Serology assays enable researchers to determine if subjects have already been infected with severe acute respiratory coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, by measuring the presence of antibodies against SARS-CoV-2 in blood (serum or plasma). Detectable anti-SARS-CoV-2 antibodies is dependent on the immune response and can be influenced by a variety of factors including dose of virus exposure, assessment stage post-symptom onset, age, gender, and health status. Therefore, specific and sensitive serological assays are important for accurate and reliable detection of anti-SARS-CoV-2 antibodies.
SARS-CoV-2 Multiplex Serology Assays
ProcartaPlex Ig Total (Up to 14-plex including variants)
The proteins that serve as primary antigens to stimulate an immune response producing IgA, IgM, and IgG antibodies during SARS-CoV-2 infection include the nucleocapsid (N), the spike (S) protein, and sub-regions of the spike protein such as the receptor binding domain (RBD) and the S1 regions. The Nucleocapsid (N) protein has the highest homology (90%) between SARS-CoV-1 and SARS-CoV-2 (1). Serology test kits available during the early phase of the SARS-CoV-2 pandemic were developed to detect antibodies against the Nucleocapsid, which displayed significant cross-reactivity, and thus higher false positive readings for subjects exposed to SARS-CoV-1 (1). The SARS-CoV spike (S) protein assembles into a trimerized structure to form a crown-like (hence corona) appearance and is composed of a S1 and S2 subunit. Within S1, the receptor binding domain (RBD), which is located in the C-terminal subdomain, has higher identity (74%) between SARS-CoV and SARS-CoV-2 than the N-terminal domain, consistent with the view that SARS-CoV-2 may use ACE2 as its receptor for entry into host cells like SARS-CoV (2). The RBD has been identified as one of the immunodominant sites of the SARS-CoV-2 spike protein, with antibodies against the spike protein correlating well with neutralization. In addition, it is important to test serologic cross-reactivity with endemic and seasonal coronaviruses to rule out false-positive results. The Human Coronavirus Ig Total 11-Plex ProcartaPlex Panel enables screening of four SARS-CoV-2 antibodies (Spike trimer, S1 subunit, RBD, and Nucleocapsid), six coronavirus strains (SARS-CoV-1, MERS, CoV-NL63, CoV-KHU1, CoV-229E, CoV-OC43 ), and one negative control in a single well using Luminex xMAP technology.
In addition, 3 variants can be plexed with this 11-plex panel to create a 14-plex assay. The 3 variant proteins include the variants originating from the UK (B.1.1.7) α, South Africa (B.1.351) β, and Brazil (P.1) γ. Simultaneous detection of anti-SARS-CoV-2 antibodies and related coronavirus antibodies in one assay can save time to provide a complete, holistic data set using plasma or serum samples.
Neutralizing Antibody 4-plex Panel
The Human SARS-CoV-2 Variants Neutralizing Antibody 4-Plex ProcartaPlex Panel enables screening of four neutralizing anti-SARS-CoV-2 antibodies—original wild type and 3 variants (B.1.1.7 (α), B.1.351 (β), and P.1 (γ)) in a single well. This serological assay is designed with SARS-CoV-2 wild type or variant proteins conjugated to the bead. Samples or controls are added where neutralizing antibodies will bind to the proteins. This is a competitive assay where the detector antibody is a biotinylated ACE-2 that will bind to any unbound protein. Streptavidin-phycoerythrin (PE) is added and the signal is indirectly proportional to the amount of specific neutralizing antibody present. Negative controls will give the highest MFI values.
Figure 1. Schematic illustrating the principle of the Invitrogen ProcartaPlex 4-plex Neutralizing Antibody panel for SARS-CoV-2 variants.
Figure 2. Screening results from 62 PCR (+) samples for neutralizing antibodies for wild-type (WT) and 3 variants. There was no significant difference between WT and B.1.1.7. However, a significant difference in % neutralization was observed for B.1.351 and P.1 variants compared to WT.
Figure 3. Screening results from 39 Covid PCR (+) and 168 healthy PCR (-) controls for SARS-CoV-2 antigens. Results show antibody levels detected for spike timer, S1, RBD, and Nucleocapsid antigens correspond to the expected results of high antibody levels for PCR (+) and low antibody levels for PCR (-) samples.
Figure 4. Screening results from 39 Covid PCR (+) and 168 healthy PCR (-) controls for 4 common cold coronaviruses. The results showed no differences between PCR (+) and PCR (-) groups, the data reflecting prior exposure of the general population to common cold coronaviruses. This highlights the ability of this 11-plex ProcartaPlex panel to also assess the relevance of common cold coronaviruses within a single assay.
Figure 5. No false positives. 110 healthy samples that were collected in 2016 and 2017 prior to the COVID-19 pandemic were below the cut-off (data shown) for the 4 SARS-CoV-2 antigens tested in contrast to the other 2 suppliers kits that gave ~10% false positives, where a total of 12 samples that were above the cutoff (data not show).
Targeted profiling of critically ill SARS-CoV-2 subjects: from transcriptome to proteome
Hear from Dr. Douglas Fraser at Western University as he demonstrates the use of the ProcartaPlex Coronavirus Ig Total 11-plex Panel to better understand the immune response to SARS-CoV-2 and to ultimately develop strategic treatment plans for critically ill patients that enter the intensive care unit (ICU) for Covid-19. Plasma samples were collected from subjects that were admitted to the ICU at day 1, 3, 7, and 10 and screened for antibody levels using the 11-plex panel.
SARS-CoV-2 ELISA Serology Assays
Ig ELISA for Serum and Plasma
The Human SARS-CoV-2 Spike (Trimer) Ig ELISA (enzyme-linked immunosorbent assay) kits are designed to measure the amount of Ig antibodies bound to SARS-CoV-2 Spike (Trimer). A trimerized Spike protein is pre-coated in the wells of the supplied microplate. Samples and controls, including a high control that can be used as a standard, are then added into these wells and bind to the immobilized (capture) Spike protein. The wells are washed, and anti-Ig conjugated to HRP are added and will bind to any captured antibodies. The wells are washed, and a substrate solution is added that reacts with the enzyme complex to produce measurable signal. The intensity of this signal is directly proportional to the concentration of antibody present in the original specimen.
Figure 6. Schematic illustrating the principle of the Invitrogen Ig Antibody ELISA for SARS-CoV-2.
Neutralizing Antibody ELISA for Serum and Plasma
The SARS-CoV-2 Neutralizing Ab ELISA kit is designed to measure the neutralizing portion of anti-SARS-CoV-2 antibodies. A receptor binding domain (RBD) protein is pre-coated in the wells of the supplied microplate. Samples or the positive control are added into the wells. Neutralizing antibodies present in a sample that specifically bind to RBD will block any interaction with biotinylated ACE2 that is subsequently added. Signal from SA-HRP is indirectly proportional to the amount of specific neutralizing antibody present.
Figure 7. Schematic illustrating the principle of the Invitrogen Neutralizing Antibody ELISA for SARS-CoV-2.
Figure 8. Ig Assay specificity and sensitivity. To establish a calibrator for cut-off levels, 39 PCR (+) and 160 PCR (-) samples were run to determine where all PCR (-) samples are below and all PCR (+) are above. Note that there were 2 PCR (+) samples that showed low antibody levels. Using another serology assay, the 2 samples were confirmed negative as well.
Figure 10. Benchmarking of our ELISA kit was done to a competitor ELISA kit, which used the Spike S1 subunit antigen and IgG. Borderline healthy control samples were included. Red arrows show 4 examples of discrepancies where in the competitor kit, a healthy sample showed false positive and 3 positive samples showed negative or borderline. With our Invitrogen kit, there was better correlation to the expected PCR results.
Figure 11. Benchmarking to other supplier’s SARS-CoV-2 surrogate Virus Neutralization Test (sVNT) and conventional VNT. The Invitrogen ELISA showed strong correlation to other supplier´s ELISA and Plaque Reduction Neutralization Test (PRNT).
Cytokine storm detection for SARS-CoV-2 research
Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can result an increase in cytokine levels, also known as a cytokine storm and is considered to be one of the major causes of acute respiratory distress syndrome (ARDS) and multiple-organ failure. This cytokine storm, or cytokine release syndrome (CRS), is characterized by an imbalance in the cytokine network toward a pro-inflammatory response combined with an insufficient anti-inflammatory response, resulting in a loss of immune system homeostasis. Measure cytokine profiles related to cytokine storm syndrome (CSS) or cytokine release syndrome (CRS) triggered by SARS-CoV-2 infections.
Related products and resources
- Okba N, Müller MA, Li W, Wang C, GeurtsvanKessel CH, Corman VM, et al. Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients. Emerg Infect Dis. 2020;26(7):1478-1488.
- Jaimes JA, André NM, Chappie JS, Millet JK, Whittaker GR. Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop.
J Mol Biol. 2020;432(10):3309-3325.
Publications using the Human Coronavirus Ig Total 11-plex Panel
- Borena, W.; Kimpel, J.; Gierer, M.; Rössler, A.; Riepler, L.; Oehler, S.; von Laer, D.; Miholits, M. Characterization of Immune Responses to SARS-CoV-2 and Other Human Pathogenic Coronaviruses Using a Multiplex Bead-Based Immunoassay. Vaccines 2021, 9, 611.
- Fraser DD, Cepinskas G, Slessarev M, Martin CM, Daley M, Patel MA, Miller MR, Patterson EK, O'Gorman DB, Gill SE, Oehler S, Miholits M, Webb B. Detection and Profiling of Human Coronavirus Immunoglobulins in Critically Ill Coronavirus Disease 2019 Patients. Crit Care Explor. 2021 Mar 12;3(3):e0369.
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For Research Use Only. Not for use in diagnostic procedures.