Institut für Mikrobiologie
der Bundeswehr

What are actually antibodies?

Antibodies, also called immunoglobulins, are protein molecules formed by the immune system to fight pathogens. There are different types of antibodies, which are found in different regions of the body and perform special tasks there.


As soon as the immune system recognizes the surface structures (so-called antigens) of a pathogen, the antibodies are produced and released. They then bind to the surface of the pathogen to be fought with an end that is specifically directed at the pathogen, thereby marking it for the body's defence or directly blocking important functions of the pathogen.

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Which antibody types are important in COVID-19?

At the beginning of the immune response to SARS-CoV-2, IgA antibodies are first formed. These are mainly found in the secretions of the mucous membranes, e.g. of the respiratory tract, the eyes and the gastrointestinal tract (saliva, gastric and intestinal juice). At the entry points of the body, they serve primarily for local defence against pathogens. IgA antibodies can also be detected in the blood.


IgM antibodies are also produced very early in many other viral infections. In SARS-1 and apparently also in SARS-2 coronavirus infections, IgM antibody formation starts almost simultaneously with IgG antibody formation. The diagnostic significance of IgM detection in COVID-19 antibody diagnostics is therefore still under discussion.

IgG antibodies are formed in the blood of most people with a SARS-CoV-2 infection after about two weeks. They are part of the so-called "immunological memory". IgG antibodies remain detectable in the blood for a very long time after many viral infections and then ensure a rapid immune response if the same pathogen enters the body again.

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What are "cross-reactive" antibodies?

Cross-reactivity in this case means the binding of an antibody to two different antigens, which however have an identical or very similar binding site. Cross-reactivity is often found in the immune response against closely related bacteria or viruses.


Cross-reacting antibodies then do not attach themselves to the original antigen (for example of a "cold" coronavirus OC-43), but also to antigens of other origin (for example of SARS coronavirus-2). This can then lead to a false positive test result when measuring antibodies against SARS-CoV-2.

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Which antibody tests are used in COVID-19 antibody diagnostics?

At the Bundeswehr Institute of Microbiology we have a wide range of laboratory techniques for investigating the immune response to virus infections. For the reliable diagnosis of antibodies against SARS-CoV-2 we are currently carrying out a step-by-step diagnostic procedure: Initially, this involves a screening test for IgG antibodies using an enzyme-linked immunosorbent assay (ELISA).


We do not currently use ELISA search tests for IgA antibodies in our diagnostics, as some of these tests have falsely indicated IgA antibodies against SARS-CoV-2 in more than 10% of the samples in blood donations from autumn 2019.

However, the IgG-ELISA screening test may also give a false-positive reaction due to cross-reactivity with antibodies against other coronaviruses (such as the "common cold" coronavirus OC-43). We therefore confirm every reactive ELISA search test result with a so-called virus neutralisation test (NT). In this test, dilutions of patient serum are mixed with standardized amounts of SARS-CoV-2 and the infectivity of cell cultures is tested. Although this test takes about 3 days due to the necessary virus culture, NT is the gold standard in antibody diagnostics. Only this test can currently ensure that the detected antibodies have a neutralizing effect specifically directed against SARS-CoV-2.

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What are the requirements for reliable COVID-19 antibody diagnostics?

The test procedures used must identify all patients who have undergone infection with SARS-CoV-2 and have produced antibodies against the virus.  The ability of a test to correctly identify all these individuals is called sensitivity. A test with 100% sensitivity correctly identifies all the patients tested. A test with 80% sensitivity identifies 80% of the patients tested (true positive), but 20% of people with the disease remain undetected (false negative).

On the other hand, the test procedures used should really only display antibodies that are actually directed against SARS-CoV-2 and not those that have already been formed after previous infections against related corona viruses (such as the "common cold" corona viruses). This property of a test is called specificity. A test with 100% specificity correctly identifies all healthy people. A test with 80% specificity identifies 80% of healthy people as test negative (right-negative), but 20% of people without disease are falsely identified as test positive (false positive).

As described above, in the current situation, the use of only one COVID-19 antibody test with high sensitivity but limited specificity can lead to people carrying only cross-reactive antibodies falsely believing that they have already gone through COVID-19 unnoticed and therefore believe they are protected. Such a procedure would not correspond to the current state of medical science and the standards of virological diagnostics.

At the Bundeswehr Institute of Microbiology we therefore use a combination of two different test procedures. First, a screening test with very high sensitivity but somewhat lower specificity (ELISA) is used. This enables us to detect with great reliability all those people who do not have SARS-CoV-2 antibodies in their blood. All samples positive in the search test are then always checked with the confirmatory test with a very high specificity (virus neutralisation test). In this way, we achieve the best possible information on the COVID-19 immune status.

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Which serious and reliable statements can currently be made with COVID-19 antibody diagnostics?

First scientific studies on SARS-CoV-2 antibody formation and its course have only been started since the beginning of this year. The Bundeswehr Institute of Microbiology has been involved in such investigations since the discovery of the first COVID-19 cases in Germany in January 2020.


The following statements can be made on the basis of the initial results:

  •     The combination of ELISA screening test and neutralisation test is suitable for confirming a healed SARS-CoV-2 infection after a positive RT-PCR result.
  •     The two test procedures can also be used to clarify whether a patient would be suitable for an "antibody donation". Some universities and blood donation institutions are currently conducting studies to investigate the effectiveness of such antibody donations (passive vaccination) in people who are seriously ill with COVID-19.

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What does COVID-19 antibody detection currently not allow for?

In the current phase of the coronavirus pandemic, many people hope that antibody detection will provide information about protective effects, as can be done for some other viral diseases, such as hepatitis B or tetanus.


Questions that are often asked are for example

  •     Can an IgG-positive person with previous diseases resume his or her normal life without having to take special protective measures?
  •     Does an IgG-positive nurse really not pose a risk of infection for patients at risk?
  •     Can soldiers with positive antibody detection be transferred directly to a foreign mission without further protection or quarantine?

All these questions cannot be answered conclusively at this point in time on the basis of the available scientific knowledge. The answers to these questions are, among other things, the subject of studies which the Bundeswehr Medical Service is also conducting. It will probably take several months before the database is sufficiently robust to provide reliable and serious answers.

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Why are coronavirus rapid tests ("pregnancy tests") currently not a good alternative?

There are two types of rapid tests: antigen and antibody tests. Antigen test would directly detect the SARS coronavirus-2. However, such rapid tests are not currently available. All rapid tests offered so far are antibody tests. They are intended to detect a corona infection and thus a possible immunity. So far, however, such rapid tests are unsuitable for medical diagnostics because they do not have sufficient sensitivity (sensitivity and specificity):

  • A positive rapid test does not prove beyond doubt that a person has undergone an infection with SARS-CoV-2, or even an immune protection against COVID-19. The tested person could therefore become infected with the virus and pass it on to other people.
  • A negative rapid test does not reliably exclude an acute or past infection with SARS-CoV-2. Antibodies will not be formed in COVID-19 until more than a week after the start of infection. The tested person can therefore be highly infectious for other people despite a negative result.

Any suspicion of COVID-19 by a physician must be reported. All supposedly positive rapid antibody test results must also be examined in a professional medical laboratory. Rapid test results are not accepted by health authorities.

 

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How does the antibody screening test work exactly?

As a screening test for the detection of SARS-CoV-2 antibodies we use the so-called Enzyme-linked Immunosorbent Assay, ELISA for short, at the Bundeswehr Institute of Microbiology. This allows the rapid testing of large numbers of samples.


This is how the ELISA search test works:

  1. A plastic plate with 96 wells is coated with artificially produced protein molecules of SARS coronavirus-2.
  2. Next, a small amount of the patient's blood serum to be tested is pipetted into each of the wells. If the serum contains antibodies against the SARS-CoV-2 protein molecules on the wall, the antibodies will bind to them. After a few minutes everything that has not bound is removed from the well.
  3. Now a specially prepared, further detection antibody is pipetted in. One end of this antibody binds specifically to the antibody type IgG. The SARS-CoV-2 patient antibodies, which have coupled to the virus protein molecules in step 2, are now bound in turn. The detection antibody carries an enzyme at its rear end, which reacts in the next step and provides the measurement signal. If SARS-CoV-2 antibodies were present in the patient sample, they are now bound between the SARS-CoV protein molecules and the detection antibody.
  4. A colourless reagent is now pipetted into these "packages", which reacts with the enzyme from step 3. The result is a staining whose intensity is measured. The stronger the staining, the more antibodies are in the patient sample.

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How does the antibody confirmation test work?

For the confirmation of positive antibody detections from the ELISA screening test we at the Bundeswehr Institute of Microbiology use a so-called Virus Neutralization Test, short VNT. With this test it can be distinguished very precisely whether antibodies against SARS-Coronavirus-2 were really formed in a patient sample and whether these antibodies are actually able to prevent an infection of body cells by SARS-CoV-2.


In order to be able to examine the largest possible number of patient samples in a short time, we use a special variant of the VNT, the microneutralisation test.

This is how the SARS-CoV-2 microneutralization test works:

  1. First, a small amount of the patient's blood serum positive in the screening test is pipetted into a well of a plastic plate with 96 wells.
  2. Half of this serum is then transferred to the next well, which already contains the same amount of dilution solution. The blood serum in the second well is now only half as concentrated as in the first.
  3. This procedure is repeated three more times, so that now different dilution levels of the patient sample are present in the wells of the plastic plate.
  4. A precisely determined amount of SARS coronavirus-2 is now added to these dilutions in the biosafety laboratory. If antibodies specifically directed against SARS-CoV-2 are present in the patient sample, these now bind specifically to the viruses. In order to represent the conditions in the body as accurately as possible, we leave the antibodies and the viruses at 37°C for a few minutes.
  5. In the next step, mammalian cells from an "immortal" cell culture are added to each well. These cells can be infected by SARS-CoV-2, but only if the virus has not previously been inactivated by the antibodies from the patient sample.
  6. The plastic plate is now kept for three days at 37°C in an incubator. The mammalian cells either begin to grow into a dense cell lawn at the bottom of the well or they are completely destroyed by the virus infection.
  7. After three days we add a blue staining agent to the wells. All living cells are coloured by the dye. At the same time, the staining solution also contains a disinfectant that safely kills all viruses.
  8. After rinsing off the excess staining, it is now possible to see with the naked eye in which wells the virus has killed the cells and in which wells the antibodies from the patient had previously inactivated the virus.
  9. Through the different dilution levels of the patient sample we can approximately estimate the amount of antibodies in the blood sample and thus the strength of the immune response.

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