Developing a Paper-Based Antigen Assay to Differentiate Between Coronaviruses and SARS-CoV-2 Spike Variants

COVID-19 first appeared in December of 2019 in Wuhan, China. Since then it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location dependent, time consuming and relatively expensive. Thus, there is a need for a more flexible method which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here we present the first steps towards a paper-based test which can differentiate between different between the Spike protein of various coronaviruses, SARS-CoV-1, SARS-CoV-2 and CoV-HKU1 with negligible cross reactivity for HCoV-OC43 and HCoV-229E in a single assay which takes less than 30 minutes. Furthermore, our test can distinguish between fractions of the same Spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration and antigen interference is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality.