* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download antibodies
Lymphopoiesis wikipedia , lookup
Hygiene hypothesis wikipedia , lookup
Major histocompatibility complex wikipedia , lookup
Human leukocyte antigen wikipedia , lookup
Immunoprecipitation wikipedia , lookup
Rheumatic fever wikipedia , lookup
Gluten immunochemistry wikipedia , lookup
Innate immune system wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Myasthenia gravis wikipedia , lookup
Complement system wikipedia , lookup
Sjögren syndrome wikipedia , lookup
Guillain–Barré syndrome wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Immune system wikipedia , lookup
Duffy antigen system wikipedia , lookup
DNA vaccination wikipedia , lookup
Adaptive immune system wikipedia , lookup
Molecular mimicry wikipedia , lookup
Immunocontraception wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Anti-nuclear antibody wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
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 1 3 Antigen bacteria viruses allergens large molecules Antigen with 3 epitopes 2 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 Fab Fc Bi-functional molecules Fab fragments bind antigen Fc fragments are used by the immune system to remove antigen-antibody complexes 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-andkey 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 1 3 Antigen 2 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? When challenged with a foreign substance, or antigen, vertebrates produce antibodies that help the system dispose of the antigen Vaccination stimulates the production of antibody-producing cells Biologists use antibodies produced in animals to localize molecules Antigen stimulates the proliferation of B lymphocyte clones that recognize epitopes on the antigen. 1 3 Antigen 2 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 Monoclonal antibodies Immunoglobin fraction from animal serum Purified from medium of cultured hybridoma cells Mixture of antibodies with different specificities Antibody recognizes a single epitope May provide greater sensitivity by binding multiple epitopes on antigen May provide lower background since less cross-reaction with other proteins Often less expensive Usually more expensive Supply is limited 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