1. Study Overview
2. What happens to the blood samples collected for this study?
3. How does an ELISA work?
4. Testing for antibodies
5. Results of antibody testing from Visit 1
6. What happens to the swab collected for this study?
7. Results of PCR from Visit 1
8. Frequently asked questions
1. Study Overview
The goal of this project is to estimate the prevalence of ongoing and resolved subclinical, asymptomatic SARS-CoV2 infections among healthcare providers at UCI Health. Details of the tests conducted for this study are described below.
2. What happens to the blood samples collected for this study?
To determine whether you had immunity to SARS-CoV-2, we first obtained peripheral blood mononuclear cells and plasma. This is accomplished by layering blood over a density separation media.
Figure 1: Depiction of PBMC and plasma isolation from whole blood (WB).
3. How does an ELISA work?
The plasma was used to test for the presence of SARS-CoV2 specific antibodies using an ELISA (Enzyme-Linked Immunosorbent Assay). Plasma is added to plates coated with an antigen. Antibodies in the plasma bind to the antigen. This binding is visualized using a second antibody directed against human IgG or IgM antibodies and coupled to a fluorophore or substrate-converting enzyme ultimately emitting a fluorescent signal that can be measured (Figure 2).
Figure 2: ELISA protocol. Image outlining stats of a typical ELISA.
4. Testing for antibodies
Plasma was first tested against the N protein of SARS-CoV-2. Any samples that showed a positive signal were then confirmed using the receptor-binding domain (RBD) of the S1 SARS-CoV2 Spike protein as a second antigen (Figure 3). This approach was used because N is very sensitive resulting in a high rate of false positives while RBD is very specific.
Figure 3: Virus Structure. 3A.) Microscopic Image of SARS-CoV-2. A.) Overall virus structure pointing out the Spike (S) Protein and N Protein. B.) Close-up of the S protein subunits: RBD (Receptor-Binding Domain), S1, and S2 proteins.
5. Results of antibody testing from Visit 1
So far only 2 individuals have confirmed antibodies against SARS-CoV-2 (Indicated by green arrows and color change)!
Figure 4: Anti-SARS CoV2 IgG seropositivity. Optical densities for positive (COVID19+) and negative (“Pre-CoV”) controls as well as surveillance samples against either the N protein or S1 RBD protein of SARS-CoV2 after seropositivity ELISA. For our negative and positive controls, we used plasma that was obtained before the Fall of 2019 (pre-COVID) and plasma from COVID19+ patients, respectively. Standard errors of the mean (SEM) are shown.
6. What happens to the swab collected for this study?
We also measured the presence of viral nucleic acids from the nasal-pharyngeal swabs using qRT-PCR.
1. The specimen is collected by swabbing the nose.
2. RNA is extracted, and reverse transcribed into complementary DNA.
3. Once the primers bind to the DNA, they provide a starting point for the
4. Amplification of the cDNA.
5. If the fluorescence crosses the threshold, the test is positive. The RNase P human gene is used as an RNA extraction procedural control to demonstrate successful recovery of RNA as well as extraction reagent integrity. The SARS2 primers were designed to detect the nucleoprotein N gene and the Spike glycoprotein S gene of the viral genome. (Figure adapted from The Worldwide Test for Covid-19, available at: globalbiotechinsights.com/articles/20247/the-worldewide-test-for-covid-19)
Figure 6: SARS2 detection by qPCR steps.
7. Results of PCR from Visit 1
No virus nucleic acids were detected in any of the samples from visit 1.
8. Frequently asked questions
Can I get reports of my test results?
Our IRB-approved protocol allows us to report suspicious PCR results to study participants within 5 days of the sample collection so that you can follow up with your primary health care provider. If you are not contacted, you can safely assume that you are tested negative. Currently, the clinical significance of the presence/absence of the antibody is unknown. Thus, our IRB-approved protocol does not allow us to report individual results to you. As more knowledge of antibodies is gained, we will work with our IRB on a mechanism for disseminating your antibody results.
Is it time for you to schedule the next visit?
Unlike some other efforts to estimate the prevalence of infection at a single time point, the goal of this study is to determine changes in COVID-19 infection risk over time. So, your ongoing participation in this study is critical for documenting any changes in infection risk that may occur over time. Please keep an eye out for reminders to fill out the follow-up survey and schedule another sample collection visit.
