The ELISA test (acronym of Enzyme Linked ImmunoSorbent Assay) is an immunological test intended to detect and/or assay a protein in a biological fluid.
It was conceptualised and developed by two Swedish scientists, Peter Perlmann and Eva Engvall at Stockholm University in 1971.
The molecules assayed by the ELISA technique are generally proteins. And the types of samples include biological fluid materials (plasma, serum, urine, perspirant), cell culture media or a recombinant protein (a protein produced by a cell whose genetic material has been modified by genetic recombination) purified in a solution.
The ELISA test is mainly used in immunology to detect and/or assay the presence of proteins, antibodies or antigens in a sample. This serological test notably detects the antibodies produced by the body in response to viral contamination.
The colour reaction confirms the identification of the isolated bacterium or the presence of the virus sought and the intensity of the colour gives an indication of the amount of antigens or antibodies in the given sample.
Methods
The ELISA technique is an immuno-enzymatic technique (a biochemical technique including enzymes and immuno agents like antibody or antigen) which makes it possible to visualise, from a biological sample, the reactions between an antigen (a body considered as foreign by the living organism) and an antibody using a colored reaction. Produced by an enzymatic label (generally alkaline phosphatase and peroxidase) previously attached to the antibody.
There are four main types of ELISA test: direct, indirect, competitive and sandwich.
In a direct ELISA, the antigen is bound to the bottom of the microplate well, then the antigen is bound by an antibody specific to it (the antibody has previously been conjugated to an enzyme, fluorophore or other molecule that allows detection).
Specific use : generally used to analyse an immunitary response to an antigen, for example for cell or tissue immunohistochemical staining.
Advantages: it is simple and fast, only one antibody is necessary and the process includes few stages. There is only one reaction that occurs because there is only one antibody, so cross-reaction can’t occur and so there is a lower risk of mistake.
Drawbacks: This test can be expensive because every test target needs a primary conjugated specific antibody. The enzyme fixation to the antibody can limit the available space where the antigen can bind on the antibody, so the reactivity can be limited.
In an indirect ELISA, the antigen is bound to the bottom of the microplate well, then an antibody specific to the antigen is added. A secondary antibody, conjugated to an enzyme or other detection molecule is then linked to the first antibody.
Specific use : it is generally used to determine the total amount of antibodies in the stamp.
Advantages: more than one marked antibody can bind to the primary antibody because every primary antibody contains several epitopes. No marker can interfere with the sites of the primary antibody and less marked antibodies are needed.
Drawbacks: one more incubation stage is needed for the second antibody. The signal isn’t specific because there is a cross-reactivity with the second antibody.
In a competitive ELISA, a reference antigen is bound to the bottom of the microplate wells. The specific antibody is infused with the sample, then sample plus antibodies are added to the wells, and if any antigen of interest is present in the sample, it competes with the reference antigen to bind to the antibody.
When the substrate is added, the remaining enzymes cause a chromogenic or fluorescent signal. The unbound antibody is eliminated. The greater the amount of antigen in the sample, the lower the amount of antibody bound by the reference antigen to the bottom of the wells, and the weaker the signal.
Specific use : the competitive assay is an attractive option if no suitable antibody pair can be identified for the sandwich ELISA, or if the analyte in question is too small to allow binding of a capture and detection antibody.
Advantages: it can be based on direct, indirect or sandwich ELISA test methods. The antigen is specifically detected and captured. It is highly robust, which lowers the effects of the dilution of the sample.
Drawbacks: very complex process with a lot of stages and the drawbacks of the chosen detection test method apply.
In a sandwich ELISA, two antibodies specific to two different epitopes on the target antigen are used. It can be direct or indirect, depending on whether the detection antibody is labelled or not.
The capture antibody is bound to the bottom of the microplate well, unbound antibody is washed off, the unbound protein is blocked, binding sides on the surface, the sample containing the target antigen is added, then it is incubated to allow the target antigen to bind to the immobilised capture antibody.
The well is washed to remove unbound antigen, the detection antibody label conjugate is added, then it incubates and is washed, the substrate is added and measured by chromogenic, chemiluminescent or fluorescent readout of the enzyme-substrate interaction or excited fluorophore.
Specific use: The sandwich ELISA test is suitable for complex samples, as the antigen does not require purification before measurement and offers 2 to 5 times higher sensitivity compared to the (In)direct ELISA test.
Advantages: direct and indirect detection methods can be used. Two antibodies are used to detect the antigen which makes this test highly specific.The purification of the antigen before the test isn’t required.
Drawbacks: more incubation stages are required and the cross-reactivity between the different antibodies must be checked.
Use ELISA test
The ELISA test is used to detect:
– Sexually transmitted diseases (STDs)
– Including hepatitis, syphilis, chlamydia and HIV.
Recommended by the health authorities, it is the main screening test for AIDS: it highlights the presence of anti-HIV antibodies and the p24 antigen six weeks after contamination.
– Regional or endemic diseases
Yellow Fever, Marburg Virus Disease (MVM), Dengue Fever, Lyme Disease, Chikungunya, Rift Valley Fever, Ebola, Lassa Fever, etc.
– Covid-19
To be carried out more than 2 to 3 weeks after the onset of symptoms, the ELISA test makes it possible to identify, in less than an hour, the presence of anti-SARS-CoV-2 antibodies.
– Viral pathogens causing prenatal infections
Toxoplasmosis, rubella, cytomegalovirus, herpes simplex for example.
But it has also found applications in detection of:
– Pregnancies;
– Autoimmune diseases;
– Food allergens: the quantitative determination of total immunoglobulins E (IgE) helps in the assessment and treatment of allergies;
– Hormonal imbalances;
– Tumour markers;
– Plant viruses.
Recently, the advantage of ELISA test has been spotlighted by the covid-19 pandemic. It has been significantly used in order to test as many people as possible.
The technique is easily transposable and used in commercial kits which is substantially servicing in case of such a widespread disease.
