One of the most sensitive immunoassays is the Enzyme-Linked Immunosorbent Assay (ELISA). An ELISA typically has a detection range of 0.1 to 1 fmole or 0.01 ng to 0.1 ng. It is an assay technique that uses plates to detect and quantify peptides, proteins, antibodies, and hormones.

In an ELISA, an antigen must first be immobilized on a solid surface before complexing with an antibody connected to an enzyme. The enzyme’s activity is assessed by allowing the conjugated enzyme to react with a substrate to create a quantifiable by-product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.

This article will review the ELISA principle, procedure, types, and applications.

What are the types of ELISA tests?

Competitive ELISA

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Competitive ELISA assays, also known as inhibition ELISA or competitive immunoassay, detect the concentration of an antigen by detecting signal interference. Each of the preceding formats is adaptable to the competitive format.

The sample antigen competes for binding to a specific amount of labeled antibody with a reference antigen. A multi-well plate is pre-coated with the reference antigen, and the sample is pre-incubated with labeled antibodies before being added to the wells. More or less free antibodies will be available to bind the reference antigen depending on the amount of antigen in the sample. This means that the more antigen in the sample, the fewer reference antigen will be detected, and the signal will be weaker.

Some competitive ELISA kits substitute labeled antigens for labeled antibodies. The labeled antigen and the unlabeled sample antigen compete for binding to the primary antibody. The stronger the signal due to the more labeled antigen in the well, the lower the amount of antigen in the sample.

Direct ELISA

In a direct ELISA, the antigen is immobilized on the multi-well plate’s surface and detected with an antigen-specific antibody. The antibody is conjugated directly to HRP or other detection molecules.

Indirect ELISA

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Indirect ELISA is a two-step detection method in which the target is bound by a primary antibody specific to the antigen. The primary antibody binds to a secondary antibody that has been labeled and directed against the primary antibody’s host species. As in direct ELISA assays, the antigen is immobilized on the surface of the multi-well plate.

The technique can also identify particular antibodies in a serum sample by replacing the primary antibody with serum.

Sandwich ELISA

The most popular ELISA format is sandwich (or sandwich immunoassay). Two antibodies with different antigen epitope specificities must be used in this format. The term “matched antibody pairs” refers to these two antibodies. One of the antibodies is used as a capture antibody and coated on the surface of the multi-well plate to help immobilize the antigen. The other antibody helps with antigen detection and is conjugated.

ELISA Procedure

Unless you use a kit that includes a pre-coated plate with an antibody, an ELISA begins with a coating step in which a target antigen or antibody is adsorbed onto a 96-well polystyrene plate.

After that, there is a blocking step (Blocking ELISA principle: all unbound sites are coated with a blocking agent.) The plate is incubated with enzyme-conjugated antibodies after a series of washes. Another round of washing removes any remaining unbound antibodies. After that, a substrate is added, resulting in a calorimetric signal. The plate is finally read.

Because the assay relies on surface binding for separation, each ELISA step includes several washes to remove unbound material. The excess liquid must be removed during this process to avoid diluting the solutions added in the following assay step. Specialized plate washers are frequently used to ensure uniformity.

When measuring protein concentration in heterogeneous samples like blood, ELISAs can be quite complex with multiple intervening steps. The detection step is the most difficult and variable in the overall process, as multiple layers of antibodies can be used to amplify signals.

ELISA Applications

Cancer Screening

Early-stage cancers, such as ovarian and breast cancer, can be clinically tested using ELISA-based techniques. It is possible by detecting and measuring cancer or tumor biomarkers in the body due to cancer growth and development. Several established tumor protein markers associated with specific types of cancer, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and anaplastic lymphoma receptor tyrosine kinase (ALK), have been identified by clinical researchers over the years. Such markers can even be used clinically to determine cancer aggressiveness and to monitor the disease’s response to various treatments.

Pregnancy Testing

During pregnancy testing, doctors look for elevated hormone human chorionic gonadotropin (hCG) in the patient’s urine sample. ELISA is commonly used to detect this hormone, which is more prevalent in pregnant women. Thus, using ELISA kits, women can easily check their pregnancy status at home.

Drug Testing

Using ELISA, the patient’s urine sample can be tested for concentrations of the most common illicit drugs, including amphetamines, cocaine, cannabinoids, opiates, benzodiazepines, and methadone. ELISA kits can also be used to monitor the levels of specific pharmaceutical drug concentrations in patients who are being treated. Anti-Drug antibodies, for example, in patients with inflammatory bowel disease and rheumatoid arthritis.


Because of the binding characteristics of the antibodies and the amplification or different read-out systems used, ELISAs are the most sensitive immunoassays. Sample volumes can also be adjusted when dealing with a low protein abundance. As previously discussed, indirect ELISAs allow for signal amplification by employing a secondary antibody. 

Other amplification systems can be used in ELISAs to create High Sensitivity ELISA Kits, which use an additional amplification step to increase sensitivity. Alkaline Phosphatase and Streptavidin HRP polymer are two examples of different amplification systems.