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

Abstract

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 NaC1 were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. • A high performance liquidd 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.