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Immune complexes of human intrinsic factor were prepared by mixing gastric juice saturated with vitamin B12, and sera from patients with pernicious anaemia that had a high proportion of binding (Type II) antibody. The complexes were isolated by sodium sulphate precipitation followed by Sephadex G-150 gel filtration. Acid conditions dissociated the immune complexes and allowed separation of specific antibody and purified antigen bound to vitamin B12 by Sephadex G-200 gel filtration. Specific antibody was covalently attached to Protein A Sepharose CL-4B by coupling with water soluble carbodiimide which allowed intrinsic factor-B12 complex to be purified directly from gastric juice. The intrinsic factor obtained after iodination, ran as a single band on SDS-polyacrylamide gel electrophoresis and was biologically active.

Type

Journal article

Journal

Clin Chim Acta

Publication Date

30/05/1984

Volume

139

Pages

155 - 165

Keywords

Anemia, Pernicious, Animals, Antigen-Antibody Complex, Biological Assay, Female, Gastric Juice, Guinea Pigs, Humans, Immunosorbent Techniques, Intrinsic Factor, Vitamin B 12