What is ELISA and what is it used for?

ELISA is an enzyme-linked immunosorbent assay, widely used in biology as a research method for quantifying proteins or other antigens in an unknown solution. The sensitive and straightforward nature of the assay makes it useful for an array of applications, such as testing blood samples for the presence of infectious disease antibodies, testing the binding between a newly isolated antibody and its target protein, or a variety of other tests that rely on antibody-antigen interactions.

ELISAs are are sandwich-type immunoassays and performed in 96-well plates, where many conditions and repeat samples can be tested at once, and then measured using a micro plate reader. A specific antibody for the antigen of interest is bound to the bottom of the well. The sample is placed in the well and if the antigen of interest is in the sample, it will bind to the immobilized antibody. The bound antigen is then detected using a second, labelled antibody. The detection antibody is conjugated to an enzyme that can cause a color change when a substrate is added, allowing detection with a plate reader, like the Ao Absorbance Microplate Reader from Azure Biosystems. For detection, HRP-secondary antibodies, like those for chemiluminescent Western blots, can also be used.

An absorbance-based plate reader, like the Azure Ao Absorbance Microplate Reader can measure the absorbance of light at chosen wavelengths relevant to whatever is produced from the enzyme reaction, providing quantitative differences between positive and negative samples. It is compatible with numerous 96-well plate assays including nucleic acid quantification, NAD/NADP conversion, and colorimetric protein assays, such as Bradford or Lowry.

What are ELISAs used to detect?

ELISAs can be used to look for the presence or absence of a protein within a mixture, to compare relative amounts of a protein, or for other experiments that rely on native protein-antibody interactions.

Diagram of an ELISA

How to use ELISAs to look for proteins

The technique starts with a 96-well plate where the material of the wells allows proteins to adhere to the surface. Immobilization allows the antigen to be bound by its detector-antibody and then washed of any unbound antibodies, followed by measurement of the antigen. An enzyme linked to the detection antibody will interact with a substrate added in the final step to produce a signal proportional to the antigen (Figure 1).

Four ways ELISAs can be set up in terms of antibodies and antigens:

  1. Direct ELISA: the unknown sample containing the antigen of interest is adhered directly to the well, followed by detection with a primary antibody.
  2. Indirect ELISA: begins the same way as a Direct ELISA, but the primary antibody is not linked with the enzyme and a secondary antibody must be used for detection.
  3. Sandwich ELISA: depicted in Figure 1. This starts with an antibody that is adsorbed to the plate first; then, the antigen-containing sample is added and the antigen
    is captured by the immobilized antibody and detected similar to the direct or indirect methods.
  4. Competitive (or inhibition) ELISA: can be setup similarly to the other three, but instead of the antibody being labeled, the antigen is labeled. The labeled antigen is added after the unknown samples to see how much unlabeled antigen in the samples competes with the labeled antigen. Unlike the other three types, higher signal from the competitive ELISA means that there is less competition and thus less antigen in the unknown.

Multi-well plates can also be imaged in the Azure Digital imagers and the Azure Sapphire Biomolecular Imager, facilitating in-cell Westerns and other applications. AzureSpot Pro software includes an analysis module that facilitates the analysis of plate-based assays including presence/absence detection and normalization.

Application note: using the Ao Absorbance Microplate Reader for measuring and analyzing results from an ELISA

Frequently Asked Questions

ELISAs are often used for qualitative and quantitative analysis of proteins in various sample types, such as serum, cell culture supernatants, and urine.

Western blotting is a powerful technique for the detection and characterization of specific proteins in a sample. Read more about the differences >>

An advantage of ELISAs offer high sensitivity, making them capable of detecting very small amounts of target proteins, even in complex mixtures and ideal choices for detecting rare or low-abundance proteins. A disadvantage of ELISAs is they can have high background due to cross reactivity of primary and secondary antibodies. Full list of advantages and disadvantages >>


  1. Goldsby RA, Kindt TJ, Osborne BA, Kuby J. ELISA. In: Immunology, Fifth Edition. Freeman, W.H. & Company. 2002. pp 148-149
  2. William J. Antigen Measurement Using ELISA. In: The Protein Protocols Handbook. Humana Press. 2009. pp 1827-1833
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