Advanced Immunoassay: A Whole Area Scanning (WAS) Enabled Direct-Counting Sandwich ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) has become the gold standard in the diagnosis of various biomarkers. With the understanding of the biological functions of these biomarkers and their impacts on public health, there is unprecedentedly need for their detection at ultralow concentration in order for early diagnosis of diseases. However, conventional ELISA is not sensitive enough to meet such requirement. Thus, to improve the performance of conventional ELISA has become a hot topic. Nowadays, there are two main strategies proposed along this direction: One is to optimize the procedures based on current ensemble assay concept such as reducing non-specific binding, while the other is to devise a new platform which can detect single molecule. This proposal aims at improving ELISA performance through exploiting an existing reporting system from a new perspective as well as developing a direct-counting enabled digital sensing platform.
Motivated by our study that long fluorescent precipitates with a pH-dependent size could be resulted from the reporting system consisting of alkaline phosphatase (ALP) and ELF substrate, a facile whole area scanning (WAS) enabled direct-counting strategy and platform for studying blocking efficiency in protein-solid surface binding was first accomplished with optimization of key parameters (e.g, substrate concentration, incubation time, lens selection for image scanning, and the quenching reagent), which shed light on mitigating the non-specific binding for the development of high-performance immunoassays. To further harness the power of the new platform, this advanced ALP-ELF solid fluorescence reporting system was successfully integrated with a conventional plate-based total mouse IgG detection ELISA kit to achieve a better accuracy, precision, and sensitivity. Compared with the ELISA kit without any modification, this new system has improved the sensitivity by 25 times with an excellent recovery rate over 97% for IgG-spiked serum samples. This dissertation demonstrates that there is a huge potential to integrate WAS with the ALP-ELP solid fluorescence reporting system to generate an advanced biosensing platform compatible with conventional plate-based immunoassay to achieve ultrasensitive detection of biomolecules of interest. It opens a new avenue in developing high-performance immunoassay as well as studying surface property.