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

• Affinity microcolumns were developed for one-site immunometric assays.
• These assays were tested for use with protein biomarkers, using HSA as a model.
• Several parameters were considered in the optimization of these assays.
• Results were obtained in the low pM range and within 35 s–2.8 min of injection.

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.