Morphology and Composition of Immunodiffusion Precipitin Complexes Evaluated via Microscopy and Proteomics

New approaches to rapid, simple, in vitro diagnostic immunoassays that do not rely on centralized laboratory facilities are urgently needed for disease diagnosis and to inform treatment strategies. The recent and ongoing COVID-19 pandemic has emphasized that rapid diagnostics are needed to help guide government policies on quarantines, social distancing measures, and community lockdowns. A common approach to developing new immunoassays is to modify existing platforms (e.g., automated ELISA and lateral flow assays) for the new analyte, even though this does not address the drawbacks of existing platforms. An alternate approach is to search for robust assays that have been superseded but could in fact solve important challenges using modern technologies. Immunodiffusion is one such platform based on unique "precipitin ring" patterns formed in gels or paper following interactions between proteins and cognate antibodies in diffusion/reaction systems. Herein, we investigate the microstructure of these precipitin rings using a combination of fluorescence and electron microscopy and also perform a mass spectrometry investigation to determine the proteomic composition of the rings. We observed that the rings were composed of microparticles, which we termed "precipitin complexes", and that these complexes were composed of at least 19 key proteins, including immunoglobulins and complement factors along with a range of plasma proteins, possibly related to immune complexes and/or high-density lipoprotein particles. This information will be useful in developing new in vitro diagnostics using reaction/diffusion systems-techniques that require a single assay step and that only require calibrated length measurements for target protein quantification.