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 applied to the purification of of recombinant proteins bearing multi-histidine domains at their N or C terminus. We have now used this procedure for the single-step purification of an anti-Hepatitis B virus surface antigen (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. Achieved 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 4B and compared 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 coupled ligand was similar for the scFv and the MAb when pure r-HBsAg was used as starting material. Preliminary results with unpurified starting material are also encouraging.
Canaán-Haden
L
L
Division of Immunotechnology and Diagnostics, Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
Ayala
M
M
Fernández-de-Cossio
M E
ME
Pedroso
I
I
Rodés
L
L
Gavilondo
J V
JV
D000911:Antibodies, Monoclonal; D003300:Copper; D003488:Cyanogen Bromide; D004797:Enzyme-Linked Immunosorbent Assay; D006514:Hepatitis B Surface Antigens / Q000276:immunology* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles; D007128:Immunoglobulin Fragments / Q000302:isolation & purification*; D007163:Immunosorbent Techniques; D009532:Nickel; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D006514:Hepatitis B Surface Antigens; D007093:Imidazoles; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D003300:Copper; D009532:Nickel; D003488:Cyanogen Bromide