IDSP-IA: Immunotriggered Double Stem-Loop Probe-Mediated Isothermal Amplification for Attomolar Protein...

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Accurate quantification of low-abundance proteins is crucial for disease management.
• Existing protein detection methods face challenges in sensitivity and specificity.

Purpose of the Study:

• To develop a novel assay for ultrasensitive and specific protein detection.
• To establish a modular and universally adaptable platform for protein quantification.

Main Methods:

• Developed an immunotriggered double stem-loop probe-mediated isothermal amplification (IDSP-IA) assay.
• Utilized a monoclonal capture antibody (mAb1) for target protein capture and enrichment.
• Employed an aptamer-oligonucleotide switch (Apt-OS) for signal transduction to nucleic acid triggers, initiating isothermal amplification.

Main Results:

• Achieved a limit of detection (LOD) of 1.3 fg/mL for alpha-fetoprotein (AFP) and 1.4 fg/mL for interleukin-6 (IL-6).
• Demonstrated a wide dynamic range (5 fg/mL to 50 pg/mL) with no cross-reactivity from other serum proteins.
• Clinical validation showed excellent correlation with established electrochemiluminescence and chemiluminescence immunoassays.

Conclusions:

• The IDSP-IA assay provides a robust, flexible, and universally adaptable tool for attomolar protein biomarker quantification.
• This assay offers high specificity, ultrasensitivity, and a modular design suitable for diagnostic and biomedical applications.