Pretreatment Methods for Human Nasopharyngeal Swabs to Increase the Signal to Noise Ratio of High Sensitivity Immunoassays

[Image: see text] Mucous samples collected through nasopharyngeal (NP) swabs are considered gold standard specimens for the detection of respiratory pathogens. Matrices of these highly viscous samples often cause significant background noises in immunoassays, especially immunoassays with high sensitivity. We demonstrated such nonspecific background signals in both a chemiluminescence enzyme-linked immunosorbent assay (ELISA) and a novel highly sensitive immunoassay called Microbubbling SARS-CoV-2 Antigen Assay (MSAA). We developed and demonstrated the effectiveness of two quick sample pretreatment methods, filtration and preadsorption, to decrease nonspecific signals and increase the signal-to-noise ratio (SNR). Using these pretreatment methods, the SNR (at 3.6 × 10(4) copies/mL of inactivated SARS-CoV-2) was increased by 42.4-fold (95% CI 41.0–43.8) and 67.1-fold (95% CI 57.9–76.3) in the MSAA, and 1.3-fold (95% CI 0.9–1.7) and 1.8-fold (95% CI 1.6–2.0) in the chemiluminescence ELISA assay. Sample pretreatment methods developed in this study are broadly adaptable for the development of immunoassays for highly viscous samples.