Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein...

The COVID-19 pandemic has had a significant impact on human health management. A rapid diagnosis of SARS-CoV2 at the point-of-care (POC) is critical to prevent disease spread. As a POC device for remote settings, a LFIA should not require cold-chain maintenance and should be kept at normal temperatures. Antigen stability can be enhanced by addressing instability issues when dealing with fragile components, such as proteinaceous capture antigens. This study used immunologically guided protein engineering to enhance the capture nucleocapsid (NP) antigen stability of SARS-CoV2. A search of the IEDB database revealed that antibodies detecting epitopes are almost uniformly distributed over NP_1-419. In contrast, N-terminal stretches of NP_1-419 are theoretically more unstable than C-terminal stretches. We identified NP_250-365 as a NP stretch with a low instability index and B-cell epitopes. Apart from NP_1-419, two other variants (NP_121-419 and NP_250-365) were cloned, expressed, and purified. The degradation pattern of the proteins was observed on SDS-PAGE after three days of stability studies at -20 °C, 4 °C, and 37 °C. NP_1-419 was the most degraded while NP_250-365 exhibited the least degradation. Also, NP_1-419, NP_250-365, and NP_121-419 reacted with purified antibodies from COVID-19 patient serum. Our results suggest that NP_250-365 may be used as a stable capture antigen in LFIA devices to detect COVID-19.