Dual amplification enabled counting based ultrasensitive enzyme-linked immunosorbent assay

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.