Immunoreagents

Polyclonal antisera that contain a mixture of antibody populations and monoclonal antibodies are the most commonly used reagents in immunoelectron microscopy where antigen |or antibody) is being localized. Immunoglobulin G (IgG) in serum frac tions is most often preferred, unless the reaction considerations specify otherwise. Purity and specificity of polyclonal antisera can be improved by affinity purification steps. The benefits of monoclonal anti body use are well known. There are also several bacterial cell wall proteins, protein A and protein G, that bind the Fc portion of y-globulin from various species and have proven useful in detecting antibody bound to antigen. Moreover, since protein A and G are proteins, they can be conjugated to markers useful in immunoelectron microscopy and applied to the detection of a variety of y-globulin classes from multiple species. Other types of biological macromol-ecules (e.g. lectins, asialoglycoprotein, etc.) unrelated to antibodies, when conjugated to markers, can be used as ligands to localize ligand-receptor interactions because of their known specificity and affinity for target molecules. Two other macromolecules, streptavidin and biotin, have been used in immuno-conjugates because of their high affinity and specificity for each other. In this case, biotin is used to directly label the specific antibody and streptavidin is conjugated to the marker. The strong interaction between streptavidin with biotin brings the marker into contact with the bound antibody.

Antibodies can be coupled to the marker by various methods. The most common method, when two protein molecules are involved, is to conjugate the antibody to the marker chemically via a low molecular weight bifunctional coupling reagent (e.g. glu-taraldehyde, metaxylylene di-isocyanate, thiocarba-mide, etc.). However, in the case of highly charged metal sols, like colloidal gold, the electrostatic forces of this material can be used to irreversibly bind antibodies, protein A and G, or streptavidin molecules to the gold particulate.

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