SARS-CoV-2 Coronavirus (COVID-19 Coronavirus) -  Neutralizing protecting SARS-CoV-2  (COVID-19) specific antibodies: neutralizing antibodies by microneutralization test.


Information 2021-01-23.


SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virus with an RNA genome (+ ssRNA) belonging to the genus Beta-Coronavirus (family Coronaviridae). The SARS-CoV-2 genome encodes four major proteins: S-spike (spike); E-envelope (envelope); M-Membrane (Membrane); and NP-Nucleoprotein (Nucleocapside) proteins. In addition, it encodes about 16 non-structural proteins (nsp 1-16), and 5 to 8 accessory proteins.

S protein (spike/spicule) has an essential role in the fixation of the virus to the cell to be infected, in the fusion of the membrane of the viral envelope with the membrane of the cell to be infected and in the penetration inside of the cell. For this reason, it is very important in the whole process of virus transmission. This protein is homotrimeric, that is, it is made up of three identical polypeptides. The S protein consist of two subunits, the S1 subunit that corresponds to the N-terminal portion of the protein, and the S2 subunit that is responsible for the fusion of both membranes (viral envelope and the membrane of the cell to be infected). The S1 subunit can be divided into the N-terminal domain (region) (NTD) and a receptor-binding domain (region) called RBD (Receptor Binding Domain). In both SARS-CoV and SARS-CoV-2, the RBD domain binds to the ACE2 receptor (Angiotensin-Converting Enzyme 2) found in pneumocytes and enterocytes, while MERS-CoV binds to the Dipeptidyl-peptidase receptor 4 (DPP4) found in the liver and lung.

The RBD domain (region) is a homotrimeric glycoprotein (three copies of the viral spike protein, S protein) that interacts with the ACE2 receptor. By binding to the ACE2 receptor, it triggers a fusion cascade that leads to penetration of the virus into the cell so that viral replication can begin.

If the interaction between RBD and ACE2 is altered, the cellular penetration of the virus is blocked. To be able to block this interaction, anti-RBD antibodies are required to bind to RBD and thus it cannot interact with the ACE2 cellular receptor.

In animal models, it has been shown that SARS-CoV-2 neutralizing antibodies (NAbs) protect against infection and are evaluated for use in human prophylaxis and therapy. In experimental studies using hamsters, neutralizing antibodies against two epitopes (aminoacid sequence) of the RBD binding domain have been shown to protect against exposure to high concentrations of SARS-CoV-2.

In humans, the majority of individuals with mild or moderate SARS-CoV-2 infection have been shown to develop a strong IgG antibody response to the viral spike protein. The amount (titre) of these antibodies detected by enzyme-linked immunosorbent methods (ELISA) is relatively stable for a period of at least 5 months and the antibody titres detected against the spike protein correlate with the antibody titres found by neutralization. Ninety percent of individuals with seroconversion, that is, they go from having no antibodies to having them, exhibit an antibody response that includes neutralizing antibodies. Cases of severe SARS-CoV-2 infections have also been shown to have antibody responses.

However, it is important to know the response in cases of mild infection and in cases of asymptomatic infection, because they correspond to the majority of infections. In these cases it is required to know the duration and functionality (neutralization in its case) of the antibody response.

SARS-CoV-2 antibodies can be targeted to structural and non-structural antigens. Two types of antigens are used in serological tests: an NP (nucleoprotein) protein that is abundant and found either within the virus or within the infected cell where the virus replicates. Due to the biological function of the nucleoprotein (NP) and because it is protected from antibodies by the viral membrane of the virus or by the cell membrane in the case of the infected cell, it is unlikely that antibodies against the nucleoprotein can neutralize or block the cellular penetration of SARS-CoV-2. The second protein that is often used as a target for detecting antibodies is the spike protein (S).

Rapid tests for IgM and/or IgG antibodies

Rapid antibody detection tests that allow results to be obtained in a maximum time of about 15 minutes, called “Qualitative Membrane Based Immunoassays”, “Solid Phase Immunochromatographic test”, “Lateral Flow test” or simply rapid IgM/IgG tests, are based on the following procedure:

A device that contains a membrane in which anti-IgM and anti-IgG antibodies are arranged along two lines, respectively, to fix the human antibodies of the IgM or IgG class that may be found in samples such as plasma, serum or whole blood. All people have these IgM or IgG class antibodies, but with different types of specificity to react to certain antigens. To know if they are specific to react to the antigens of Coronavirus SARS-CoV-2 (cause of COVID-19), it is necessary to use an antigen of this Coronavirus. The different tests (kits) available can be differentiated depending of the antigen they use. Most tests do not indicate what type of antigens of this virus they include. These antigens are usually recombinant and may correspond to the virus nucleoprotein (NP), the spike (S) or some specific region of the spike. To reveal whether the antigen used has been bound by antibodies previously bound to the membrane detection systems, the antigens used are bound to colloidal gold particles, forming a conjugate called "antigen-colloidal gold conjugate". If among a person antibodies bound to the membrane of the detection system and there are specific antibodies for this Coronavirus antigens, they will bind this complex and a colored band will appear on the membrane.

It is important to know what type of proteins is used in rapid tests (kits) for the detection of IgM and/or IgG antibodies, since if these tests contain nucleoprotein antigen (NP), a correlation with the existence of nucleoprotein (NP) cannot be established or predicted the existance of neutralizing antibodies. On the contrary, if the rapid tests contain an antigen from the S1 region of the spike glycoprotein, it can be concluded that in 90% of cases, when they are positive, they can detect antibodies that could be functionally neutralizing.

Neutralizing antibodies by microneutralization

In the neutralizing antibody tests by microneutralization, a SARS-CoV-2 strain isolated in cell culture in our laboratory from a patient sample is used, which is replicated in our laboratory by cell cultures passages in Vero E6 cells and has been quantified to be able to use a given concentration of virus in the tests. These viruses are confronted for a set time with the serum or plasma of infected or vaccinated individuals, so that if they have neutralizing antibodies, they will bind to the spike proteins. Finally, to demonstrate if the viral spike of the virus and serum or plasma mixture have been bound and blocked, it is inoculated into susceptible cell cultures to determine if the virus is active or blocked, according to the effects produced in the cell cultures. The test is performed with different dilutions (concentrations) of serum or plasma in order to determine the amount (titre) of neutralizing antibodies that the evaluated person may possess.

Tests carried out at IVAMI:

  • Neutralizing antibody test by viral microneutralization using the SARS-CoV-2 strain isolated in Valencia and blood serum or plasma of the infected or vaccinated person.

Recommended sample:

  • Serum or plasma from peripheral blood of the person analyzed (minimum 1 mL) separated in a sterile tube.

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days. Longer, frozen.