Immunological Techniques

• uses of antibodies in research and diagnosis include:

  • affinity chromatography and Immunoprecipitation
  • immunochemistry
  • western blotting
  • radioimmunoassay and ELISA
  • flow cytometry
• an antibody can bind to a specific epitope on an antigen through the variable regions
• Antibody Structure

  A) variable region

  B) constant region

  C) light chain

  D) heavy chain

  E) hinge region

  
• an epitope is a specific region of an antigen that is recognised by an antibody
• there are 5 classes of antibodies:

  • IgG, IgD, IgF
  • IgA (dimer)
  • IgM (pentamer)
• the basic Y-shape structure is the same in all classes of immunoglobulin however each class has a unique amino acid sequence in the heavy chain constant region
• each antibody has a variable region specific to a particular epitope, which is where it will bind.
• valence is the number of antibody molecules that can bind to an antigen at one time
• IgG antibodies have a flexible hinge region which is located between the 2 arms and the stem of the Y
• antibodies are highly specific - can be thought of as a receptor for a particular molecule
• IgG is the most commonly used antibody in immunological methods
• the constant region of the antibody is important for mediating the effects of the antibody in vivo - with the different classes of immunoglobulins having different biological functions
• the proportion of each class of antibody can vary and may change in certain diseased states
• the surface of an antigen possesses many potential antigenic determinants or epitopes - however, the number which can bind is bound by the valence of the antigen
• steric conditions can limit the number of different antibodies that bind to the surface of an antigen at any one time so that the umber of epitopes on an antigen is always greater than or equal to its valence
• an antibody can be thought of as a specific label for a molecule - and so can be detected by the antibody when it is present, the antibody is labelled in the constant region
• common antibody labels include:

  • magnetic beads (antibodies can then be "selected" by a magnet)
  • agarose beads (antibodies mobilised in a column or centrifuged due to the bulky nature of the bead)
• fluorophores are common antibody labels and are molecules that give off light and can be seen under a microscope revealing the location of the antibody and hence the antigen
• enzymes such as horseradish peroxidase (HRP) or alkaline phosphate (AP) are common antibody labels and can be coupled with an antibody and will catalyse a reaction at the site at which the antibody is found
• a primary antibody binds to epitopes on the antigen whereas a secondary antibody binds to epitopes on the primary antibody
• instead of labelling the primary antibody that recognises the molecule of interest, it is possible to label a secondary antibody which recognises the primary antibody
• it is possible to detect an antibody using another antibody, with the secondary antibody being conjugated
• chromatography is the collective term for an analytical technique which is used to separate the components of mixtures of molecules to identify and possibly estimate their concentration in the mixture of interest
• affinity chromatography is a powerful method of purification of a specific molecule or a group of molecules from complex mixtuers
• affinity chromatography utilises highly specific biological interactions between two molecules such as interactions between:

  • enzyme and substrate
  • receptor and ligand
  • antibody and antigen
• the specific biological interactions used in affinity chromatography are usually reversible and are used for purification by placing one of the interacting molecules (affinity ligand) onto an insoluble matrix to immobilise the ligand (sold phase) while the target molecule is in the mobile phase (molecules in solution)
• the specific antigen used in affinity chromatography is then eluted by altering the pH which usually disrupts the antibody-antigen bonds. antibodies can be purified in the same way on beads coupled to antigen and protein may be detected by staining or by western blotting
• immunoprecipitation is when antibodies are used to capture a specific protein, with the entire reaction taking place in solution instead of using an antibody column
• for immunoprecipitation an antibody with magnetic beads attached as a secondary antibody can be used to pull the antibodies to the bottom of the tube bringing the primary antibody and antigen with it
• immunoprecipitation can also be used with the secondary antibody bound to agarose beads and subsequently centrifuged to bring the antibody beads to the bottom of the tube
• protein A is a preparation of bacterial cell walls that interacts with antibodies causing them to precipitate
• Any protein that interacts with and binds to antigen A will end up at the bottom of the tube with the antibody complex (called co-immunoprecipitation) and by separating the proteins by SDS-PAGE and detecting the proteins by western blotting interactions can be determined
• Using protein A during immunoprecipitation can also detect interacting proteins.
• Immunocytochemistry (ICC), Immunohistochemistry (IHC) and Immunofluorescence (IF) all utilize antibodies to visualise proteins which can indicate the location of a protein and how abundant it is.
• IHC, ICC, and IF are similar techniques but ICC is carried out on cells, IHC on tissue (histology – microscopic study of tissue) but normally using chromogenic reagents. The difference for IF is it uses fluorescent labels.
• tissues are cut into thin sections (3-10um) and antigens are detected with a specific antibody which will bind to the tissue section where the antigen is found, multiple antigens can be detected using differently labelled antibodies
• In a direct method of IHC/IF, the primary antibody is conjugated to a fluorophore or chromophore and visualised directly
• in IHC/IF indirect methods either a conjugated secondary antibody or labelled protein A is used. an advantage of this is that more label is associated with the primary antibody (amplification of the signal) compared to direct labeling
• immunofluorescence is using antibodies labelled with a fluorescent dye such as fluorescein are used to reveal the presence of their corresponding antigens in cells or tissues
• the stained cells in immunofluorescence are then examined in a microscope that exposes them to blue or green light to excite the fluorescent dye which emits light at a characteristic wavelength which is captured by viewing the sample through a selective filter.