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
Autor: | Raymond V. Henry, Alan J. Cumber, Geoffrey D. Parnell, Edward J. Wawrzynczak |
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Rok vydání: | 1990 |
Předmět: |
endocrine system
Immunology Ricin High-performance liquid chromatography Chromatography Affinity Sepharose chemistry.chemical_compound Mice Affinity chromatography Tumor Cells Cultured Immunology and Allergy Animals Gelonin N-Glycosyl Hydrolases Chromatography High Pressure Liquid Plant Proteins Chromatography Momordin Chemistry Elution Ribosome-inactivating protein Immunotoxins Antibodies Monoclonal Ribosome Inactivating Proteins Type 2 Biochemistry Ribosome Inactivating Proteins Type 1 Electrophoresis Polyacrylamide Gel Rabbits Ribosomes |
Zdroj: | Journal of immunological methods. 135(1-2) |
ISSN: | 0022-1759 |
Popis: | 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. |
Databáze: | OpenAIRE |
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