Development of a microcolumn one-site immunometric assay for a protein biomarker: Analysis of alpha₁-acid...

A one-site immunometric assay based on affinity microcolumns was developed for the analysis of alpha₁-acid glycoprotein (AGP) as a model protein biomarker. In this assay, a sample containing AGP was incubated with an excess amount of a labeled binding agent, such as fluorescein-labeled anti-AGP antibodies or F_ab fragments. The excess binding agent was removed by passing this mixture through a microcolumn that contained an immobilized form of AGP, while the signal was measured for the binding agent-AGP complex in the non-retained fraction. Theoretical and practical factors were both considered in selecting the concentration of labeled binding agent, the incubation time of this agent with the sample, and the application conditions for this mixture onto the microcolumn. The effects of using various labeling methods and intact antibodies vs F_ab fragments were also considered. The final assay was performed with fluorescein-labeled anti-AGP antibodies and a 2.1 mm i.d. × 1.0 cm AGP microcolumn operated at 0.30 mL min^-1. This method required only 1 µL of serum or plasma, had a detection limit of 0.63 nM AGP, and gave a potential throughput of 2 min per sample. This assay was used to measure AGP in normal serum and plasma from patients with systemic lupus erythematosus, giving good agreement with the literature and a reference method. The same approach and guidelines can be used to create assays for other protein biomarkers by changing the labeled binding agent and immobilized protein within the microcolumn.