Cov19 FluoBolt™-DAT

Quantitative Duplex Antibody Test (DAT) for antibodies to SARS-CoV-2
Summary and Explanation
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus strain responsible for the Coronavirus Disease 2019 (COVID-19) and pandemic. SARS-CoV-2 emerged in China in December 2019 and is transmitted mainly through droplets and surface contact routes. Symptoms can include signs and symptoms of acute respiratory illness, such as fever, cough, shortness of breath, but the infection can also be asymptomatic.
The virus infects human cells through interaction with angiotensin converting enzyme 2 (ACE2) on the surface of respiratory cells and spike (S) protein on the outer envelope of the virion particle, specifically with its receptor binding domain (RBD). The Spike (S) and nucleocapsid protein (NC), are the main immunogens of SARS-CoV-2. Antibodies against the RBD of the S protein are considered to have neutralizing activity as they can block the interaction with the ACE2 receptor, thereby blocking cellular infiltration.
While the scientific community has focused for some time now on those neutralizing antibodies, the occurrence of immune-evasive variants and the first studies to also develop vaccines using the nucleocapsid of SARS-CoV-2 will generate the need to determine both antibody species.
Therefore, we decided to use our FluoBolt™-Technology to develop the first

Quantitative Duplex Antibody Test (DAT) for antibodies to SARS-CoV-2.

The “Cov19 FluoBoltTM-DAT” (Art. Nr. FIA-1707-FC5) allows the simultaneous and quantitative measurement of antibodies against the S1RBD AND nucleocapsid antigen of SARS-CoV-2 within one single measurement by using only 10 µl of sample and generates results within 60 minutes.

In contrast to other antibody assays, which detect all antibodies generated against the S1- or NC-protein, the “Cov19 FluoBoltTM-DAT” is epitope specific and detects immune-dominant antibodies species, i.e. the most important ones generated by either vaccination or infection. This is also nicely demonstrated by the fact, that it detects anti-S1RBD against all dominant virus variants identified so far (see chart below):
Therefore, the “Cov19 FluoBoltTM-DAT” assay may be valuable tool to establish an antibody based “protective correlate”, i.e. what amount and type of antibody gives protection for a certain period of time.

Principle of the assay:

Mef-fia for sars-cov-2 s1 and nc antibodies
Anti-S1RBD and anti-NC antibodies present in serum or plasma samples from patients compete with analogous fluorescence labelled antibodies (FITC and Cy5 labelled) for the binding sites of the NC-protein and the S1RBD domain coated onto a metal enhanced fluorescence microtiter plate (MEF-MTP). A direct relationship exists between the amount of SARS-CoV-2 antibodies present in the sample and the amount of fluorescence units (FUs) measured with a fluorescence microplate reader. Calibrators with a given amount of anti-S1RBD and anti-NC antibodies are used to construct calibration curves to quantify the antibody concentration of an unknown sample.
Impact of SARS-CoV-2 variant-associated RBD mutations on the susceptibility to serum antibodies elicited by COVID-19 infection or vaccination. Chen LL et al., Clin Infect Dis. 2021 Jul 26: doi: 10.1093/cid/ciab656. Online ahead of print.

Neutralizing antibodies against SARS-CoV-2 variants induced by natural infection or vaccination: a systematic review and pooled meta-analysis. Chen X et al Clin Infect Dis. 2021 Jul 24:ciab646. doi: 10.1093/cid/ciab646. Online ahead of print.

Antibody response to SARS-CoV-2 infection in humans: A systematic review. Post N et al., PLoS One. 2020 Dec 31;15(12):e0244126.

The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients. Smits VAJ et al., Biochem Biophys Res Commun. 2021 Mar 5;543:45-49 Anti-spike, Anti-nucleocapsid and Neutralizing Antibodies in SARS-CoV-2 Inpatients and Asymptomatic Individuals. Brochot E. et al., Front Microbiol. 2020 Oct 19;11:584251

The SARS-CoV-2 spike protein: balancing stability and infectivity. Berger I, Schaffitzel C. Cell Res. 2020 Dec;30(12):1059-1060. doi: 10.1038/s41422-020-00430-4. PMID: 33139926; PMCID: PMC7604330

Ju B, Zhang Q, Ge J, et al. Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature. 2020 Aug;584(7819):115-119. doi: 10.1038/s41586-020-2380-z. Epub 2020 May 26. PMID: 32454513