In the initial stages of the immune response, small numbers of immature B lymphocytes are able to bind foreign antigen molecules weakly via the antibodies expressed on their
surfaces. Antigen binding stimulates the lymphocytes to proliferate and to differentiate into mature lymphocytes that secrete antibodies. An amazing series of transformations occur as B lymphocytes mature in response to antigen. Antigen binding stimulates responding lymphocytes to rearrange segments of their antibody-encoding genes, producing new potential antigen- binding sites. Most rearrangements are unproductive, but some rearrangements generate antibodies with greater affinity for the antigen. Antigens act as selective agents. The lymphocytes that bind the antigen with the highest affinity receive the greatest growth signal and proliferate most rapidly, because a higher fraction of their surface antibodies are bound to antigen at any one time. In the latter stages of differentiation, a hypermutation process further increases the range of potential antibody sequences. Mature B lymphocytes that have survived the selection process are known as plasma cells. Each plasma cell secretes a single antibody with high affinity for antigen. Plasma cells are virtual antibody factories that can be identified in electron micrographs by their extensive rough endoplasmic reticulum. The scope of antibody diversity
is immense - vertebrates are capable of producing billions of antibody molecules with distinct specificities.
Polyclonal vs. monoclonal antibodies
For our western blots, we will be using both monoclonal and polyclonal antibodies. As their names imply, monoclonal antibodies bind to the same epitope on an antigen. Polyclonal antibodies are actually mixtures of antibodies that bind to different epitopes on an antigen. An animal’s response to antigen is polyclonal, because antigens stimulate the proliferation of multiple lymphocyte clones, each of which produces a different antibody to the antigen. Consequently, the serum collected from an immunized animal contains a mixture of antibodies with different specificities. The polyclonal antibodies used in the lab are purified from the sera of animals that have been inoculated with antigen.
By contrast, monoclonal antibodies are produced in the lab from cultured hybridoma cells. Hybridoma cells are generated by fusing a lymphocyte from an immunized animal, most commonly a mouse, with a cancerous myeloma cell that can divide indefinitely in culture (right). Because the lymphocytes from the spleen of an immunized mouse recognize a range of different epitopes on an antigen, the hybridomas resulting from the fusion secrete a variety of different antibodies. Standard culture techniques are then used to isolate individual hybridoma cell lines, each of which secretes a unique antibody that binds to a single epitope.
Hybridoma technology has revolutionized biomedical research since its description (Kohler & Milstein, 1975), both because monoclonal antibodies recognize well-defined epitopes and because monoclonal antibodies can be produced indefinitely by cultured hybridoma cells. Investigators often use both monoclonal and polyclonal antibodies at different steps in western blots.