Antibodies are blood proteins produced in the immune system in response to a specific antigen. This specific antigenicity is what makes antibodies such a powerful tool in immunochemistry. The earliest reference to antibodies goes back to the 1890s. Emil von Behring and Shibasabura Kitasato discovered that transferring serum from a diphtheria-immunized animal to animals infected with diphtheria altered the course of the disease (1). Since the 1890s groundbreaking strides have been made in the world of antibodies and their applications in life science and health science fields. With a wide variety to choose from, there are important considerations to make when selecting your own primary antibody. Keep reading to learn how to choose a primary antibody for your workflow.
An antibody is classified as a primary antibody if it binds directly to the target antigen of interest. In comparison, a secondary antibody is an antibody whose antigen is another antibody. The primary antibody must be raised against the target antigen, otherwise, there would not be any successful detection. The sample processing should also be addressed. For example, some antibodies only recognize proteins in their denatured state, thereby revealing the epitope that would otherwise be obscured. Other antibodies may require proteins in their folded state. Additionally, some primary antibodies may require an antigen retrieval step to reverse cross-links introduced by formalin-fixed, paraffin-embedded tissues. These characteristics of the antibodies should be considered during experimental design.
Next, it is important to choose the clonality of the primary antibody—whether it will be monoclonal or polyclonal. In monoclonal antibody production, individual B-cells are isolated from an immunized animal, cloned, and grown to produce homogenous antibodies (2). These monoclonal antibodies not only target the same antigen, but a single epitope, aka binding site, on the antigen. In comparison, polyclonal antibodies are purified from the serum of an immunized animal, so the antibodies have a mixture of epitope targets of the same antigen (3). A monoclonal primary antibody will give more specificity to the target with less crosstalk with other proteins. Additionally, since the monoclonal antibodies are derived from cultured cells, there is less variability from lot to lot. The benefit of polyclonal antibodies is greater tolerability in target protein conformation (fixation type, temperature, pH, etc.) and higher sensitivity.
The host species of the primary antibody will have important implications if a secondary antibody is used in the detection system. In this case, it is most common to choose a host species different from the species of tissue being tested. For example, when detecting an antigen in rabbit tissue, a mouse-raised primary antibody is an appropriate host species option. Specific considerations must be taken when the primary antibody is raised in the same species as the tissue to prevent cross reactivity between the secondary antibody and the tissue. For example, if a mouse-raised primary antibody is used on mouse tissue, the secondary antibody, which must be anti-mouse, will detect endogenous mouse Ig in the tissue sample, leading to non-specific detection and potential false positives. This is addressed with certain blocking reagents, but experimental designs can be simplified by choosing an alternate host species of primary antibody.
In mammals there are 5 main classes of antibodies: IgG, IgM, IgA, IgD, and IgE, which each have a unique structure. Physiologically, each class of antibody has unique biological properties, locations, and antigenicity (4). In immunochemistry staining, it is important to note the class of the primary antibody because the secondary antibody must be directed against the class of the primary antibody. This is especially important in monoclonal antibodies.
Labels are conjugated to antibodies to make visualization of the target antigen possible. Labeling can occur on the primary antibody, secondary antibody, or subsequent amplifying reagents. The most typical labels are either a reporter enzyme for immunohistochemistry or a fluorophore for immunofluorescence. These allow for either color development or fluorescent signal at the site of the antigen.
A primary antibody can be directly conjugated with a label for visualization, removing the need for secondary antibodies. While possible, it is more likely to use a conjugated secondary antibody or amplification reagent, so the primary antibody can remain unconjugated. This increases sensitivity and allows the primary antibody to be used in lower concentration, therefore saving money on an expensive reagent.
There are many factors to consider when choosing a primary antibody and this is not an exhaustive list. Understanding each of these characteristics is important when developing an immunochemistry protocol and selecting a primary antibody.