Direct ELISA

Direct ELISA is is typically getting used to analyse an immune response to an antigen, e.g. for 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 getting 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 workflow consisting of a few steps only
  • Less error-prone – relatively small number of pipetting steps compared to other ELISA methods and due to single antibody requirement no risk of antibody cross-reactivity

Disadvantages of direct ELISA

  • Limited sensitivity – no signal amplification step
  • Time-consuming assay development – each assay target requires a specific conjugated primary antibody, which makes the assay relatively expensive, too
  • 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

Indirect ELISA – the most popular ELISA method - uses a 2-step detection process. First, an unlabelled primary antibody binds specifically to the antigen. Second, an enzyme conjugated secondary antibody binds specifically to the primary antibody. This method is typically getting used to determine total antibody concentration in samples.

Advantages and disadvantages of indirect ELISA

Advantages of indirect ELISA

  • Flexibility – secondary antibody can be used as long as it matches the host species of the primary antibody and wide variety of labeled secondary antibodies are available commercially
  • Sensitivity – more than one labelled antibody can bind the primary antibody because each primary antibody contains several epitopes
  • Maximum immunoreactivity – no label does interfere with the sites on the primary antibody
  • Economical – fewer labeled antibodies are required

Disadvantages of indirect ELISA

  • More complex workflow – additional incubation step for the secondary antibody is required
  • Potential for cross-reactivity – nonspecific signal due to cross-reactivity with the secondary antibody

Sandwich ELISA

In Sandwich ELISA a so-called capture antibody is used to coat the wells which then binds the antigen from the sample. Then the antigen is detected either in direct or indirect ELISA configuration. This method is often used to analyse 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 – purification of the antigen prior to measurement is not required

Disadvantages of sandwich ELISA

  • Complex workflow – requires more incubation steps vs other ELISA formats
  • Requires more optimisation – cross-reactivity between the different antibodies used must be checked

Competitive ELISA

Each of the above assay types can be adapted for competitive ELISA, also known as inhibition ELISA. The basic concept of this method is the sample antigen competing with an inhibitor antigen, binding to a limited amount of labelled antibody. The inhibitor antigen is pre-coated on a microplate. Then the sample pre-incubated with labelled antibody is added to the wells. The signal detected will be weaker the more antigen was in the sample as less antibody is able to bind to the antigen in the wells. This method is typically getting used to analyse 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 – minimises 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