Kinetic chromatographic sequential addition immunoassays using protein A affinity chromatography

Area of Science:

• Analytical Chemistry
• Biochemistry
• Immunochemistry

Background:

• Chromatographic immunoassays offer sensitive detection but can be complex and time-consuming.
• Existing methods may suffer from desorption buffer interferences and require tagged molecules.
• There is a need for faster, simpler, and more robust immunoassay techniques.

Purpose of the Study:

• To introduce a new chromatographic immunoassay technique named kinetic immunochromatography sequential addition (KICQA).
• To evaluate the impact of flow rate on KICQA performance.
• To demonstrate the applicability of KICQA for quantifying analytes in human serum.

Main Methods:

• Sequential injection of antibody, sample (antigen), and known antigen onto a protein A column.
• Kinetic immunochromatography sequential addition (KICQA) technique.
• Optimization of flow rate and antibody concentration to control dynamic range.

Main Results:

• KICQA successfully shortens analysis times and reduces assay complexity.
• The dynamic range of KICQA is selectable over 2.5 orders of magnitude by adjusting antibody concentration.
• Accurate determination of transferrin and albumin in human serum was achieved, with albumin assay completed in under 1 minute.

Conclusions:

• KICQA is a promising new technique for rapid and simplified immunoassays.
• The kinetic nature of KICQA allows for tunable dynamic range and efficient analysis.
• KICQA demonstrates high accuracy and efficiency for clinical analyte quantification in human serum.