Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Dynamics Tracking

Abstract

Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples. Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients. Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections. Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.

Debulking SARS-CoV-2 in saliva using angiotensin converting enzyme 2 in chewing gum to decrease oral virus transmission and infection

“…Similarly, recombinant CTB-ACE2 and increasing concentrations of ACE2 gum powder effectively inhibited entry of VSV expressing S protein, compared with controls, when measured using red fluorescence. 42 Incubation of COVID samples with ACE2 gum significantly reduced the microbubble counts at femtomolar concentration, 40,41 confirming that ACE2 gum dramatically decreased the amount of nucleocapsid antigens. Unlike the microbubble assay where actual viral particles are measured, ddPCR quantifies viral RNA, which may not necessarily be associated with virions and has been shown to persist after clearance of active infection.…”

“…The microbubble SARS-CoV-2 antigen assay detects the nucleocapsid antigen of SARS-CoV-2 at femtomolar concentration. 40,41 The number and size of microbubbles correlates with the amount of nucleocapsid antigen in the sample. Because of the limited volume of swab samples, a dose-dependent study on ACE2 gum could not be done using the same patient sample or in duplicates/triplicates.…”

“…The lysed sample was then tested with the Microbubble SARS-CoV-2 Antigen Assay. 40,41 In brief, sample solutions (100 mL) were incubated with suspensions of 500,000 capture antibody functionalized magnetic beads, on a roller (12 rpm) at room temperature for 30 min. The beads were then separated using magnets and washed three times with PBS buffer (pH 7.4) containing 0.01% Tween 20, then resuspended in 100 mL of 250 ng/mL biotinylated detection antibody in PBS containing 1% BSA, on a roller (12 rpm) at room temperature for 30 min.…”