Antigen Retrieval: Methods, Tips and Techniques

By Dr. Surat P, Ph.D.Reviewed by Dr. Jennifer Logan, MD, MPH

Antigen retrieval techniques are used to enhance signals in immunohistochemical testing.

What are antigen retrieval methods needed?

Fixation is a key step performed during immunohistochemistry to preserve tissue morphology. However, this method may also have adverse impacts on immunohistochemistry, as it often changes the protein biochemistry and masks the epitope of protein of interest.

Due to this, the primary antibody is unable to bind to the protein, and low signal intensity results. This masking effect may be caused by cross-linking of amino acids in the epitope or cross-linking of peptides that are unrelated but are present near the epitope. This changes the conformation or the electrostatic charge of the antigen. During the process of antigen retrieval, this masking is reversed so that binding between antigen and epitope may occur.

What are ways to retrieve antigen?

The antigen retrieval is based on several variables, such as target antigen, antibody, tissue type, and duration and method of fixation. Oftentimes, polyclonal antibodies may work better without the use of antigen retrieval methods, as they recognise multiple epitopes. However, monoclonal antibodies rely on the recognition of a single specific epitope to function.

Multiple strategies are being used to retrieve antigens. Some methods are as simple as changing the pH or concentration of cations in the antibody diluent, which can also change the affinity of antibody and its epitope.

In cases where the epitope is partially masked, the primary antibody incubation can be increased before trying other antigen retrieval methods. However, these parameters of incubation time, temperature, and concentration must first be optimised. Two primary methods are currently used to retrieve antigens.

Protease-induced epitope retrieval (PIER)

In this method, different enzymes, including proteinase K, trypsin, and pepsin, are used to restore binding between antigen and epitope.

This process is supposed to work by cleaving of peptides that form the epitope. However, PIER has a low success rate and tissue morphology and antigen may be destroyed in the process.

Heat-induced epitope retrieval (HIER)

In this technique, heat is used to reverse cross-linkages and restore the original and tertiary epitope structure. This method is optimised for each tissue, fixation method, and antigen of interest.

The heat is supplied in this case using microwave ovens, pressure cookers, steamers, autoclaves, or water baths. This method has a higher success rate than the PIER method. During the process, samples are subjected to heat for short periods before buffer replacement.

Certain labs use a water bath that has been set to a temperature of 60°C, then incubate slides overnight in the retrieval solution.

Such techniques may be useful in case sections fall off the slide at high temperatures (possible for bone, cartilage, and skin). In this method, slides are cooled before subjecting them to heat. HIER is subject to variations in time, temperature, buffer, and pH, and the exact protocol is determined using trial and error.

What are the limitations of antigen retrieval?

The process antigen retrieval often requires the sample to be adhered strongly to the slide. Also, this method is often too harsh for certain samples, such as cryostat tissues and alcohol-fixed tissues.

There is also a need to optimise temperature, time, and pH for each sample; these conditions need to be tested before deciding on optimum conditions for each sample.

There is also a possibility of artifact that may emerge during this method. For these reasons, proper controls should always be used.

Further Reading

• All Antigen Content
• Antibody Selection using DNA Origami Scaffolds
• The Characteristics of Antigens