Development of an on-line immunoextraction/entrapment system for protein capture and use in drug binding studies by high-performance affinity chromatography

Elliott L. Rodriguez, Saumen Poddar, Meera Choksi, David S. Hage

Research output: Contribution to journalArticle

Abstract

An on-line purification and entrapment system was developed that could extract a protein from a sample such as serum and entrap this protein within a small column for use in high-performance affinity chromatography. Human serum albumin (HSA) was employed as a model protein for this work. Immunoextraction columns containing polyclonal anti-HSA antibodies were developed to capture and isolate HSA from applied samples. This was followed by the use of a strong cation-exchange column to recapture and focus HSA as it eluted from the immunoextraction columns. The recaptured HSA was entrapped within 1.0 cm × 2.1 mm I.D. columns containing hydrazide-activated silica and in the presence of oxidized glycogen as a capping agent. The binding and elution properties of HSA on the various components of this system were examined and optimized. The entrapped columns produced by this system were then evaluated for their use in binding studies with several sulfonylurea drugs. The HSA columns created by this approach typically contained 0.3–0.6 nmol HSA and were stable over several weeks and more than 50–60 sample injections. Drug binding constants could be determined with these columns in 8 min or less by zonal elution and gave good agreement with literature values. The same system could be used for the capture and entrapment of other proteins by utilizing antibodies against the given target for immunoextraction.

Original languageEnglish (US)
Article number121812
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1136
DOIs
StatePublished - Jan 1 2020

Keywords

  • Drug-protein binding
  • Entrapment
  • High-performance affinity chromatography
  • Human serum albumin
  • Immunoextraction
  • Protein immobilization

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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