Enzyme-linked immunosorbent assay (ELISA) is also known as an enzyme immunoassay (EIA). ELISA is defined as a biochemical technique used in many applications, including microbiology, blood screening, veterinary, and immunology, to detect antigens and antibodies present in a sample.
ELISA technology is considered an essential diagnostic tool in plant and medicine pathology. It is also used for quality control checks in food industries. ELISAs depend on characteristic antibodies to fix the target antigen. A detection system is then used to mark the existence and the number of antigen-bindings.
In Enzyme-linked Immunosorbent Assay (ELISA), different antigen-antibody combinations are used, including an antibody or enzyme-labelled antigen always, and enzyme activity is calculated colourimetrically. The enzyme activity is measured using a substrate that changes colour when modified by the Enzyme. The light absorption of the product formed after substrate addition is measured and converted to numeric values. Depending on the antigen-antibody combination, the different types of Elisa is called a direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, etc.
Basic Principle of ELISA
The reagents within the ELISA test are immobilized, and this makes the procedure easy to perform. The assay features an antibody coat on the microtiter plate. The well-liked antibody is IgG, which is purified and is employed in conjugate to avoid interference from other proteins when binding with the Enzyme. When the blood sample is added, the precise antibody (primary antibody) adheres to the protein of interest (e.g., a cytokine).
A secondary antibody binds to a unique epitope on the protein. The assay is labelled with biotin, which allows for subsequent binding of a protein like streptavidin-conjugated Enzyme. Commonly used enzymes during this procedure are peroxidase (HRP) and alkaline phosphatase (AP).
Any unbound reagents/serum components are eliminated by a thorough washing of the plate. PBS-T (Phosphate buffered saline with Tween) is employed because of the diluent for removing unbound molecules.
A chromogenic substrate, like Tetramethylbenzidine (TMB), is employed for staining. It’s added to the assay, which develops a colour supported by the enzymatic reaction (it is directly proportional to the antigen bound quantity). The substrate selection depends on the sort of instrumentation (spectrophotometer, fluorometer, and luminometer) used. The Enzyme features a fluorescent tag that converts the substrate to a detectable product by a fluorometer.
The concentration of the protein is decided by a typical curve of known protein concentrations. Mean absorbance is calculated for the quality, controls, and therefore the samples. A standard curve is made by plotting the mean absorbance on the Y-axis vs concentration on the X-axis or using computer software programs. The optical densities are often measured at different target wavelengths using an ELISA plate reader.
Common ELISA Workflow, a stepwise ELISA protocol:
Step2- Primary Antibody Reaction
Step3- Application of Secondary Antibody
Step4– Substrate Preparation
VARIATIONS OF ELISA PROCEDURES
Several variations of the ELISA procedure can be formulated. The selection depends on the sensitivity, specificity, and convenience required, interfering factors, and the type and activities of the antisera available. In three variations, a, b and c, the solid-phase (ELISA plate) is coated with antibody to the antigen to be detected. This antibody, identified as a trapping antibody (TA in the figure), traps its corresponding antigen (identified as V) from suspension or solution.
1. Double antibody sandwich
An inexperienced user should start with the double antibody sandwich (DAS) if possible. This has been the most commonly used form of ELISA for plant virus detection antibodies. DAS can be done with a single good quality polyclonal antiserum. The immunoglobulins present are partially purified, and one portion is saved for use as a trapping antibody while another is conjugated to an enzyme.
Alkaline phosphatase is commonly used as the Enzyme, and the conjugation can be done in the presence of dilute glutaraldehyde antibodies for coating and detection do not have to come from the same source, e.g., monoclonal antibodies could be used for layer, and a polyclonal antiserum could be used to prepare the enzyme-labelled secondary antibody.
2. Double antibody sandwich indirect
DAS can be converted to an indirect procedure (DAS-I). The first two steps are the same as in DAS. However, the antigen bound to the trapping antibody is detected by an unlabeled intermediate antibody (IA), which is specific to the same antigen but is from an animal species different from the one used to prepare the trapping antibody.
DAS-I ELISA involves an additional step but is more sensitive and allows using a commercially prepared enzyme-labelled antibody to the IA. A single LA can also be used for multiple virus detection systems. The intermediate antibody does not have to be purified and is needed in only a limited quantity. If the intermediate antibody in particular, e.g., most monoclonals, then a highly specific antiserum is not required for coating.
The DAS-I procedure can be further modified to amplify the reaction achieved. Users should be aware of the possibility of employing amplification when additional sensitivity is needed, but regular procedures should be tested before amplification is attempted.
3. Plate-trapped antigen
Another basic approach to ELISA is the plate-trapped antigen procedure. The method is to trap the antigen on the plastic exterior and then react to the trapped antigen with an unlabeled intermediate antibody (IA) specific to it.
The IA is then detected in DAS-I using an enzyme-labelled antibody (LA) particular to the IA. This procedure, called plate-trapped antigen indirect (PTA-I) ELISA, is relatively simple and involves no advance purification of antisera or conjugate preparation if a commercially prepared enzyme-labelled antibody to the unlabeled IA is used.
The PTA-I procedure is usually less sensitive than DAS or DAS-I for crude plant extracts and may not be effective when antigen concentration in the sample is low. Since binding to the plate is non-specific, the target antigen and other proteins present in the extract compete for the plate’s available binding sites.
Plate-trapped antigen tests can be conducted as a direct assay using an enzyme-labelled antibody to the antigen. However, sensitivity is even lower than for the indirect method, and the conjugate must still be prepared. As described for DAS-I, amplification procedures can also be used for the PTA-I procedure to increase sensitivity.
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