The immune response Antibodies Antigens Epitopes (antigenic determinants) the part of a protein antigen recognized by an antibody Haptens small molecules that can elicit an immune response when linked to a protein carrier (often keyhole limpet hemocyanin) Antibody-producing cells (B lymphocytes) see Nelson and Cox Table 5-2
Light chain Heavy Chain Antigen binding site Figure 5-6. Schematic diagram of IgG.
Figure 5-24
Figure 5-27c
Figure 5-23b
constant regions are the same in all IgG, variable regions are different for IgG's directed against different antigens Figure 5-23a
origin of diversity of variable regions V-D-J recombination recombination joins a particular combination of V, D and J coding regions together Berg et al., Biochemistry, Figure 33.17 somatic hypermutation further variation by mutations in variable region during B cell proliferation clonal selection each B cell produces one antibody, presence of antigen induces one clone of B cells to proliferate
From Berg et al., Biochemistry (available online) Figure 33.17. V( D ) J Recombination. The heavy-chain locus includes an array of 51 V segments, 27 D segments, and 6 J segments. Gene rearrangement begins with D-J joining, followed by further rearrangement to link the V segment to the DJ segment.
classes of antibodies IgA, IgD, IgE, IgG, IgM Figure 5-25 IgM
Polyclonal antibodies an antiserum containing antibodies directed against different epitopes on the antigen Monoclonal antibodies a single type of antibody recognizing a single epitope
Proteins used to detect antibodies Secondary antibodies antibody from one species directed against the constant region of antibodies from another species Protein A binds to all IgG Biotin-avidin (or streptavidin) avidin and streptavidin bind biotin with very high affinity, antibodies can be biotinylated (have biotin covalently attached)
Figure 5-28a
Detection methods Radioactive detection usually by iodinating tyrosines on secondary antibody with radioactive iodine Enzyme detection horseradish peroxidase and alkaline phosphatase covalently bound to secondary antibody (Nelson and Cox figure 5-28a) give products that are: Colored -- e.g. NBT/BCIP detection system Figure 5-8 of lab manual Chemiluminescent enzyme produces a phosphorescent product
2 Br Cl BCIP OPO 3 2 N NBT H ox PO 4 3 NBT red (blue) Br Cl O H N N H O Dehydroindigo Cl Br Figure 5-8. Alkaline-phosphatase catalyzed dephosphorylation of BCIP and subsequent colorreaction.
Figure 5-28a -- ELISA
Figure 5-28b
Western Blot (a) Membrane A B C Top Bottom SDS-PAGE Conjugated second antibody AP (c) (side view) A B C (b) Primary antibody NBT/BCIP (d) Blue Color Figure 5-7. Immunoblot procedure. (a) Protein is electrophoretically transferred from gel to PVDF membrane; (b) binding of primary antibody; (c) binding of second antibody, conjugated to alkaline phosphatase (AP); (d) color development with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-indoyl phosphate (BCIP).
Figure 5-28c
Antibody specificity and titer "Titer" refers to the dilution of the antibody Antibodies bind strongly to the target antigen but may bind weakly to other proteins, weak binding is promoted by high concentrations Find a titer which is concentrated enough to give a strong signal but dilute enough to avoid nonspecific binding
Blocking Antibodies can bind nonspecifically to hydrophobic surfaces Before adding an antibody, incubate the surface (plastic well in an ELISA, membrane in a blot) with an inexpensive protein solutioin (BSA, gelatin, non-fat dry milk) to block the nonspecific binding sites
Information you can get with Western blots In which tissues is a given protein expressed at high levels? Monitor synthesis or degradation of a protein Monitor proteolytic cleavage of a protein Monitor covalent modification of a protein covalent modification giving a shift in gel mobility use of modification-specific antibodies (e.g. antibodies that recognize only phosphorylated proteins) Monitor presence of a protein during purification Is the protein you purified the one you were trying to purify?
Other experiments with antibodies Immunoprecipitation detect protein-protein interactions if proteins A and B form a complex in solution, adding an antibody to protein B gives a precipitate with both proteins Immunoaffinity chromatography protein purification covalently link an antibody to an affinity column
Immunocytochemistry localize proteins within cells peroxidase labeling protein with iron atoms is electrondense and gives black staining in micrographs of tissue sections immunofluorescence use antibody with a fluorescent label, fluorescence shows how the antigen is localized in cells Immunoelectron microscopy use antibodies with colloidal gold attached (typically 5 nm diameter), shows location of antigen at an ultrastructural level (electron micrographs)
Figure 16-49. Focal contacts and stress fibers in a cultured fibroblast. (A) Focal contacts are best seen in living cells by reflection-interference microscopy. In this technique, light is reflected from the lower surface of a cell attached to a glass slide, and the focal contacts appear as dark patches. (B) Immunofluorescence staining of the same cell (after fixation) with antibodies to actin shows that most of the cell's actin filament bundles (or stress fibers) terminate at or close to a focal contact. (Courtesy of Grenham Ireland.)
Figure 19-12. Focal adhesions. (A) In these immunofluorescence micrographs, cells in culture have been labeled with antibodies against both actin (green) and the intracellular anchor protein vinculin (red). Note that vinculin is located at focal adhesions, which is also where bundles of actin filaments terminate at the plasma membrane. (B) Some of the proteins that form focal adhesions. The transmembrane adhesion protein is an integrin heterodimer, composed of an and a subunit. Its extracellular domains bind to components of the extracellular matrix, while the cytoplasmic tail of the subunit binds indirectly to actin filaments via several intracellular anchor proteins. (A, from B. Geiger, E. Schmid, and W. Franke, Differentiation 23:189 205, 1983.)
Figure 19-54. Immunoelectron micrograph of a fibroblast cell showing plectin cross-links between intermediate filaments and microtubules. Microtubules are highlighted in red; intermediate filaments, in blue; and the short connecting fibers between them, in green. Staining with gold-labeled antibodies to plectin (yellow) reveals that these fibers contain plectin. See Figure 5-17 for a description of this microscopic technique. [From T. M. Svitkina, A. B. Verkhovsky, and G. G. Borisy, 1996, J. Cell Biol. 135:991; courtesy of T. N. Svitkina.]
Recent developments Tobacco antibodies single-chain antibodies Recombinant antibodies Catalytic antibodies