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ELISA Principles: Explained

Posted by The Protein Man on Sep 13, 2022 11:15:00 AM
The Protein Man

What is ELISA?

ELISA, or enzyme-linked immunosorbent assay, is a plate-based assay technique designed to detect and quantify target molecules such as peptides, proteins, antibodies, and hormones in biological samples. Like other types of immunoassays, ELISA relies on specific antigen-antibody interactions to detect a target antigen.

In an ELISA, either the antigen or antibody is immobilized to a solid surface (usually a 96-well or 384-well polystyrene plate), either directly or using a capture antibody immobilized on the surface and conjugated to an enzyme-linked detection antibody (usually an enzyme or a fluorophore). Non-specifically bound materials such as free enzyme-linked antibodies are removed by washing and a substrate is added to produce a colored product that can be measured via spectrophotometry. ELISA supplies such as antibodies, blocking agents, buffers and detection reagents are offered by a wide range of vendors. 

This biomolecular technique has a multitude of applications in clinical diagnostic and forensic science and plays a vital role in food research as well. Specifically, this assay can be used to detect:

  • early stages of cancer, including breast and ovarian cancer  
  • platelet antibodies in blood serum of patients with systemic lupus erythematosus and idiopathic thrombocytopenic purpura
  • viral infections, such as COVID-19, Ebola, HIV, Newcastle disease virus (NDV), West Nile virus, and Zika virus disease
  • other infections and medical conditions such as Lyme disease, Rocky Mountain spotted fever, pernicious anemia, syphilis, and toxoplasmosis

ELISA can also be used for pregnancy and drug testing and proves to be a powerful tool in detecting the presence of allergens, potential contaminants, oils, and other substances (egg whites, milk, etc.) in food and food products. Many pre-made ELISA kits are commercially available for various research and testing and diagnostic applications mentioned here.

Different Types of ELISA

Basically, there are four main types of ELISA, each having its own distinct features, advantages, and disadvantages.

Direct ELISA

In direct ELISA, the antigen is immobilized to the surface of the polystyrene plate and detected using an antibody that has been directly conjugated to a reporter enzyme such as horse radish peroxidase (HRP), alkaline phosphatase (AP) or other suitable detection molecules.

Since it only uses one antibody and requires fewer steps, direct detection is fairly quick and has a lower risk of cross-reactivity. However, there are also several disadvantages to using this technique.

  • Non-adherent cells may adhere to the coverslip.
  • Labeling with enzymes or tags may adversely affect the immunoreactivity of the primary antibody. 
  • Limited flexibility in choosing primary antibody labels.
  • Minimal signal amplification.

Indirect ELISA

This technique involves a two-step detection process. The antigen or target molecule is attached to the plate, incubated with blocking buffers containing neutral blocking agent, such as BSA or milk proteins to block the remaining protein-binding sites. A suitable primary antibody is added to bind with the antigen or target molecules and a secondary antibody that has been conjugated with a reporter enzyme capable of binding with the primary antibody is added after washing away the free primary antibodies. A specific substrate is then added to each well to form a colored product, and the absorbance is measured by spectrophotometry.

While it requires an extra incubation step, there are several advantages to using indirect ELISA:

  • Commercial availability of labeled secondary antibodies.
  • Highly versatile. Aside from the possibility of making many primary antibodies in one species, the same secondary antibody can also be used for detection.
  • Since the primary antibody is not labeled, maximum immunoreactivity is retained.
  • Highly sensitive since the primary antibody contains several epitopes that can bind with the labeled secondary antibody.

Sandwich ELISA

This requires the use of two antibodies (or matched antibody pairs) specific for different epitopes of the antigen. Prior to the addition of the sample solution, the microtitre wells are coated with the capture antibody to immobilize the target antigen. After incubation, the wells are washed to remove unbound antigens and a second antibody (or detection antibody) is added to form a colored product. 

Sandwich ELISAs are the most used format in the lab due to their high specificity, suitability for use with complex and/or crude samples, and flexibility. 

Competitive ELISA

In competitive ELISA, the microplate is coated with the antigen solution, incubated overnight at 4oC, and washed with a suitable wash buffer to remove the coating solution. The remaining protein-binding sites in the coating wells are then blocked with a suitable blocking buffer (containing BSA or milk protein/PBS or 5% non-fat dry milk), incubated, and washed with a wash buffer.

After preparing the antigen-antibody solution, the mixture is added to the wells and the plates are incubated for 90 minutes at 37oC. An enzyme-conjugated secondary antibody (diluted in wash buffer) is added to the wells and the plates are incubated and washed accordingly. 

Finally, an appropriate ELISA substrate or detection solution is added, and the plates are again incubated for 30 minutes at room temperature or until a color (blue) precipitate is observed. Note: Absorbance values should be read at the appropriate wavelength within 30 minutes.

Alternatively, an acid stop solution (this changes the color of the precipitate from blue to yellow) may also be added and the optical densities (O.D.) value is read at 450 nm to measure the amount of the molecule of interest.

In competitive ELISA, the amount of free antibodies depends on the amount of antigen in the sample. Thus, if there is more antigen in the sample, there will be less of the reference antigen and the signal will be weaker.

Topics: Assay Development (ELISA)

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