Orthogonal Aptamer-Lectin Co-Recognition Enables Sandwich-Type Assays for Glycoproteins

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Aptamer-based sandwich assays are crucial for protein detection.
• Challenges exist in finding two aptamers that bind distinct epitopes on target glycoproteins.
• Glycoprotein analysis requires methods that can address complex epitope binding.

Purpose of the Study:

• To develop an orthogonal co-recognition strategy for glycoprotein analysis.
• To combine aptamers targeting non-glycan epitopes with lectin-mediated glycan recognition.
• To establish a high-performance sandwich-type assay for glycoproteins.

Main Methods:

• Selected a high-affinity aptamer against non-glycosylated Ara h1.
• Developed a lectin-based enzyme-linked aptamer binding assay.
• Advanced to an aptamer-captured protein assembly assay using lectin-mediated target-enzyme complex capture.

Main Results:

• Demonstrated aptamer binding to both glycosylated and non-glycosylated Ara h1.
• Identified significant epitope overlap in conventional aptamer-only assays.
• Achieved sensitive Ara h1 detection (0.14 ng/mL) with the aptamer-captured protein assembly assay.
• Extended assay to multiple analytical readouts (HRP, urease, GOx).

Conclusions:

• The aptamer-lectin co-recognition strategy overcomes limitations in glycoprotein detection.
• The aptamer-captured protein assembly assay offers a sensitive, versatile, and simplified workflow.
• This framework is practical and generalizable for high-performance glycoprotein assays.