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

Abstract

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 An alternate approach is to search for robust assays that have been superseded but could in fact solve important challenges using modern technologies.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.

Evaluation of techniques used for visualisation of hydrogel morphology and determination of pore size distributions

“…Agarose hydrogels form upon cooling, and the final gel structure and pore size distribution are affected by the cooling rate. 38,39 In the current study, the gel was formed with rapid cooling over 30 min from 72 to 30 °C, followed by a very slow cooling phase until the hydrogel reached 22 °C (the cooling curve was previously published 9 ). Separate agarose gels prepared from agarose solutions of various concentrations (0.38–2.0% w/v) are referred to as agarose gels of the respective concentration.…”

“…2 As the transport properties within gels are largely dominated by diffusion gradients, the pore size and pore size distribution of hydrogel materials are important design features for developing biomaterials with applications spanning diagnostic, therapeutic, drug delivery, and cell encapsulation. [3][4][5][6][7][8][9] Common to these applications is a need to control diffusive properties of small molecules and/or proteins through the hydrogel structure. In addition, the hydrogel morphology including the pore size distribution influences the mechanical properties such as compressive modulus and elongation.…”

Automated proteomic sample preparation: The key component for high throughput and quantitative mass spectrometry analysis

“…The pandemic has accelerated the need for rapid and accurate automated RNA based assays in the clinical laboratory (Tauschmann & Hovorka, 2018). Most recently, a few reports were published in using mass spectrometry‐based for COVID‐19 related screening in clinical laboratories (Cardozo et al, 2020; Jayawardena et al, 2021; Rajczewski et al, 2021; Renuse et al, 2021). One study by Cardozo et al developed an automated sample preparation method using a robotic liquid handler combined with a high throughput targeted LC‐MS/MS assay to detect SARS‐Cov‐2 nucleoprotein peptides from nasopharyngeal and oropharyngeal swabs (Cardozo et al, 2020).…”