PURIFICATION AND APPLICATION OF A SINGLE-CHAIN FV ANTIBODY FRAGMENT SPECIFIC TO HEPATITIS-B VIRUS SURFACE-ANTIGEN

Immobilized metal affinity chromatography (IMAC) has been recently app lied to the purification of recombinant proteins bearing multi-histidi ne domains at their N or C terminus. We have now used this procedure f or the single-step purification of an anti-Hepatitis B virus surface a ntigen (HBsAg) single-chain Fv (scFv) antibody fragment. Adjusting the metal ion (Cu+2 or Ni+2) and elution conditions (pH or imidazole), we efficiently separated active scFv forms from inactive molecules. Achi eved purity was 93%, with a 20% yield with respect to the scFv content in the initial material. The pure scFv was coupled to CNBr-activated Sepharose(R) 4B and compared to the original monoclonal antibody (MAb) CB-Hep.1 in the immunoaffinity purification of a vaccine recombinant HBsAg (r-HBsAg). Results indicate that eluted antigen per mg of couple d ligand was similar for the scFv and the MAb when pure r-HBsAg was us ed as starting material. Preliminary results with unpurified starting material are also encouraging.