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for robust assays that have been superseded but could in fact solve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.