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

作者：Pfaunmiller Erika L; Anguizola Jeanethe A; Milanuk Mitchell L; Carter NaTasha; Hage David S^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2016, 1021: 91-100.

DOI：10.1016/j.jchromb.2015.12.055

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

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.

• 出版日期2016-5-15