Development of Absolute Quantification Methods for Digital Immunoassays: Theoretical Framework and Establishment of Droplet-Based Digital Immunoassay Techniques

This paper analyzes the current digital Immunoassays techniques and their theoretical foundations. Traditional single-molecule immunoassay models based on a single discrete Poisson distribution cannot explain or achieve absolute quantification in theory. Therefore, this paper interprets the process of target protein capture by magnetic beads in digital immunoassays as a Poisson distribution process, using the magnetic beads themselves as discrete objects to calculate microvolume (Vd). Then, it models the secondary sampling process using hypergeometric or multivariate hypergeometric distributions to achieve absolute quantification of the target proteins. The paper includes a simulated analysis of the entire process. Additionally, we introduce a droplet-based absolute quantification method using digital immunoassay, establishing two technical approaches: the bead-counting method and the external calibration method. These are compared with the Single Molecule immunoassay and the electrochemiluminescence Immunoassay method. Finally, the feasibility of the internal calibration method is discussed through simulation and modeling. The theories introduced in this paper can effectively solve the problem of absolute quantification in protein immunoassays, providing theoretical support for the establishment of precise protein quantification methods.