Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex.
Factor IX is a vitamin K-dependent blood clotting zymogen that is functionally defective or absent in patients with hemophilia B. A method of immunoaffinity chromatography has been developed for a one-step high yield purification of factor IX directly from plasma. The technique utilizes conformation-specific antibodies that bind solely to the metal-stabilized factor IX conformer, but not to the conformer of factor IX found in the absence of metal ions. Anti-factor IX-Ca(II) antibodies were immobilized on an agarose matrix. Human plasma in the presence of 7.5 mM MgCl2 was applied to the antibody-agarose column. The factor IX that binds to these antibodies was specifically eluted by metal chelation with EDTA. This immunopurification resulted in a 10,000-fold one-step purification of the fully functional zymogen. Purified factor IX yielded a single band upon gel electrophoresis in Na-DodSO4 and had a specific activity of 120-150 units/mg. The purified factor IX was separated from other vitamin K-dependent blood clotting proteins and hepatitis virus; no activated factor IX was detected. This method has application for the large scale purification of factor IX for the treatment of hemophilia B.
H A
HA
Liebman
S A
SA
Limentani
B C
BC
Furie
B
B
Furie
D000906:Antibodies / Q000276:immunology*; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005164:Factor IX / Q000302:isolation & purification*; D002836:Hemophilia B / Q000097:blood; D006515:Hepatitis B virus / Q000302:isolation & purification; D006801:Humans; D008670:Metals / Q000378:metabolism*; D011487:Protein Conformation
D000906:Antibodies; D000939:Epitopes; D008670:Metals; D005164:Factor IX; D002122:Calcium Chloride; D002118:Calcium