Demonstration of a direct capture immunoaffinity separation for C-reactive protein using a capillary-based...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Biomedical Engineering

Background:

• C-reactive protein (CRP) is a key biomarker for inflammation and cardiovascular disease (CVD) risk.
• Accurate and rapid CRP quantification is crucial for clinical diagnostics.
• Existing methods may lack the sensitivity, speed, or portability required for widespread point-of-care use.

Purpose of the Study:

• To develop and validate a direct labeling/direct capture immunoaffinity separation method for CRP.
• To demonstrate the utility of a miniaturized immunoaffinity chromatography device for biomarker analysis.
• To assess the performance of the developed system in terms of accuracy, precision, and detection limits.

Main Methods:

• Constructed a miniaturized immunoaffinity chromatography device using syringe pumps, a microchip, and a laser-induced fluorescence detector.
• Utilized biotinylated monoclonal anti-CRP immobilized on streptavidin-coated silica beads as the solid support.
• Employed a one-step fluorescent labeling of CRP in a simulated serum matrix followed by direct injection and elution with an acid gradient.

Main Results:

• Achieved a limit of detection of 57.2 ng/mL for CRP.
• Demonstrated quantitative accuracy within 17.0% and precision within 17.0% relative standard deviation across a concentration range of 0.47 to 95.0 µg/mL.
• Completed chromatographic runs in under 10 minutes, highlighting the assay's speed.

Conclusions:

• The developed miniaturized immunoaffinity chromatography system provides a simple, rapid, and potentially portable method for CRP quantification.
• The assay demonstrates good accuracy and precision, suitable for cardiovascular disease risk assessment.
• The platform's modular design allows for adaptation to measure other clinically relevant biomarkers by changing the capture antibody.