Last week our blog looked at immunohistochemistry and its history. This week we thought we’d delve a bit deeper.
As we’re an antibody search engine we’ll look first at the different factors involved in selecting a primary antibody for IHC. We’ll look at IHC methods and protocols for sample preparation, antigen retrieval and immunostaining in another post very soon.
When choosing an antibody for IHC there are advantages and disadvantages to selecting a monoclonal or a polyclonal antibody. Understanding which of these pros and cons is most important to you is essential.
Monoclonal antibodies are the product of a single cloned B lymphocyte and while production is initially expensive to establish, the use of immortalised cells creates a potentially permanent resource. This also allows production of antibodies that are highly consistent from batch to batch, while variation can be a potential limitation of polyclonal antibodies.
Polyclonal antibodies avoid the use of expensive lymphocyte/tumour cell hybridomas, instead utilising a hosts natural immune response. The antibodies produced as a result of immunization are harvested and purified, however as they are secreted by multiple B cell clones they can be more likely to show non-specific reactivity.
However, in situations when the effect of the tissue fixation and processing on the antigen is unknown, polyclonal antibodies might be the better option. Because polyclonals are capable of binding multiple epitopes within an antigen they are less likely to be affected by changes in an antigens tertiary structure, while monoclonals may find their antigen buried.
Whichever type of antibody you chose, it is essential that its properly validated, an important subject we will return to in the future.
On the subject of validation, those working on human tissues, and in particular cancerous tissues might find The Human Protein Atlas particularly useful. Funded by the Knut and Alice Wallenberg Foundation, the project has been setup to “allow for a systematic exploration of the human proteome using Antibody-Based Proteomics.” What makes the site particularly useful from our point of view is that the database contains a great deal of example IHC images, demonstrating antigen localisation in both normal and cancerous tissues.
CiteAb lists both polyclonal and monoclonal antibodies, and has over 15000 citations that reference the use of these in IHC, so you can read how others have used a particular antibody in their research.
In our previous blog we list some useful resources which are worth taking a look if you’re interested in developing a better understanding of which antibodies to use for IHC. If there are any you’ve found useful that we haven’t listed here then please do post in the comments section, we’d love your input!
– The CiteAb team