In the previous units of this series, the immunohistochemical (IHC) methods of immunofluorescence and immunoperoxidase were described and discussed. These methods use antibodies to localize cellular proteins in tissue sections, which can then be visualized in either a dark field fluorescence microscope or routine bright field microscope. It is important to understand antibody structure and function in order to fully appreciate these immunohistochemical (IHC) techniques.
Antibodies are proteins made by mammals in response to foreign “invaders” such as bacteria, viruses and other protein elements. Each antibody is made in response to specific antigenic determinants on those proteins, that are called antigens. Each antibody is made by specialized B cells, which divide and form clones which provide long term “memory” of the antigen. This makes up part of the mammalian immune system which provides protection against such “invaders”.
Antibodies themselves are made of four polypeptide chains: two identical copies of a heavy chain and two identical copies of a light chain, which contain a variable region. The variable region binds a unique, specific epitope region on the antigen protein. The immunoglobulin class of an antibody is defined by the heavy chain makeup, while the light chain is classified as a kappa or lambda type. Figure 1 shows a schematic diagram of a typical antibody. Figure 2 shows the “protein” configuration of an antibody.
Figure 3 shows the protein configuration of an antigenic protein. The five different colors depict five different antigenic determinants on the protein. Each determinant would be recognized by a different antibody clone. If an antibody preparation is “monoclonal”, it contains only one of the antibody clones. In this scenario, the monoclonal preparation would contain only the red antibody (i.e. red “Y”). If an antibody preparation is “polyclonal”, it contains many antibody clones – each directed to a specific antigenic determinant on the protein. In this scenario, the preparation would contain all of the different colored antibodies, as shown in the diagram.
When performing IHC methods, the histotechnician and pathologist must determine the best type of antibody for the project. There are many vendors which provide many primary antibody preparations for use in IHC. Some are provided in concentrated “neat” forms, which must be optimized in terms of concentration and antigen retrieval conditions. Other primary antibodies are provided in “ready to use” form: already diluted and optimized for the specified procedural conditions. Whether to use a monoclonal or polyclonal antibody is up to the pathologist.
Once the decision has been made on which primary antibody to use, the procedure must be optimized by the laboratory and validated prior to utilization on patient specimens. This is done by running experimental staining procedures using a positive control tissue. When the pathologist is able to visualize the proper staining pattern and intensity in the positive control tissue, the stain is ready for use in the laboratory. It is important to remember that the stain procedure must be validated each time a new lot of primary antibody is ordered and received – even if it is the same. It is possible that lot-to-lot variation can occur and may have an effect on the final stain.
Schematic representation of an antibody.
Antibody, space-filling model. Atoms are coloured according to convention.
3D configuration of a protein, showing different colored antigenic determinants within the protein. The different antibody clones are shown as corresponding colored “Y”’s.
Tell us which IHC techniques work in your laboratory.
- Chapman CM. The Histology Handbook. Amazon CreateSpace Independent Publishing Platform; 2017.
- Chapman CM, Dimenstein IB. Dermatopathology Laboratory Techniques. Amazon CreateSpace Independent Publishing Platform; 2016.
- https://www.ncbi.nlm.nih.gov/books/NBK26884/ B cells and antibodies.