COVID-19 antibody testing and ELISA (Part 1)
The coronavirus outbreak was first seen in Wuhan city, China. The outbreak came into light when WHO declared the COVID-19 disease, a pandemic. COVID-19 is caused by SARS CoV virus. As of 29 May 2020, 5.9 million people were affected by the virus, and more than 362 thousand deaths were recorded. Many countries in the world are struggling to control the spread of the disease.
As of today, there is no specific drug or treatment that can be used to treat the disease. Many theories are being applied across the globe for the testing procedures, different procedures came into light to test the presence of viruses. There is an augmented demand of various diagnostic tests by countries across the world.
The Centre for Disease Control and Prevention (CDC) classified the testing methods into two categories. The test includes the methods – viral testing and antibody testing. Viral testing – to detect the presence of virus itself, includes methods such as rt-PCR, isothermal nucleic acid amplification, antigen test etc. The antibody testing – to detect the antibodies produced , as an immune response to the infection.
Viral test :
The viral test method involves the use of a swab kit for collection of samples. The sample is taken from your respiratory system to check for the presence of SARS CoV-2 virus. The test kits are sent to laboratories for the analysis and interpretation, it takes 1-2 days to get the results.
RT-PCR (real time polymerase chain reaction) is the most widely used method. SARS CoV virus contains RNA as genetic material. After the samples (oropharyngeal/nasopharyngeal) are collected from the individuals, RNA is extracted and used for the RT-PCR assay. The RT-PCR method is an effective technique, with an automated, high-throughput workflow, reliable instrumentation, and has become the preferred method.
Antibody tests can be used to determine the percentage of the population that has contracted the disease and that is therefore presumed to be immune.However, there is no evidence that proves if a person is immune to disease even after developing the antibodies. The test involves coating viral proteins (in this case of SARS COV2 virus, the spike protein) on a plate. The samples collected are then applied on viral protein, and if the patient has antibodies to the viral protein they bind together. The antibody-antigen bound can then be detected with another wash of antibodies that produce a color that can be seen in fluorescence.
Benefits of antibody testing:
- Antibody testing is a quick method and used as first-line screening for COVID, to enable population wide screening by testing on a larger scale.
- This helps the government/organisation understand the true extent of the spread of the virus.
- Antibody testing is also useful during organizational surveillance. Asymptomatic individuals (who are infected with the virus) can be identified through antibody testing.
- Other benefits of antibody testing are selection of convalescent plasma donors (for therapy) and evaluation of host response to vaccines.
The Indian Council of Medical Research (ICMR) and National Institute of Virology (NIV) announced that ELISA test can be used for the detection of coronavirus. Scientists have developed the indigenous IgG ELISA test for the antibody detection for the SARS CoV-2. The antibody test kits were tested in Mumbai and found to have high sensitivity and specificity.
What is an ELISA test?
Enzyme linked immunosorbent assay (ELISA) is a serological test (blood test) based test that can be used to detect if a person is exposed to COVID or not. ELISA is a highly sensitive test that detects and measures the presence of antibodies, neurobiological analysis, cytokines, and other immune cells in blood.Antibodies are immunological proteins produced by the body in response to various pathogenic antigens that enter the body.
Why ELISA and how is it different from other diagnostic tests?
The currently used RT-PCR is the frontline method for the clinical diagnosis for COVID, the diagnosis validates the presence of the virus, if any. The test does not subsequently report about the infection and the recovery of the person. The collection of samples requires expertise.
The antibody test works on the same basic principle of the ELISA test. It takes 2-5 hours to get the results. The ELISA test helps know the frequency of the disease in the population. The test detects the presence of IgG antibodies. The IgG-ELISA provides a useful alternative to immunofluorescence to detect the presence of SARS-CoV2.
The World Health Organisation (WHO) recommends the ELISA test because of high sensitivity and specificity compared to other antibody tests. The test is specifically designed for surveillance purposes, screening large numbers of specimens at a time.
Basic principle of ELISA:
ELISA is performed in polystyrene microtiter plates. These plates consist of 96 wells or 384 wells. The reagents used in the ELISA test are immobilized which makes the procedure easy to perform. The assay has a monoclonal antibody coat (IgM, IgG antibodies are used). The most preferred antibody is IgG, which is purified and used in conjugate to avoid interference from other proteins when binding with the enzyme on the microtiter plate. When the blood samples are added, the primary antibody adheres to the protein.
A secondary antibody binds to a different epitope on the protein. The assay is labelled with biotin which allows for subsequent binding of a protein such as streptavidin– conjugated enzyme(commonly used enzymes are horseradish peroxidase and alkaline phosphatase). Any unbound serum components or the reagents are eliminated by thorough washing of the plates, PBS-T (Phosphate buffered saline along with Tween20) is generally used as the diluent, for removing any unbound molecules.
A chromogenic substrate (which develops a color based on the enzymatic reaction) is used for staining. Substrates such as Tetramethylbenzidine (TMB) are used for the staining. The choice of the substrate mostly depends on the type of instrumentation (spectrophotometer, fluorometer and luminometer) used. The enzyme has a fluorescent tag that converts the substrate to a product that can be detected by a fluorometer. The concentration of the proteins is determined by the standard curve of known protein concentrations. Mean absorbance is calculated for the standard, controls and the samples. The standard curve is constructed by plotting the mean absorbance on the Y axis vs concentration on the X axis or by using a computer software program. Optical densities can be measured at different wavelengths using an ELISA plate reader.
So how is ELISA performed in a laboratory? Are the results foolproof? What does a positive/negative result mean? Stay tuned for Part 2 – where you can read all about the science behind this method.