Immunocytochemistry - The only technique which can identify an antigen in its tissue or cellular location. In Situ.

Immunocytochemistry originated with Albert H. Coons and colleagues in 1941. First to label an antibody with a fluorescent dye and use it to identify and antigen in tissue sections with a fluorescence microscope. Helped remove uncertainty in histopathology which were previously dependent on special stains. Due to antigen-antibody specificity, positive identification of tissue constituents can now be achieved. First fluorescent dye attached to antibody was fluorescein isocyanate. Fluorescein isothiocyanate soon became the label of choice because the molecule was easier to conjugate to antibody. Fluorescein compounds emit a bright apple-green fluorescence when excited a a wavelength of 490 nm. New fluorophores and enzyme labels have been produced. First enzyme to be used as horse-radish peroxidase. Other enzymes include alkaline phosphatase, glucose oxidase, and beta-D-galactosidase. Electron-dense particles like ferritin and colloidal gold particles are used for electron microscope immunolabeling. Radioactive elements have also been used in autoradiography. Other subsequent modifications have been made including two-layer indirect method, unlabeled antibody-enzyme methods and others. ----Antibody production Antibodies, gamma-globulins, are raised by immunizing animals with antigen. Antigen must be completely pure to ensure the antibody is specific. Polyclonal antibodies are directed to different parts of the antigen or even to the larger carrier protein. If an antigen is small as many bioactive peptides, or if the peptide is not imunogenic, it

must be combined with a larger carrier protein such as bovine serum albumin, thyroglobulin, or limpet haemocyanin. If antibody is specific for part of carrier protein, using an addition of the carrier protein to antibody before use can be used to cause it to be absorbed out. A second booster injection is used to introduce a larger antibody response. Testing Testing for the presence of an antibody can be done with ELISA against pure antigen, RIA, or blotting technique. The most satisfactory way is to use immunocytochemistry on known positive tissue. The antibody can be tested for quality of the specific staining against the "background". If background is too high, antibody must be abandoned. Aviditity of the antibody is also tested because the antibody must combine strongly to not be washed off. Region Specific antibodies Glutaraldehyde attaches primarily to amino groups. The free portion of the happen molecule, distant from the combining site, is most likely to stimulate antibody formation. thus if only free amino group on happen molecule is the NH2-terminal, then immunization with that hapten after coupling with gluaraldehyde is likely to produce antibodies to the free C-terminal. Carbodiimide, on the other hand, will react with free amino or carboxyl groups. thus immunization with a carbodiimide-coupled happen, having these groups only at either end of the molecule, is likely to produce a mixture of antibodies directed to the C- and Nterminals. Knowledge of the structure of the happen is thus essential if the coupler is to be chosen intelligently so that the likely region-specificity of the resulting antibody may be foreseen. Monoclonal antibodies The in vitro production of pure antibodies was introduced by Kohler and Milstein (1975).

Produced in mice, activated by B-lymphocytes from spleen are fused with myelomas. Hybrid cells which continue to grow and divide in culture and also produce antibodies. The hybridoma cells are gradually cloned by limiting dilution into cell lines derived from a single cell, in which all cells produce the same antibody. Alternatively, the hybridoma cells can be grown as an ascitic tumor inside a host mouse. The fluid around the tumor will contain concentrated antibody, about 10 ug/ml. Disadvantage is it may contain other secreted proteins. The great advantage of monoclonal antibodies is their absolute specificity for a single sequence or epitope on the antigen molecule. Disadvantages can be weaker staining. The one particular epitope of interest can be altereted and become unavailable for reaction, resulting in no staining. Characteristics of a 'good' antibody The main requirement for a good antibody is that it shall be of high affinity for its antigen, in other words that its binding sites fit well with the antigen sites on its specific antigen and do not attach to other antigens. The avidity, or binding strength, is a connected property depending on the number of fitting sites. Immunocytochemistry requires antibodies of high avidity, so that they are not washed off the preparations during the staining process. Damaged tissue containing areas of necrosis and with whole cells in smears or unwashed cultures or cytospins can cause antibodies to bind non-specifically. The concentration of the antibody is very important. A high concentration means the polyclonal antiserum can be diluted to get rid of unwanted antibodies.