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ELISA and Immunodetection Assay in 1971, Two Swedish Scientists, Eva

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ELISA and Immunodetection Assay in 1971, Two Swedish Scientists, Eva ELISA and immunodetection assay In 1971, two Swedish scientists, Eva Engvall and Peter Perlman invented a test that revolutionized medicine called the ELISA, which stands for enzyme-linked immunosorbent assay also often referred to as enzyme immunoassay (EIA). An ELISA, like other types of immunoassays, relies on antibodies to detect a target antigen using highly specific antibody- antigen interactions. ELISA can readily detect and quantify less than a nanogram of a specific antigenic protein and is typically performed in a multi-well microtitre plate (96- or 384-wells) as shown below – ELISA is routinely used for the diagnosis of infectious agents such as viruses, and other substances in blood. The antigen is the substance or agent to be measured. In this technique the antigen is immobilised on to a solid phase, either the reaction vessel (ELISA plate) or a bead. Immobilisation is achieved by the use of a coating antibody which actively traps antigen to the solid phase. A second antibody (antibody enzyme conjugate) which is labelled with a reporter enzyme is allowed to bind to the immobilised antigen. The enzyme substrate is then added to the antigen/ antibody/enzyme complex and a colour change, is seen. These assays rely on a stepwise addition of layers with each one being linked to the one before. The antigen is central to the assay as it provides the bridge between the solid phase and the signal generating molecule. According to how it works, ELISA can be divided into five major types: direct, indirect, sandwich (it can be of two types -Double antibody sandwich (DAS) or Triple antibody sandwich (TAS) ELISA), Enhanced and competitive but all of them rely on the antibody–antigen complex being formed and the presence of it being confirmed by the reactions of the reporter enzyme. Direct ELISA - In direct ELISA, only an enzyme-labeled primary antibody is used, meaning that secondary antibodies are not needed. The enzyme-labeled primary antibody "directly" binds to the target (antigen) that is immobilized to the plate (solid surface). Next, the enzyme linked to the primary antibody reacts with its substrate to produce a visible signal that can be measured. In this way, the antigen of interest is detected. As shown in the diagram below – Direct ELISA Ag = Antigen Indirect ELISA - In indirect ELISA, both a primary antibody and a secondary antibody are used. But in this case, the primary antibody is not labeled with an enzyme. Instead, the secondary antibody is labeled with an enzyme. The primary antibody binds to the antigen immobilized to the plate, and then the enzyme-labeled secondary antibody binds to the primary antibody. Finally, the enzyme linked to the secondary antibody reacts with its substrate to produce a visible signal that can be measured. Indirect ELISA Sandwich ELISA - In direct and indirect ELISA, it is the antigen that is immobilized to the plate. In sandwich ELISA, however, it is the antibody that is immobilized to the plate, and this antibody is called capture antibody. In addition to capture antibody, sandwich ELISA also involves the use of detection antibodies, which generally include the unlabeled primary detection antibody and the enzyme-labeled secondary detection antibody. It can be of two types - Triple antibody sandwich (TAS) ELISA and Double antibody sandwich (DAS) ELISA. TAS ELISA is often used to identify antibodies in patient blood which may be there as the result of infection. The system is used to test patient blood for the presence of hepatitis B virus (HBV) antibodies as a diagnostic test for this disease. In this test HBV coating antibody is bound to the wells of a microtitre plate and HBV coat protein added to them. The live virus is not used as antigen as this would be too dangerous to use in the laboratory. HBV coat protein is made synthetically specifically for use as antigen in this type of test. After incubation and washing, patient serum is added which if it contains antibodies reacts to the antigen. Anti-human antibody conjugated to an enzyme marker is then added which will bind to the patient antibodies. Substrate is then added to identify samples which were positive. Diagrammatic representation of TAS is shown below – or Diagrammatic representation of TAS ELISA DAS ELISA is probably the most widely used immunochemical technique in diagnostics. It is rapid, robust, and reliable and can be performed and the results interpreted with minimal training. The principle is the same as other ELISA techniques in that the antigen is immobilised to a solid phase by a primary antibody and detected with a second antibody which has been labelled with marker enzyme. The antigen creates a bridge between the two antibodies and the presence of the enzyme causes a colour change in the chromogenic (colour-producing) substrate (as shown below). The marker enzyme used is usually either horseradish peroxidase (HRP) or alkaline phosphatase (AP). Diagrammatic representation of TAS ELISA In some systems the enzyme is replaced with a radioactive label and this format is known as the immunoradiometric assay (IRMA). DAS ELISA is used extensively in horticulture and agriculture to ensure that plant material is free of virus. Potato tubers that are to be used as seed for growing new crops have to be free of potato viruses and screening for this is carried out by DAS ELISA. There are many potato viruses but potato leafroll virus (PLRV) in particular causes considerable problems. PLRV antibodies are coated onto the wells of ELISA plates and then the sap to be tested is added. After incubation, the plates are washed and PLRV antibody conjugated to alkaline phosphatase is added. The plates are incubated and after washing, substrate is used to identify the positive wells. The system again requires the presence of the antigen (PLRV) for the sandwich of antibodies to be built up. Enhanced ELISA systems - There are two basic ways that signal can be amplified in ELISA. More enzyme can be bound by using multivalent attachment molecules. Systems using the avidin– biotin binding system allow amplification through this route. Both avidin and biotin are tetravalent (i.e. they have four binding sites) and it is this property that produces the amplification. The detection antibody is labelled with biotin and the reporter enzyme with avidin. The high affinity and multivalency of the reagents allows larger complexes of enzyme to be linked to the detection antibody, producing an increase in substrate conversion and improved colour development in positive samples. Competitive ELISA - Compared with all of the ELISA types explained above, competitive ELISA is relatively complex because it involves the use of inhibitor antigen, so competitive ELISA is also known as inhibition ELISA. In competitive ELISA, the inhibitor antigen and the antigen of interest compete for binding to the primary antibody. Here is a procedure of competitive ELISA: Firstly, the unlabeled primary antibody is incubated with the sample containing the antigen of interest, leading to the formation of antigen-antibody complex (Ag-Ab). In this step, the antibody is excessive compared with the antigen, so there are free antibodies left. Secondly, the Ag-Ab mixture is added to the plate coated with inhibitor antigen that can also bind to the primary antibody. The free primary antibody in the mixture binds to the inhibitor antigen on the plate, while the Ag-Ab complexes in the mixture do not and are therefore washed off. Thirdly, the enzyme-labeled secondary antibody is added to the plate and binds to the primary antibody bound to the inhibitor antigen on the plate. Finally, a substrate is added to react with the enzyme and emit a visible signal for detection. Through this procedure, you may find that the final signal is inversely associated with the amount of the antigen of interest in the sample, meaning that the more antigen in the sample, the weaker the final signal. This is because primary antibodies bound to sample antigen will be washed off, while free primary antibodies left will be captured by inhibitor antigen immobilized to the plate and be measured by an enzymatic reaction. This ELISA is used in assays for small molecules such as hormones in blood samples where often only a single epitope is present on the antigen. It is quantitative when used in conjunction with a standard curve (as shown in diagram below). This routinely used for testing blood samples for thyroxin. Thyroxin is a hormone that is responsible for regulating metabolic rate and deficiencies (hypothyroidism) and excesses (hyperthyroidism) of it will slow or speed up the metabolism. Patients can be given additional thyroxin if required if they are deficient but it is important to establish the baseline level before treating the condition. In some assays the enzyme is replaced with a radioactive label and this form of competitive ELISA is known as the radioimmunoassay (RIA). Applications of ELISA – 1. Presence of antigen or the presence of antibody in a sample can be evaluated. 2. Determination of serum antibody concentrations in a virus test. 3. Used in food industry when detecting potential food allergens. 4. Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc. Advantages of ELISA – 1. High sensitivity and specificity: it is common for ELISAs to detect antigens at the picogram level in a very specific manner due to the use of antibodies. 2. High throughput: commercial ELISA kits are normally available in a 96-well plate format. But the assay can be easily adapted to 384-well plates. 3. Easy to perform: protocols are easy to follow and involve little hands-on time. 4. Quantitative: it can determine the concentration of antigen in a sample.
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