A novel method to purify immunotoxins from free antibodies using modified recombinant toxins

Mohanraj Dhanabal, Daniel K. Fryxell, Sundaram Ramakrishnan

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Monoclonal antibodies linked to toxin polypeptides (immunotoxins) are developed for clinical application against cancer and graft rejection. Immunotoxins prepared by many conventional methods often contain a trace amount of free antibody. Present studies describe a method to purify immunotoxins from free antibody in conjugation mixtures. Recombinant ricin A chain and a truncated form of diphtheria toxin (385 residues) containing ten consecutive histidine residues at the amino terminus were prepared. The modified toxin polypeptides retaining full biological activity were chemically linked to monoclonal antibodies (317G5 and 454C11) reactive to breast cancer cells. The high affinity of consecutive histidine residues for nickel-based resin (Ni-NTA) was exploited to purify immunotoxins from unreacted free antibodies. SDS-PAGE analysis of conjugates eluted from nickel column contained trace amounts of detectable free antibody whereas conjugates purified by other conventional methods using phenyl Sepharose or Cibacron blue Sepharose chromatography contained significant amounts of unconjugated antibody. Furthermore, the immunotoxin fraction containing predominantly two toxin molecules linked to one antibody can be separated from stoichiometric conjugates by Ni-NTA column. Cytotoxicity experiments showed that the complex of two toxin molecules linked to an antibody was more cytotoxic to tumor cells in vitro than the fraction enriched with immunotoxin containing equimolar stoichiometry.

Original languageEnglish (US)
Pages (from-to)165-175
Number of pages11
JournalJournal of Immunological Methods
Volume182
Issue number2
DOIs
StatePublished - Jun 9 1995
Externally publishedYes

Fingerprint

Immunotoxins
Antibodies
Nickel
Histidine
Monoclonal Antibodies
Diphtheria Toxin
Ricin
Agarose Chromatography
Peptides
Graft Rejection
Polyacrylamide Gel Electrophoresis
Neoplasms
Breast Neoplasms

Keywords

  • (Antibody)
  • Diphtheria toxin A chain
  • Immunotoxin
  • Ricin A chain

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

A novel method to purify immunotoxins from free antibodies using modified recombinant toxins. / Dhanabal, Mohanraj; Fryxell, Daniel K.; Ramakrishnan, Sundaram.

In: Journal of Immunological Methods, Vol. 182, No. 2, 09.06.1995, p. 165-175.

Research output: Contribution to journalArticle

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