Single-chain Fv antibody fragment for the immunoaffinity purification of a recombinant hepatitis B virus surface antigen particle

A single-chain Fv (scFv) antibody fragment specific for the ''a'' determinant of the Hepatitis B surface antigen (HBsAg), was expressed at high levels (10-18% of the total cell protein) as an insoluble protein aggregate in the periplasm off. coli MM-294 cells grown in 5-liter fermenters, The scFv was extracted from the insoluble cell material with 8 M urea and purified by immobilized metal ion affinity chromatography (IMAC) using Sepharose-IDA-Cu+2. Elution was done with 200 mM imidazole at neutral pH, with a final purity of 87%. Urea was removed by dialysis against a phosphate buffer to restore the biological activity of the scFv, This protocol yielded approximately 10 mg of pure active scFv/1 of culture. After concentration by ultrafiltration, the scFv was coupled to BrCN-activated Sepharose CL-4B at different protein/gel ratios (0.5-4.5 mg/ml of gel), and compared with the original mouse monoclonal antibody (MAb; 5.0 mg/ml of gel) as ligand for the immunoaffinity purification of a crude preparation (20% purity) of a recombinant HBsAg (r-HBsAg; average 22 nm particles) from yeast, At 4.5 mg scFv/ml of gel the antibody fragment column exhibited similar performance to the column prepared from the intact antibody, in terms of mg of eluted antigen/ml of gel, and comparable r-HBsAg purity (70-80%), Also, the scFv column had a similar performance to that of a MAb gel, after repetitive cycles of antigen purification, These results indicate that scFv are a viable alternative for industrial immunoaffinity purification of large particulate antigens.