria surface antigens equivalent t o that of the native MAbs, Nucleotide sequence analysis of the VL genes fro m hybridomas 5D11 and 2-1 and genomic DNA revealed vestiges of gene convers ion with Vh pseudogenes, These recombinant scFv antibodies will prove usefu l for further characterization of natural Eimeria surface antigens as poten tial vaccine candidates.