Purification of immunotoxins containing the ribosome-inactivating proteins gelonin and momordin using high performance liquid immunoaffinity chromatography compared with blue sepharose CL-6B affinity chromatography.
Comparable amounts of the ribosome-inactivating proteins (RIPs) ricin A chain, gelonin and momordin were allowed to bind to Blue Sepharose CL-6B (immobilised Cibacron Blue F3GA) in phosphate buffer, pH 7.5, and were then eluted quantitatively with buffer containing 0.5 M NaCl. Differences in the elution profiles indicated that the RIPs possessed different affinities for the Cibacron Blue F3GA dye. Conjugation of the RIPs to the monoclonal antibody Fib75 resulted in decreased affinity for Blue Sepharose. Under conditions allowing the complete separation of the Fib75-ricin A chain immunotoxin from unconjugated Fib75, the Fib75 immunotoxins made with gelonin and momordin failed to bind completely to the Blue Sepharose column. The Fib75-gelonin and Fib75-momordin fractions that eluted from the column with 0.5 M NaCl were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. A high performance liquid immunoaffinity chromatography procedure based on the selective binding of conjugated RIP to immobilised affinity-purified anti-RIP antibody permitted the complete separation of the gelonin and momordin immunotoxins from unconjugated Fib75 without altering the composition, molecular integrity or cytotoxic activity of the immunotoxins.
Cumber
A J
AJ
Section of Medicine, Institute of Cancer Research, Sutton, U.K.
Henry
R V
RV
Parnell
G D
GD
Wawrzynczak
E J
EJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000922:Immunotoxins / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D011817:Rabbits; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; D012270:Ribosomes / Q000187:drug effects; D012276:Ricin / Q000302:isolation & purification / Q000494:pharmacology; D012685:Sepharose / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000922:Immunotoxins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; C017842:cibacron blue-sepharose; C025674:GEL protein, Gelonium multiflorum; D012276:Ricin; D012685:Sepharose; D009699:N-Glycosyl Hydrolases