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

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Erika PfaunmillerDavid S Hage

Abstract

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 8-19ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10mL/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 va...Continue Reading

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Sep 30, 2014·Journal of Chromatography. a·Erika L PfaunmillerDavid S Hage

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Citations

Sep 2, 2020·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Elliott L RodriguezDavid S Hage
Jul 2, 2021·The Analyst·Sagar DhamaneRichard C Willson
Apr 3, 2018·Methods : a Companion to Methods in Enzymology·Jeanethe A AnguizolaDavid S Hage

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