Covid-19 serology tests research applications

A serology test is a powerful tool to be used in epidemiological studies, they help understand viral behaviours and monitor the effectiveness of the different virus control measures as vaccination.

Anti-SARS-CoV-2 Assays can qualitatively and sometimes quantitatively show the concentration of antibodies against the virus that is present in a particular sample, and the assessment of these antibodies’ titers provide useful information on virus circulation, exposure, and replication.

PCR tests reveal the present infection and helps avoiding the viral transmission, on the other hand, serological assays determine the production of antibodies the system generate to protect from the virus in the future. Both are vital tools to be used in the management of the pandemic, and in particular, serology antibody tests are fundamental to reveal the acquired immunity of patients who have suffered the disease or have been vaccinated against it, which is vital in order to monitor the level of immunity in the population.

Why is this testing important? Because it allows to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Without data on COVID-19 we would not understand how the pandemic is progressing, testing is our way to obtain information about how the pandemic is progressing and how the virus it is spreading, which is fundamental.

▌What does serology testing means?

Serology tests detect a certain form of immune response acquired in response to a disease antigen. Serology relies on the immune system identifying an antigen (usually a protein) as unusual and triggering a humoral response.

Anti‐SARS‐CoV‐2 assays can evaluate and quantity the level of IgG, IgM, IgA antibodies present in a sample; therefore, they can identify the presence of neutralizing antibodies. In this sense, a serology test positive means that the individual has developed antibodies against the virus, because antibodies against one or more of the different proteins of the virus has been detected.

A large number of different serological assays have been developed so far. These assays usually detect antibody responses against protein S or against nucleoprotein N in COVID-19 patients, because the two proteins are highly immunogenic, but the detection of other proteins has proven to be very usefulin the interpretation of the different immunological profiles after infection and vaccination, such as the Mpro protein, which is a essential protein in the viral replication process.

Based on this information, what does a SARS-CoV-2 serology test could show? First of all is important to underline that the specificity of the assay detecting SARS-CoV-2 antibodies must be high to offer quality information

When deciding which protein to analyse will depend on the information to be extracted: The nucleocapsid protein is the virus’ most abundant protein‐antigen. However, cross‐reactivity with other coronaviruses might be an issue.

The SARS‐CoV‐2 spike protein has been the most common target because it deviates most among coronaviruses with a number of unique epitopes. The spike protein shares only 75% genome sequence identity with SARS‐CoV and the similarity with the common cold coronaviruses spike proteins is only 50% to 60%. Spike protein is critical in viral entry to the host cell by the receptor‐binding domain (RBD). (Anti-SARS-CoV-2 Immunostep ELISA Spike)

It should be noted that some assays are only qualitative, and the manufacturer may recommend that positive results must be confirmed with another method. In this sense, at Immunostep we recognized the importance of extracting the most volume of information possible with one assay and we developed a quantitative assay for the identification of 4 proteins against 3 immunoglobulins simultaneously (Anti-SARS-Cov-2  Immunostep Multiplex Assay).

FIGURE 2: HISTOGRAM IN CYTOMETRY COMPARING RESULTS OF ANALYSIS OF A POSITIVE VS. A NEGATIVE SAMPLE FOR 4 VIRAL PROTEINS. (MPRO: MAIN VIRUS PROTEASE OR 3C-TYPE PROTEASE (MPRO/3CLPRO). | NP NUCLEOCAPSID PROTEIN (N). | S: STABLE TRIMER OF THE SPICULE GLYCOPROTEIN (S). | RBD: RECEPTOR-BINDING DOMAIN (RBD) OF S-GLYCOPROTEIN) AND 3 IMMUNOGLOBULINS (IGA, IGG, AND IGM).

▌Research Applications of COVID‐19 Serology tests

At this point, there have been very few guidelines on serological testing, these studies have been mainly used to confirm the prevalence of COVID-19 infection in certain populations or sectors.

However, serological tests can be used for several purposes, these serological tests can be used to quantitatively assess antibody response in patients with COVID-19, to help identify potential plasma donors, to help identify immunity and assess antibody response to vaccines, and to help monitor the progression of herd immunity, among others.

In this sense, serological testing offers a wide range of possibilities to understand COVID-19. For this reason, seroprevalence surveillances of COVID-19 have been performed worldwide, for example, In the US, nationwide repeated cross-sectional seroprevalence research was conducted using a total of 177,919 serum samples from all 50 states.

►  Immune biomarkers to predict SARS-CoV-2 vaccine effectiveness

One of the main possible applications of serology tests for covid 19 is the study of the vaccine effectiveness in different patient’s profiles and suffering from different malignancies.

A positive serological test result means that the test has detected some antibodies to SARS-CoV-2 (COVID-19) in the blood sample, and this indicates that you are likely to have developed antibodies to the coronavirus, which could be the result of natural infection, vaccination, or both.

The study of different antibody titers in different time periods, different profiles of individuals and characteristics help researchers confront the virus and its different manifestations with more information, especially when patients suffer from other diseases, when this knowledge could be vital.

In this regard, a recent study using serology covid assays concluded that there is emerging evidence suggesting reduced effectiveness of SARS-CoV-2 vaccine in patients with hematological malignancies.

For this study, antibody response at day 7 after second-dose vaccination and at day 100 after first-dose vaccination was assessed using a CE-IVD Immunostep serological SARS-CoV-2 multiplex bead-based flow cytometry immunoassay.

Figure 3: A Peripheral blood and serum from 83 patients with hematological malignancies and 102 health care practitioners (HCP) were analyzed. B Antibody response at day 7 after the second-dose vaccination was measured using a CE-IVD serological SARS-CoV-2 multiplex bead-based flow cytometry immunoassay (See Figure).

The used of this serology test allowed the simultaneous and quantitative detection of specific IgM, IgG, and IgA antibodies to four different viral antigens present in serum: the receptor-binding domain (RBD) of the S-glycoprotein the stable trimer of the spicule (S) glycoprotein; the nucleocapsid (N) protein; and the main virus protease (Mpro). In this sense, the detection of antibodies against the N and Mpro antigens allowed the identification of individuals infected with SARS-CoV-2 before or during vaccination. Thus, the assay enabled the accurate discrimination between vaccinated and naturally infected individuals (i.e., negative and positive for both antigens, respectively).

►  Impact of SARS-CoV-2 infection analysis

Another possible application of these type of assays is to determine the impact of SARS-CoV-2 infection, as well as the concentration, and persistence of the virus, in order to generate knowledge about the level of protection and transmissibility that different individuals obtain after suffering from the infection.

In this line, a prospective multicentre study in Spain develop and validate a specific protocol for SARS-CoV-2 detection in breast milk to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration, and persistence of specific SARS-CoV-2 antibodies.

In this study, researchers generated serology reports for 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19 and for a control group of 13 women before the pandemic.

As a result of this study, all breast milk samples showed negative results for presence of SARS-CoV-2 RNA. However, they observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM, and IgG. On the other hand, main Protease (Mpro) domain antibodies were also detected in milk.

Levels of antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the non-structural main protease Mpro or 3C-like protease (3CLpro) were analysed and Mpro-reactive antibodies were quantified using ELISA kit (Immunostep Anti-SARS-CoV-2 ELISA Mpro).

The study confirmed the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer, providing crucial data to support official breastfeeding recommendations based on scientific evidence.