Direct ELISA is typically used to analyse an immune response to an antigen, e.g. immunohistochemical staining of cells or tissues. This ELISA method requires an antigen coated to a multi-well plate. For detection, an antibody that has been directly conjugated to an enzyme is used. The workflow of this method is relatively simple with only a few steps required.
Advantages and disadvantages of direct ELISA
Advantages of direct ELISA
- Simple and fast – just a single antibody required and the workflow consists of only a few steps.
- Less error-prone – relatively small number of pipetting steps compared to other ELISA methods and due to a single antibody there is virtually no risk of antibody cross-reaction.
Disadvantages of direct ELISA
- Limited sensitivity – there is no signal amplification step.
- Time-consuming assay development – each assay target requires a specific conjugated primary antibody which can also makes the assay relatively expensive.
- Risk of limited antibody reactivity – attachment of the enzyme to the antibody may limit the spatial availability of sites on the antibody that should bind to the antigen.
Indirect ELISA – this is the most popular ELISA method and uses a 2-step detection process. First, an unlabeled primary antibody binds specifically to the antigen. Second, an enzyme conjugated secondary antibody binds specifically to the primary antibody. This method is typically used to determine total antibody concentration in samples.
Advantages and disadvantages of indirect ELISA
Advantages of indirect ELISA
- Flexibility – a secondary antibody can be used if it matches the host species of the primary antibody and a wide variety of labeled secondary antibodies are available commercially.
- Sensitivity – more than one labeled antibody can bind the primary antibody because each primary antibody contains several epitopes.
- Maximum immunoreactivity – no label interferes with the sites on the primary antibody.
- Economical – fewer labeled antibodies are required.
Disadvantages of indirect ELISA
- More complex workflow – an additional incubation step is required for the secondary antibody.
- Potential for cross-reactivity – nonspecific signal due to a cross-reaction with the secondary antibody.
Sandwich ELISA uses a so-called capture antibody to coat the wells which binds the antigen from the sample. Then the antigen is detected in either direct or indirect ELISA configurations. This method is often used to analyze complex samples.
Advantages and disadvantages of sandwich ELISA
Advantages of sandwich ELISA
- Flexibility and sensitivity – both direct and indirect detection methods can be used.
- High specificity – two antibodies are used to detect the antigen.
- Suitable for complex samples – it is not required to purify the antigen prior to measurement.
Disadvantages of sandwich ELISA
- Complex workflow – requires more incubation steps versus other ELISA formats.
- Requires more optimization – cross-reactivity between the different antibodies used must be checked.
Each of the above assay types can be adapted to a competitive ELISA, also known as inhibition ELISA. The basic concept of this method is that the sample antigen is competing with an inhibitor antigen and is binding to a limited amount of labeled antibody. The inhibitor antigen is pre-coated on a microplate and then the sample is pre-incubated with labeled antibody which is added to the wells. The signal detected will be weaker when less antibody was able to bind to the antigen. This method is typically getting used to analyze small antigens that have just a single epitope.
Advantages and disadvantages of competitive ELISA
Advantages of competitive ELISA
- Highest flexibility – can be based on direct, indirect or sandwich ELISA format
- Highest specificity – the antigen is specifically captured and detected
- Very robust – minimizes the effects of sample dilution and sample matrix effects
Disadvantages of competitive ELISA
- Rather time consuming – very complex protocol
- Disadvantages of the detection format selected applies