Mapping population immunity: How protease and antibody fragmentation shape wastewater findings

Wastewater-based infection surveillance relies on nucleic acids detection, however incorporating antibody detection would allow deeper insights into population immunity. Antibodies serve as biomarkers of past infections and offer a longer timeline for surveillance compared to nucleic acids. However, the complex composition of wastewater presents challenges for the sensitivity of ELISA-based immunoassays. In our study, antibody profiles in stool and upstream wastewater samples were analyzed to evaluate the feasibility of detecting antigen-specific antibodies through immunoassays. In wastewater antibodies in were present in liquid fractions and were present in multiple distinct forms with the majority being proteolytically cleaved fragments that retain antigen binding domains. However, antigen-specific ELISA performance was affected by wastewater proteases due to their altering antigens, not the antibodies, as the proteases interfered in an antigen-dependent manner that was alleviated with protease inhibitors. Moreover, by employing an anti-CH1-based ELISA, detection sensitivity was improved, but diminished recovery for standardized spiked antibodies was observed. Using a modified protocol including concentration and protease inhibitors, antibodies specific to SARS-CoV2 RBD and N protein were detected and quantified in bio banked wastewater samples. Our findings highlight the challenges and potential solutions for antibody detection in wastewater, advancing the field of wastewater-based serology as a viable tool for population-level infection surveillance.