Diagnostic Immunology and Serology PDF

Diagnostic Immunology and Serology LM335 Dr. Hassan Kofahi  Part I Revision of the basic Immunological concepts Introduction Immunity is the resistance to diseases Immunity Innate Immunity...

Diagnostic Immunology and Serology LM335 Dr. Hassan Kofahi  Part I Revision of the basic Immunological concepts Introduction Immunity is the resistance to diseases Immunity Innate Immunity Adaptive immunity 1. Non specific 1. Specific 2. Present at birth 2. Acquired later in life 3. Always ready 3. Activated and become ready only after the encounter with the microbe 4. Immediate response 4. Delayed response 5. Composed of the first and 5. Humoral and cellular responses. second lines of defense. 6. No memory 6. Has memory 1st line of defense 2nd line of defense Plasma cells Antibodies Th1 Th2 CD4+ T cells (Helper) Th17 Tfh CD8+ T cells (Cytotoxic /CTL)  Innate immunity 1st line: External defenses 2nd line: Internal defenses  1st Line of Defense: External Defenses Composed of physical, chemical, and biological barriers Prevent microbes from entering the body. Skin Physical barrier Lactic acid and fatty acids maintain the skin at a pH of approximately 5.6, which inhibits the growth of pathogens Psoriasin, a small protein produced by skin cells, has antibacterial effects Lysozyme—an enzyme found in many bodily secretions such as tears and saliva—attacks the cell walls of microorganisms 1st Line of Defense: External Defenses (continued) Respiratory tract Mucous traps microbes and prevents them from adhering to epithelial cells Cilia: expel mucus and the trapped microbes Coughing/sneezing moves pathogens out of tract Genitourinary tract: The flushing action of urine helps to remove many potential pathogens Digestive tract Stomach’s acidity (pH = 1) kills microorganism. Intestines: Normal flora (microbiota) prevent pathogens from establishing themselves. 2nd line of defense: Internal defenses  The second line of defense acts if the microbe do succeed in entering the tissues or circulation. Main Components Cells Macrophages, Dendritic cells. Granulocytes: neutrophils, eosinophils, basophils NK cells. Mast cells Proteins Complement proteins Acute phase proteins Cytokines Macrophages Main functions: Ingestion and destruction of microbes. Production of inflammatory cytokines. Clearance of dead cells and initiation of tissue repair. Antigen-presenting cell (APC). Dendritic cells Main functions: Production of inflammatory cytokines. The most efficient antigen-presenting cell (APC). Neutrophils Present in the blood in high numbers. Functions: The first cell to respond to infection. Phagocytose and destroy microbes. Generate neutrophil extracellular traps (NETs) that help in killing the bacteria Important for the defense against bacterial infections. Eosinophils Functions: Defense against parasitic infections. Play a role in allergic reactions Basophils Circulate in the blood Very rare Functions: Inflammation and allergy Mast cells Reside in the tissues Resemble basophils Functions: Inflammation and allergy Natural killer Cells (NK cells) Lymphocyte-like cells Functions: A. Kill virus-infected cells and tumor cells B. Activate the macrophages to kill phagocytosed microbes Complement system Series of proteins that normally present in an inactive form. Infection triggers a cascade of cleavage and activation of these proteins. The complement system can be activated by three pathways: 1. The classical pathway: Initiated by antibody-antigen reaction. 2. The alternative pathway: Initiated by the cleavage and activation of complement proteins directly on the surface of the microbe. 3. The lectin pathway: Initiated when mannose-binding lectin (MBL: a carbohydrate binding protein) which binds to mannose on the surface of the microbe. Complement activation results in Inflammation Opsonization and phagocytosis Lysis of the microbe Acute phase proteins Present normally in the serum. Produced by the liver. Increase rapidly after infection. Examples: Protein Functions C-reactive protein Opsonization, complement activation Complement C3 Opsonization, lysis of the microbe Mannose binding lectin Complement activation Fibrinogen Clot formation Inflammation Inflammation is a tissue response to infection or damage. The purpose of inflammation is to bring the mediators of host defenses (cells & proteins) to the site of infection or tissue damage. Events: Capillary widening (vasodilation) which increases the blood supply to the infected area Increased capillary permeability Extravasation and migration of the white blood cells to the site of infection. Systemic responses: Fever and increasing the proliferation of WBCs. Signs: Heat, Redness, Swelling and pain.  Adaptive immunity Humoral Cell-mediated Antibodies T-lymphocytes Cell-mediated immunity Mediated by T lymphocytes. CD4+ T lymphocytes (helper T cells) Recognize peptide antigens displayed by MHC class II molecules. Have several subsets, each perform distinct functions Th1 activate the macrophages to kill the ingested microbe. Th2 important in the defense against helminths. Th17 induce inflammatory reactions to destroy extracellular bacteria. Tfh Help in antibody production. CD8+ T lymphocytes (Cytotoxic T lymphocytes, CTLs) Recognize peptide antigens displayed by MHC class I molecules. Main functions: Kill virus-infected and tumor cells. Humoral immunity: Antibodies Antibodies form the immunoglobulin family of proteins. Produced by B-lymphocytes and plasma cells. Antibodies exist in two forms: Membrane-bound form act as B cell receptors. Secreted form  effector molecules of the humoral immunity. 5 isotypes/classes: IgM, IgG, IgA, IgE and IgD. Each isotype/class performs distinct effector functions. . . . . . milk Antibody structure Y-shaped molecule Composed of 2 identical light chains and two identical heavy chains. Each monomeric antibody molecule has two identical antigen-binding sites. Regions: Fab region: the arms of the Y shape. Contains the antigen binding site. Fc region: the stem of the Y shape. Responsible for most of the effector functions (e.g. complement activation). Antigen, immunogen, hapten and epitope Antigen: any substance that binds specifically to an antibody or a T-cell receptor. Immunogen: any substance capable of inducing an immune response. All immunogens are antigens but not all antigens are immunogens. Haptens are small molecules that are antigens (i.e. can bind to the antibody) but are not immunogenic unless they are attached to a carrier protein. Epitope: The part of the antigen that is recognized by the antibody. Antibody production for the lab use The majority of the tests in the immunology/serology Lab uses antibodies as one of the reagents. Two types of Abs are used in the immunology/serology lab Polyclonal antibodies A collection of antibodies from different B cell clones that recognize multiple epitopes on the same antigen. Monoclonal antibodies Antibodies produced by a single B cell clone. Recognize a single epitope on the antigen. Production of polyclonal antibodies Other types of animals are also used including goats, hoarses and mice. Production of monoclonal antibodies Steps: 1. A mouse is immunized with a certain antigen (antigen X). 2. After a time, the mouse’s spleen cells are harvested. Some of these cells are B cells that produces anti-X antibodies. 3. Spleen cells are fused with myeloma cells in the presence of polyethylene glycol (PEG) to produce hybridomas (i.e. cells that resulted from the fusion between spleen cells and myeloma cells).  The myeloma cell are immortal (can grow indefinitely).  The fused hybridoma cells have the ability to: A. Grow indefinitely in culture (acquired from the myeloma cell). B. Produce antibodies (acquired from the spleen B cells). Production of monoclonal antibodies (continued) 4. All cells are placed in selection culture medium. This medium separates out the hybridoma cells by allowing them to grow selectively and not allowing unfused myeloma cells or spleen cells to survive. 5. Single hybridoma cells are placed in separate wells of a microtiter plate and each is allowed to grow to generate a clone, each derived from a single hybridoma cell. 6. The supernatant of each clone is screened for the presence of the desired antibody. Once identified, a hybridoma is maintained in cell culture indefinitely and produces a permanent and uniform supply of monoclonal antibody that reacts with a single epitope.  Polyclonal antibodies Monoclonal antibodies Advantages Inexpensive Large quantities of Quick to produce antibody can be produced Recognize multiple Batch to batch epitopes on the antigen. homogeneity High specificity and less cross-reaction Disadvantages Limited quantities Expensive Batch to batch variation Require more time to Less specificity and produce higher cross-reaction Recognize only one epitope on the antigen.