Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats

Erika L. Pfaunmiller, Jeanethe A. Anguizola, Mitchell L. Milanuk, Na Tasha Carter, David S. Hage

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8-19 ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.

Original languageEnglish (US)
Pages (from-to)91-100
Number of pages10
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
StatePublished - May 15 2016


  • Affinity microcolumn
  • Chromatographic immunoassay
  • Competitive binding immunoassay
  • Human serum albumin
  • Protein G

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology


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