Optimization of HPLC detection for acridinium ester-labeled antibodies

Wanda S. Reiter, David S. Hage

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


In this study we focused on the optimization of reaction conditions for the online chemiluminescent detection of acridinium-ester labeled antibodies in immunoassay schemes based on high-performance liquid chromatography (HPLC). Factors that were examined in this work included the effect of hydrogen peroxide, sodium hydroxide, and surfactant concentrations on maximum and total measured luminescence. We performed initial studies on a benchtop luminometer which gave a maximum signal when using 0.05% hydrogen peroxide and 1.0 M sodium hydroxide, whereas a post-column detection scheme gave maximum light production when using 0.08% hydrogen peroxide and 0.5-1.0 M sodium hydroxide. The addition of a surfactant (Triton X-100) enhanced the chemiluminescence signal in both cases by three-to four-fold. The general trends observed agreed with earlier data obtained for low molecular weight acridinium ester conjugates, but with the optimum reagent concentrations occurring at higher levels for the labeled antibodies. Based on the final optimized detection conditions, we used a post-column reactor and protein G column to generate calibration curves for injection of the labeled antibodies onto an HPLC system.

Original languageEnglish (US)
Pages (from-to)246-252
Number of pages7
JournalJournal of Clinical Ligand Assay
Issue number4
StatePublished - Dec 1996


  • Acridinium ester
  • Chemiluminescence
  • Chromatographic immunoassay
  • HPLC
  • Post-column detection

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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