Enzyme-linked immunosorbent assay (ELISA) is a popular plate-based assay technique utilising one or more specific antibodies for the detection of analytes such as proteins or antibodies. This sensitive immunoassay allows quantitative or qualitative analysis of an analyte from within a complex mixture. Multiple ELISA formats are commonly used within the laboratory, designed with varying levels of complexity. However, they are all based on the immobilisation of an antibody or antigen to the surface of a microplate, followed by the specific binding of an enzyme-linked antibody. The antigen/antibody complex is detected via enzymic activation of a substrate and measured on a microplate reader.
The simplest ELISA method is direct ELISA, where an immobilised antigen of interest is probed by conjugated primary antibody, and any bound primary antibody is detected via its enzyme-conjugate. Indirect ELISA employs an additional antibody step, where bound unconjugated primary antibody is detected via a conjugated secondary antibody. As suggested by the name, sandwich ELISA captures the antigen between an immobilised capture antibody and a conjugated detection antibody. Sandwich ELISA is highly specific, as two different primary antibodies must successfully to bind to the antigen before detection. It is the method of choice for analysis of complex samples containing a mixture of proteins. Direct ELISA is the quickest and easiest ELISA method, but indirect ELISA has the added advantage of signal amplification, as multiple secondary antibodies can bind to a single primary antibody.
Below are some basic considerations to take into account when setting up or troubleshooting a standard ELISA protocol:
Use flat-bottomed microplates specifically designed for ELISA. These are usually clear polystyrene or polyvinyl chloride plates for assays with a colourimetric readout on a spectrophotometer. For assays with a fluorescent or chemiluminescent readout, black or white opaque plates may be required for use on a fluorometer or luminometer.
The antibody or antigen coated on the plate should ideally be as pure as possible, without contaminants which could outcompete the intended coating protein for binding sites on the plate surface. For example, avoid coating antibodies with BSA added as a stabiliser. Plates should be covered during incubations and not stacked as this can lead to inconsistent temperatures between wells.
Blocking and Buffers
Blocking is an important step which if not optimised can lead to increased background readings or reduced signal. Standard blocking buffer usually contains BSA, a protein which readily binds to areas of the plate not covered by coating protein. In subsequent steps of the assay, the BSA block prevents antibody binding non-specifically to the plate causing a high background signal. Conversely, excessive blocking can interfere with the binding between antibody and antigen, thus reducing specific signal. In addition to blocking buffer, a number of other buffers are required in a standard ELISA protocol such as wash buffer, sample diluent and coating buffer. These buffers can be prepared in the laboratory or if preferred, pre-made buffers are commercially available. When dealing with complex samples or problems with sensitivity or background, commercially available buffers can help rule out human error, and some are designed to tackle specific issues such as matrix effects.
Antibodies should be validated for use in ELISA, as not all antibodies are suitable for every application. If the manufacturer does not provide a suggested dilution for use in ELISA, a rough guide is a concentration of between 1-10 ug/ml for a coating antibody and 1-5 ug/ml for a detection antibody. For sandwich ELISA, the use of validated matched pair antibodies is vital. These antibodies are specific for the same target but are not competing for the same epitope on an antigen, and so have demonstrated that they can bind to the antigen simultaneously. Commercially available ELISA kits contain validated matched pairs, appropriate buffers and optimised protocols for ease of use.
Thorough washing and blotting of the ELISA microplate removes unbound reagents and excess liquid. High background signal can be caused by insufficient washing. Poor blotting could lead to cross contamination of the wells, but never allow a plate to dry out completely during the procedure.
Always include a positive control of known concentration, a negative control and a blank sample control. In complex samples, spiking of a known concentration of analyte into the matrix can indicate the level of sample recovery by the assay and therefore assay performance.
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