Development of microcolumn-based one-site immunometric assays for protein biomarkers

Erika L. Pfaunmiller, Jeanethe A. Anguizola, Mitchell L. Milanuk, Efthimia Papastavros, Na Tasha Carter, Ryan Matsuda, Xiwei Zheng, David S. Hage

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1. mm i.d. ×. 5. mm and contained 8-9. nmol of immobilized HSA. These microcolumns were used from 0.10 to 1.0. mL/min and gave results within 35. s to 2.8. min of sample injection. Limits of detection down to 0.10-0.28. ng/mL (1.5-4.2. pM) or 25-30. pg/mL (0.38-0.45. pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ±0.1-4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers.

Original languageEnglish (US)
Pages (from-to)92-100
Number of pages9
JournalJournal of Chromatography A
StatePublished - Oct 31 2014


  • Affinity microcolumn
  • Chromatographic immunoassay
  • Human serum albumin
  • Near-infrared fluorescence
  • One-site immunometric assay
  • Protein biomarker

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry


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