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

•Protein G microcolumns were developed for use in chromatographic immunoassays.•Human serum albumin was used as a model protein target for this work.•Several solution- and column-based competitive binding formats were compared.•These methods were compared in terms of their response, speed, and precision.•The guidelines that were created can be used to extend this approach to other targets.

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.