immunology

• The cells and molecules of innate immunity recognize and respond to common patterns shared by a wide range of pathogens.
• The immune system provides the human host with specific protective responses to microorganisms
• The immune system has a memory and responds more rapidly and effectively to pathogens encountered previously
• Components of the immune system include:

  • Soluble components like antibodies (Immunoglobulins - IgG, IgA, IgM, IgD, IgE)
  • Cellular components like B Lymphocytes, T Lymphocytes, and Natural Killer Cells
• Antigens are chemical substances of high molecular weight that stimulate the immune response in animals
• Bacterial antigens interact with the host immunological system in various ways and are composed of proteins, nucleoproteins, polysaccharides, and some glycolipids
• An antigen may contain several epitopes that are recognized by immune cells
• Antibodies are glycoproteins produced by plasma cells in response to an antigen and circulate in the host's blood and secretions
• Antibodies are specific and bind to antigens, marking them for immunological attack (Opsonization)
• Functions of antibodies include complement activation and neutralization of pathogens
• Cellular components of the immune response include B Lymphocytes, T Lymphocytes, and Natural Killer Cells
• The immune system has two arms: Antibody-Mediated (humoral immunity) and Cell-Mediated (cellular immunity)
• Antibody-antigen reactions are characterized by specific binding, firm but reversible binding, and involve non-covalent bonds between antigenic determinants and variable regions of antibodies
• Antibodies may show cross-reactivity by binding to other antigens that share chemical properties with the original antigen
• Monoclonal antibodies are highly specific, recognize a single epitope on an antigen, and are produced in vitro through hybridoma cell technology
• Characteristics of a single antibody:

  • Expensive production
  • Long production time
  • Large quantities of specific antibodies
  • Production is continuous and uniform once the hybridoma is made
• Antibody Types:

  Monoclonal:

  • Expensive production
  • Rapid production
  • Large quantities of nonspecific antibodies
  • Different batches vary in composition
  • Target a single epitope on an antigen

  Polyclonal:

  • Heterogeneous
  • Less specific - recognize multiple epitopes on an antigen
  • Produced in vivo by injecting animals with antigen to produce different antibodies with different specificities
  • Antibody idiotype (antigen binding site) variation is due to alterations in the nucleotide sequence during antibody production
• Production of a monoclonal antibody:

  • Process for harvesting polyclonal antibodies produced in response to an antigen
  • Antigen and antibody reactions in vitro are known as serological tests
  • Most serologic tests are adapted to detect IgM
  • Bacterial (Direct) agglutination tests: specific antibodies bind to bacterial antigens in a thick suspension to cause visible agglutination
  • Particle (Indirect) agglutination tests: detects antibody via the agglutination of an artificial carrier particle bound with antigen
  • Precipitation tests: soluble antigen interacts with antibody, resulting in a visible precipitate
• Bacterial (Direct) Agglutination Test:

  • Measures the antibody level produced by a host infected with a specific pathogen
  • Uses whole pathogen as a source of antigen
  • Is a direct agglutination assay, since the bacterial cells themselves agglutinate
  • Used as a visual indicator of the antigen-antibody reactions
• Latex Agglutination:

  • Antibodies are bound to latex beads
  • Antigen in the specimen/sample binds to the combining sites of the antibody, forming cross-linked aggregates of latex beads and antigen
  • Can detect as little as 0.1 ng/ml bacterial polysaccharides
• Coagglutination:

  • Antigen-specific antibodies are treated with inactivated cells of Staphylococcus aureus, which contain protein A
  • Protein A binds to the base of the antibody heavy chain, leaving antigen-binding ends free to bind antigen
  • Used for identification of streptococci (Lancefield groups A, B, C, D, F, G, and N), Salmonella Enterotoxin
• Precipitation Tests:

  • These tests measure an antigen or antibody in body fluids by the degree of visible precipitation of antigen-antibody complexes within a gel (agarose) or in solution
  • Most precipitin tests use a polyclonal antiserum rather than monoclonal antibodies
  • Detect soluble antigen using Classic Ouchterlony method (Double Immunodiffusion) and Counterimmunoelectrophoresis
• Double Immunodiffusion:

  • Classic method for detecting soluble exoantigen (pathogen detection)
  • Small wells are cut out of agar in petri dishes
  • Antigen-specific antibody is put in one well, and the sample/specimen is put in the other well
  • Antigen and antibody diffuse toward each other in agarose gel and produce a visible precipitin band at the zone of equivalence
  • Used to detect fungal exoantigen, but is time-consuming (18-24 hours)
• Counterimmunoelectrophoresis (CIE):

  • Electric current is applied
  • Involves simultaneous electrophoresis of antigen and antibody in gel in opposite directions, resulting in precipitation at a point between them
  • Used only if antigen and antibody have opposite charges
  • Used in the diagnosis of disease-causing viruses, producing precipitation lines within 30 minutes
• Immunomagnetic Separation (IMS):

  • Used for detecting and isolating biomolecules
  • Pre-enriched sample is mixed with uniform, superparamagnetic, monodisperse, polymer beads coated with a ligand directed against the specified target
  • Magnetic beads bind to the target, forming a target-bead complex that can be easily isolated using a magnet particle concentrator (MPC)
• Direct Fluorescent Antibody Identification (DFA):

  • Dye is directly conjugated to the antibody
  • Faster than IFA
• Indirect Fluorescent Antibody Identification (IFA):

  • Antigen-specific antibody is unlabeled; the second antibody is conjugated with dye
  • Two-step technique
  • More sensitive than DFA
• Immunochromatographic Assays:

  • Also known as lateral flow tests
  • Allow testing of antigen in a dilute solution
  • Antibodies conjugated to small particles bind the antigen in the first stripe and flow onto the second stripe, producing a line of color
• Western Blot:

  • Steps: electrophoretic transfer of proteins, blocking of nonspecific protein binding sites, incubation with primary antibody, incubation with secondary antibody
• Coagglutination:

  • Antigen-specific antibodies are treated with inactivated cells of Staphylococcus aureus, which contain protein A
  • Protein A binds to the base of the antibody heavy chain, leaving antigen-binding ends free to bind antigen
  • Used for identification of streptococci (Lancefield groups A, B, C, D, F, G, and N), Salmonella Enterotoxin