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Updated: Oct 3, 2025
Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Published on: June 14, 2018
Dual amplification enabled counting based ultrasensitive enzyme-linked immunosorbent assay
Haomin Liu1, Yu Lei2
1Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT, 06269, USA.
Insights
A novel dual amplification ELISA method enhances sensitivity for detecting low-abundance biomarkers. This technique significantly improves the detection limit for mouse IgG, offering a new approach for ultrasensitive biomarker analysis.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biotechnology
Background:
- Enzyme-linked immunosorbent assay (ELISA) is a primary method for biomarker detection.
- Detecting low-abundance biomarkers with ELISA requires enhanced sensitivity.
Purpose of the Study:
- To develop an ultrasensitive ELISA technique for detecting low-abundance biomarkers.
- To improve the sensitivity of conventional plate-based ELISA.
Main Methods:
- Developed a dual amplification strategy using tyramide signal amplification (TSA) and alkaline phosphatase.
- Enabled fluorescent precipitate formation for counting-based detection.
- Applied the method to detect mouse total IgG as a model biomarker.
Main Results:
- Achieved a lower limit of detection of 54.5 pg/mL for mouse IgG, a significant improvement from the commercial kit's 1.56 ng/mL.
- Demonstrated good linearity between precipitate count and IgG concentration.
- Recovery tests confirmed the reliability of the developed ultrasensitive ELISA method.
Conclusions:
- The dual amplification ELISA provides ultrasensitive detection of biomarkers.
- This method enhances the sensitivity of conventional plate-based ELISA, opening new avenues for biomarker analysis.
Abstract:
ELISA is a predominant technique in the detection of biomarkers. Notwithstanding its ubiquity and numerous advantages, its application for detection of low abundant biomarkers requires the ultrasensitivity. To bridge the gap between the need and availability, an innovative dual amplification enabled counting based ELISA was developed for ultrasensitive detection of mouse total IgG (a model biomarker). The dual amplification strategy, which is compatible with conventional plate-based ELISA, was realized through tyramide signal amplification (TSA) and alkaline phosphatase enabled formation of fluorescent precipitates. The counting of fluorescent precipitates in 25 images of each well can correlate the number of precipitates to the concentration of IgG with good linearity and lower the limit of detection of the commercial mouse IgG kit (1.56 ng/mL) to 54.5 pg/mL. Recovery tests further demonstrate the reliability of the developed method. This study opens a new avenue to improve the sensitivity of conventional plate-based ELISA.
