Antibodies and Immunology

• The cells of the immune system express molecules on the surface, many of which are functional components of immune cells, and these are called MARKERS, usually on the surface of the cell but can be expressed internally.
• In 1982, a group of scientists established the 'CD' system (Cluster of Differentiation) for naming these markers, and now there are over 370 CD molecules.
• Not all markers/CD molecules are specific for one cell type, some may be expressed by a number of different cells.
• Monocyte, T cell, and B cell markers include CD3, CD19, CD20, and CD14, which can be used to identify and classify these cells.
• Antibodies can detect the presence of these markers, and naïve B cells express antibodies/membrane Immunoglobulin (IgM and IgD) as part of the surface B cell receptor (BCR).
• Each B cell recognizes a different antigen, and when the BCR encounters antigen (with T cell help), they secrete IgM antibodies against the antigen.
• Later in the immune response, the same antibody variable region may be expressed in IgA, IgG, or IgE antibody classes, known as 'class switching'.
• Antibody classes can be further divided into subclasses, such as IgG1, 2a, 2b, and 3 in mice and IgG1, 2, 3, and 4 in humans, also called 'isotypes'.
• The structure of antibodies varies slightly between isotypes, with the Constant Region responsible for Effector Function (binding FcRs, activation of complement).
• Commercial Antibody preparations can be produced from one specific B cell (monoclonal Ab: MAb) or from a pool of B cells specific for different epitopes of the same antigen (polyclonal Ab).
• Antibodies can be tagged with fluorescent markers, Enzymes, or Radioactive isotopes and used on cells, soluble/secreted/biological products, and tissues.
• Antibody detection of cell surface (or internal) molecules can be used to identify/count cells, purify cells/tissues/secreted products, examine cellular activation/differentiation status, and examine pathology and expression of abnormalities.
• Techniques that use Antibodies include Affinity purification, Immunoprecipitation, ELISPOT, Immunofluorescence, Histology (immunohistochemistry), ELISA, and Flow cytometry.
• Flow cytometry uses the light scattering properties of single cells in suspension in conjunction with laser-excited fluorescence from surface marker-specific Antibodies with fluorescent tags (fluorophores) to detect expression of surface or internal markers and cellular products.
• Key features of Flow cytometry include hydrodynamic or acoustic focussing of a single cell suspension, cell size and granularity detection by Sheath fluid light scattering patterns, and the ability to 'stain' or 'label' cells for specific markers with fluorescently tagged antibodies or dyes.
• Fluorophores are excited by a laser and emit fluorescence at a longer wavelength, which is filtered, quantified, and displayed/stored by the software.
• Multiple, simultaneous measurements can be made on the same cell using different fluorophores with different excitation/emission profiles.
• Flow cytometry allows for the measurement of large numbers of cells quickly and accurately, including rare cell types.

Test your knowledge on antibodies, including their structure, classes, subclasses, and functions. Learn about antigen binding sites, class switching, and effector functions. Discover how antibodies are used as scientific tools and how they can be produced.