Direct detection of 4-dimensions of SARS-CoV-2: infection (vRNA), infectivity (antigen), binding antibody, and functional neutralizing antibody in saliva

Abstract

We developed a 4-parameter clinical assay using Electric Field Induced Release and Measurement (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. The 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 pre-pandemic (n = 81) with AUC of 0.9481, 1.000, and 0.9962, respectively. The NAb assay detected NAbs with a 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%. Our combo assay represents a significant technological advancement to simultaneously address SARS-CoV-2 infection and immunity, and it lays the foundation for tackling potential future pandemics.

Fig. 1

Schema and biorecognition elements of saliva SARS-CoV-2 viral RNA, N antigen, binding antibody, and neutralizing antibody assay.

Fig. 2

The analytical performance of RT-LAMP vRNA assay with extracted viral RNA. The N2 + NL RT-LAMP assay performance using quantitative PCR (qPCR) control SARS-CoV-2 viral RNA from BEI resources (cat# NR-52346) at (a) 25 copies/reaction, (b) 12.5 copies/reaction, (c) 6.25 copies/reaction, and (d) no-template negative control. The assays were conducted with SYTO-9 dye for monitoring the reaction on qPCR machine. 12 replicate reactions were performed at each concentration. The LOD of the assay was further determined with colorimetric RT-LAMP reaction on 20 replicates with 12 (e) and 6 (f) copies/reaction of SARS-CoV-2 RNA.

Fig. 3

Analytical and clinical performance of LAMP-EFIRM direct Saliva SARS-CoV-2 vRNA assay. (a) The LOD was determined with saliva spiked with heat inactivated SARS-CoV-2 virus (USA-WA1/2020 strain). NTC, no-template control. (b) Viral RNA test analysis results for RT-qPCR-positive samples of acutely infected hospitalized patients (n = 10) vs. vaccinated infection-naïve patient samples (n = 33). Box plot of vRNA test results corresponding to EFIRM measurement. The dotted line indicates cutoff of mean + 3 × SD. (c) ROC analysis of vRNA assay performance within 15 days post onset of symptoms resulted in an AUC of 0.9818.

Fig. 4

Analytical and clinical performance of EFIRM direct Saliva SARS-CoV-2 N Antigen assay. (a) Analytical linearity with NR-52,287 (gamma inactivated virus) from 0–300 TCID₅₀/mL. (b) LOD determined by 24 replicates at LOD, 2 LOD and ½ LOD. (c) Antigen test analysis results for RT-qPCR-positive samples of acutely infected hospitalized patients (n = 10) vs. vaccinated infection naïve patient samples (n = 33). Box plot of antigen test results corresponding to Log10 genome equivalence. The dotted line indicates cutoff of mean + 3 × SD. (d) ROC analysis of antigen assay performance within 15 days post onset of symptoms resulted in an AUC of 1.000. (e) Box plot of antigen test corresponding to EFIRM antigen level (TCID₅₀/mL).

Fig. 5

Analytical and Clinical performance of EFIRM direct saliva SARS-CoV-2 antibody assay. Linear range determination for (a) anti-RBD IgG, (b) IgM, and (c) IgA assays. Box plot of antibody test results corresponding to measured IgG/IgM/IgA in ng/mL for (d) total anti-RBD Immunoglobulins, (e) anti-RBD IgG, (f) anti-RBD IgM, and (g) anti-RBD IgA antibody. ELISA serum-positive samples were from acutely infected hospitalized patients (n = 35, COV+), vaccinated recovered COVID-19 outpatients (n = 13, COV + VAC+), and vaccinated infection naïve patient samples (n = 13, COV- VAC+) vs. pre-pandemic samples (n = 81). (h–j) ROC analysis of antibody test performance resulted in AUC of 0.9481, 1.000, and 0.9962 for COV+, COV + VAC+, and COV- VAC + groups, respectively.

Fig. 6

Reference range of saliva anti-RBD antibody assay of 81 pre-pandemic subjects in normalized current (ΔnA) and ng/mL of (a,b) IgG, (c-d) IgM, and (e-f) IgA assays.

Fig. 7

Analytical and clinical performance of EFIRM saliva and plasma SARS-CoV-2 neutralizing antibody assay. (a) SARS-CoV-2 NAb Calibration Curve and calculated LOD. (b) NAb test results for saliva samples of vaccinated recovered COVID-19 outpatients and vaccinated infection naïve patients (n = 31) vs. pre-pandemic SMC saliva samples (n = 60). Box plot of NAb test results corresponding to measured %inhibition. (c) ROC analysis of saliva NAb test performance resulted in an AUC of 0.923. (d) NAb test results for plasma samples of vaccinated recovered COVID-19 outpatients and vaccinated infection naïve patients (n = 30) vs. pre-pandemic plasma samples (n = 60). (e) ROC analysis of plasma NAb test performance resulted in an AUC of 1.000. (f) A correlation of r = 0.98 was found between NAb titers in cPass and EFIRM plasma NAb assays. (g) A correlation of r = 0.75 was observed between NAb titers in paired saliva and plasma measured on EFIRM platform. (h) A correlation of r = 0.77 was found between NAb titers in paired saliva and plasma measured on EFIRM and cPass platforms, respectively.