Multiparametric SARS-CoV-2 VRNA, Antigen, Binding and Neutralizing-Antibody Saliva-Based Diagnostic Assay

Objectives: Electric Field Induced Release and Measurement (EFIRM) is a novel platform that can quantify molecular targets in biofluids. We developed a 4-parameter clinical assay using EFIRM technology to simultaneously assess SARS-CoV-2 RNA (vRNA), nucleocapsid antigen, host binding (BAb) and neutralizing antibody (NAb) levels from a drop of saliva with performance that equals or surpasses current EUA approved tests.
Methods: The vRNA and antigen tests were developed using saliva samples from acutely infected and infection-naïve patients. BAb and NAb tests were developed using saliva samples collected from COVID-19 recovered and/or vaccinated patients, and control saliva samples collected before 2019.
Results: EFIRM SARS-CoV-2 vRNA and antigen assays achieved lower limit of detection (LOD) of 100 copies/reaction and 3.5 TCID50/mL, respectively. The vRNA assay differentiated between acutely infected (n=10) and infection-naïve patients (n=33) with an AUC of 0.9818, sensitivity of 90%, and specificity of 100%. The antigen assay similarly differentiated these patient populations with an AUC of 1.000. The BAb assay detected BAbs with an LOD of 39 pg/mL and distinguished acutely infected (n=35), vaccinated with prior infection (n=13), and vaccinated infection-naïve patients (n=13) from control (n=81) with AUC of 0.9481, 1.000, and 0.9962, respectively. The NAb assay detected NAbs with an LOD of 31.6 Unit/mL and differentiated between COVID-19 recovered or vaccinated patients (n=31) and pre-pandemic controls (n=60) with an AUC 0.923, sensitivity of 87.10%, and specificity of 86.67%.
Conclusions: Our multiparameter assay represents a significant technological advancement to simultaneously address SARS-CoV-2 infection and immunity.