ELISA

What is ELISA and what is it used for?

ELISA is an enzyme-linked immunosorbent assay, that is widely used as a research method for quantifying proteins or antigens in an unknown solution, or in medicine as a diagnostic tool. The sensitive and straightforward nature of ELISAs makes them useful for 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 Ag-Ab binding (antibody-antigen interactions).

Read: History of the antibody

Examples of use cases for ELISAs include: testing for pregnancy, diagnosis of HIV infection, measuring histamine levels in food levels, and measurement of cytokines in cell supernatant or serum.

ELISA technique

The ELISA 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.

Four major types of ELISA

There are four ways that ELISAs can be set up in terms of antibodies and antigens: direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA (Table 1).

Table 1. How each ELISA method is conducted

ELISA MethodHow it is performed
Direct ELISAThe unknown sample containing the antigen of interest is adhered directly to the well, followed by detection with a primary antibody.
Indirect ELISABegins 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.
Sandwich ELISA (Figure 1)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.
Competitive (or inhibition) ELISACan 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.
Diagram of a sandwich ELISA test
Figure 1. Diagram of a sandwich ELISA test

The difference between indirect ELISA and direct ELISA

The only difference in indirect and direct ELISAs (Figure 2) is the detection antibody used.

In a direct ELISA, only one antibody is used. This single, primary antibody is conjugated directly to the detection enzyme. The indirect ELISA begins the same way as a direct ELISA, but requires two antibodies, a primary antibody and a secondary antibody for detection.

Difference between Direct vs Indirect ELISA
Figure 2. Direct detection [left] immunoassays are the most rapid but are less stringent and so are ideal for tests which require a rapid readout, but where ultimate accuracy is not required. Indirect detection [right] immunoassays improve upon the specificity of direct detection immunoassays by introducing a conjugated secondary antibody.

What are indirect ELISAs used for?

Indirect ELISAs are used for detecting antibodies in a sample to quantify immune responses.

What are direct ELISAs used for?

Direct ELISAs are suitable for epitope mapping, quantitative and qualitative antigen detection, and antibody screening.

What is the purpose of the sandwich ELISA test?

One of the most commonly used ELISA tests is the sandwich type (Figure 1), which is commonly used for allergy testing or to test allergen proteins in food for quality control purposes. Sandwich ELISAs are 2-5 times more sensitive than their direct or indirect ELISA counterparts. They are performed in 96-well plates, where many conditions and repeat samples can be tested at once. 

Steps / Method of Sandwich ELISA

Sandwich ELISAs are 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.

Azure Biosystems Ao Absorbance Microplate Plate Reader is used for ELISA, Bradford Assays, and more
The Ao Microplate Reader has a multitude of features to help you save time with arrays like ELISA, Bradford, and Lowry assays with wavelength ranging from 340-750nm. Each system comes with an 8 position filter wheel with 8 filters: 405, 450, 492, 595, and 630 standard, plus 3 from the filter catalogue, built-in shaker with speed selection, single and dual-read modes, and analysis software.

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

Can ELISA be used to detect proteins?

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. The results of ELISA tests can be quantitative, qualitative, or semi-quantitative. Results are usually graphed and compared with other results in order to come to a definitive result.

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.

Multi-well plates can also be imaged in the Azure Digital imagers and the Azure Sapphire FL Biomolecular Imager, which can facilitate in-cell Westerns and other related 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

Both ELISA and Western Blotting are indirect tests to analyze a wide variety of samples. ELISAs are simpler and faster to perform than Western blots, which are also less specific.

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 >>

REFERENCES

  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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