Antibodies Cells of the vertebrate acquired immune system produce antibodies with an exquisite specificity for molecules Biologists use antibodies to localize molecules of interest in complex preparations

Antibodies bind tightly and specifically to their target molecules Bound antibodies are visualized with additional treatments that render the bound antibody visible CIL:10233 Hippocampal neurons Actin (red); tubulin (green) western blot of bacterial cell extracts

What is the relationship between antibodies, antigens and epitopes? What happens during an immune response? How are monoclonal antibodies generated? How are antibodies used to visualize molecules?

Antigens are foreign substances that stimulate the immune system to produce antibodies 2 1 3 bacteria viruses allergens large molecules Antigen with 3 epitopes Epitopes refer to the portions of the antigen recognized by the immune system Lymphocytes produce antibodies capable of binding epitopes Most antigens have multiple epitopes (1-3 in the figure above)

Antibodies are composed of two identical heavy chains (red and blue) and two identical light chains (yellow and green) Fab Fc Bi-functional molecules Fab fragments bind antigen Fc fragments are used by the immune system to remove antigen-antibody complexes

Antigens bind hyper-variable regions at the tips of Fab fragments Lymphocytes can generate millions of different antigen binding sites by DNA rearrangement and mutation - processes restricted to immune cells!! Antigens bind hyper-variable regions at the tips of Fab fragments antigen binding has been compared to a lock-and-key fit (complementary surfaces) A limited number of (Fc) regions determine how other cells will process antibody-antigen complexes Antibodies used in molecular biology have a gamma heavy chain (IgG immunoglobins)

Fab regions of antibodies bind specifically to epitopes on antigens 2 1 3 Antibody binding leads to the removal of the antigen from the system mediated by the Fc fragment

What is the relationship between antibodies, antigens and epitopes? What happens during an immune response? How are monoclonal antibodies generated? How are antibodies used to visualize molecules?

Biologists use antibodies produced in animals to localize molecules When challenged with a foreign substance, or antigen, vertebrates produce antibodies that help the system dispose of the antigen Biologists use antibodies produced in animals to localize molecules Vaccination stimulates the production of antibody-producing cells

Antigen stimulates the proliferation of B lymphocyte clones that recognize epitopes on the antigen. 2 1 3 Each lymphocyte secretes large amounts of a SINGLE antibody molecule.

Antibodies make their way to the animal’s bloodstream. Serum contains antibodies that recognize many different epitopes. Polyclonal antibodies are semi-purified fractions derived from animal serum (antiserum) Polyclonal antibodies may recognize multiple epitopes on the same antigen Limitations of polyclonal antibodies: A limited amount of serum can be obtained from an animal It is often useful to have antibodies with a defined specificity

What is the relationship between antibodies, antigens and epitopes? What happens during an immune response? How are monoclonal antibodies generated? How are antibodies used to visualize molecules?

Monoclonal antibodies recognize a single, well-defined epitope produced by cultured hybridoma cells hybridoma cells are formed by fusing antibody-secreting lymphocytes from an animal with myeloma cells hybridoma cells can be maintained indefinitely in tissue culture hybridoma cells secrete large amounts of antibody that can be harvested from the culture medium Lots of commercial interest in monoclonal antibodies!

Select hybridomas producing antibody of interest Culture cells

Comparison: polyclonal vs. monoclonal Polyclonal antibodies Immunoglobin fraction from animal serum Mixture of antibodies with different specificities May provide greater sensitivity by binding multiple epitopes on antigen Often less expensive Supply is limited Monoclonal antibodies Purified from medium of cultured hybridoma cells Antibody recognizes a single epitope May provide lower background since less cross-reaction with other proteins Usually more expensive Supply is theoretically limitless

What is the relationship between antibodies, antigens and epitopes? What happens during an immune response? How are monoclonal antibodies generated? How are antibodies used to visualize molecules?

Detection protocols often use a sequence of antibodies Primary antibodies often a mouse monoclonal antibody for an epitope of interest Secondary antibodies animals are injected with Fc fragments from a different species polyclonal antibodies are common enzyme or a chromochrome is often covalently attached to the secondary antibody signal is amplified

Secondary antibodies amplify the signal 2. Secondary antibodies recognize multiple sites on primary antibody Enzyme or fluorochrome amplifies the signal 1. Primary antibody (stoichiometric binding) Antigen molecules

Western blot Primary antibody: mouse monoclonal antibody Secondary antibody: Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) HRP produces a colored reaction product western blot of bacterial cell extracts