Antibody Structure and Function PDF

ANTIBODY STRUCTURE AND FUNCTION CHAPTER 5 PREAMBLE  PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY.  PowerPoints DO NOT cover the details needed for the Unit exam  Each student is responsible for READING the TEXTBOOK for detai...

ANTIBODY STRUCTURE AND FUNCTION CHAPTER 5 PREAMBLE  PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY.  PowerPoints DO NOT cover the details needed for the Unit exam  Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES  Unit Objectives are your study guide (not this PowerPoint)  Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! CHAPTER OVERVIEW General Structure of Immunologic characteristics of Antibody classes immunoglobulins memory antibodies Antibody Immunoglobulin Monoclonal specificity and genes antibodies diversity ANTIBODIES  Are immunoglobulins  Glycoproteins found in the serum  82% to 96% polypeptide and 2% to 14% carbohydrate  Five major classes: IgG, IgM, IgA, IgD, IgE  Are the key element of the humoral immune response ANTIBODIES (CONTINUED)  Appear primarily in the gamma (γ) globulin band with serum electrophoresis at pH 8.6  TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS  Basic four-chain polypeptide  Two large peptide chains = heavy (H) chains  Two small peptide chains = light (L) chains  Held together by noncovalent forces and disulfide interchain bridges  Heavy chains Denoted by Greek letters for each Ig class (e.g., Gamma [g] for IgG) HEAVY AND Light chains — two types LIGHT Kappa (κ) CHAINS Lambda (λ) Have a molecular weight of ~22,000 daltons and differ by just a few amino acids Found in all five classes of Igs but only one type is present in a single molecule  TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS (CONTINUED)  Unique variable region (amino- terminal end) allows for specific binding to a particular antigen.  Three to four constant regions (carboxy-terminal end) are responsible for biological functions of antibody. HINGE REGION  Segment of H chain between the CH1 and CH2 regions  Allows for flexibility  Two antigen-binding sites can operate independently  Assists in initiation of complement cascade and Fc receptor binding CARBOHYDRATE PORTION OF IGS  Found in all classes of immunoglobulins  Localized between the CH2 domains of the two H chains  Increases the solubility of immunoglobulin  Provides protection against degradation  Enhances functional activity of the Fc domains TREATMENT OF IG WITH PAPAIN  Two Fab fragments  One L chain plus one-half of an H chain held together by S=S  Contains antigen-binding sites  One Fc fragment  The carboxy-terminal halves of two H chains held together by S=S  Important in effector functions of Igs TREATMENT OF IG WITH PEPSIN  Pepsin cleaves the Ig above the set of disulfide bonds that hold the heavy chains together  F(ab’)2 created  Contains two antigen-binding sites held together  Fc’ portion in nonfunctional pieces ISOTYPES, ALLOTYPES, AND IDIOTYPES Amino acid sequences in immunoglobulins that are antigenic determinants Can react with antibodies produced by immunization of heterologous species ISOTYPES  Unique amino acid sequences common to all Igs of a given class or subclass  Identical in all individuals of a species and differ between species  Located in constant regions of heavy chains and are denoted by Greek letters for each Ig class: IgG: γ chain, IgM: μ chain, IgA: α chain, IgD: δ chain, IgE: ε chain ALLOTYPES  Minor variations of amino acid sequences that are present in some individuals of the same species but not others  Located in the constant regions of the IgG subclasses, one IgA subclass, and the l light chain IDIOTYPES  Variations in variable regions that give individual antibody molecules specificity  Located in amino terminal regions of heavy and light chains THREE-DIMENSIONAL STRUCTURE OF ANTIBODIES  Peptides folded into compact globular subunits stabilized by intrachain disulfide bonds.  Creates a cylindrical structure called an immunoglobulin fold, which captures antigen.  Antigen binds to hypervariable regions containing CDRs. MECHANISMS OF ANTIBODY ACTION Complement fixation is the main mechanism used against cellular antigens Antibodies bound to cells change shape and expose complement binding sites This triggers complement fixation and cell lysis Complement activation: Enhances the inflammatory response Uses a positive feedback cycle to promote phagocytosis Enlists more and more defensive elements MECHANISMS OF ANTIBODY ACTION  Neutralization – antibodies bind to and block specific sites on viruses or exotoxins, thus preventing these antigens from binding to receptors on tissue cells  Agglutination – antibodies bind the same determinant on more than one antigen  Makes antigen-antibody complexes that are cross-linked into large lattices  Cell-bound antigens are cross-linked, causing clumping (agglutination)  Precipitation – soluble molecules are cross-linked into large insoluble complexes Figure 20.13 IGG Predominant immunoglobulin in humans (70%–75% of total serum immunoglobulin) Longest half-life of any immunoglobulin (23 days) Monomer with a molecular weight of 150,000 daltons and a sedimentation coefficient of 7S Four subclasses: IgG1: 66%, IgG2: 23%, IgG3: 7%, IgG4: 4%   Have slight differences in constant region amino acid sequences  Differ in number and position of disulfide bridges between the γ chains  Differ in functions IGG SUBCLASSES IGG SUBCLASSES IgG1 and IgG3 Produced in response to protein antigens IgG3 Has the largest hinge region Has the largest number of interchain disulfide bonds Is the most efficient at binding complement IgG2 and IgG4 Are involved in responses to polysaccharide antigens Have shorter hinge segments Are poor mediators of complement activation MAJOR FUNCTIONS OF IGG  Binding to complement  Leads to inflammation and destruction of foreign cells  IgG3 is most efficient, followed by IgG1   Neutralization of MAJOR toxins and viruses FUNCTIONS  Only Ig that can cross OF IGG the placenta – provides immunity to newborn (CONTINUED)  Lab Testing: Precipitation and agglutination MAJOR FUNCTIONS OF IGG Providing Fixing Coating Neutralizing Participating Providing immunity Fixing complement Coating antigen for Neutralizing toxins Participating in for the newborn (important in enhanced and viruses agglutination and (because IgG can Blood Bank) phagocytosis precipitation cross the placenta) (opsonization) reactions IGM Known as a macroglobulin Has a molecular weight of about 900,000 d Accounts for 5% to 10% of all serum immunoglobulins Has half-life of 6 days (much shorter than that of IgG) Can exist as: Monomer (on surface of B cells) Pentamer (found in blood) IGM (CONTINUED)  Pentamer form  Held together by a J chain, which forms disulfide bonds that link ends of adjacent monomers  Has a star-like shape with 10 antigen- binding sites FUNCTIONS OF IGM Complement fixation Agglutination Neutralization of bacterial toxins and viruses Known as the primary response antibody Appears first after antigenic stimulation and in the maturing infant Synthesized only as long as antigen remains present and may be used to diagnose acute infection IGM: PRIMARY VERSUS SECONDARY RESPONSE IGA  Accounts for 10% to 15% of all circulating immunoglobulins in serum  Serum IgA: A monomer with three constant regions and a molecular weight of 160,000 d  Two subclasses — IgA1 and IgA2 IgA1 IgA2 Acts as an anti-inflammatory agent Predominantly found in secretions Downregulates IgG-mediated at mucosal surfaces along the phagocytosis, chemotaxis, respiratory, urogenital, and intestinal bactericidal activity, and cytokine tracts release  SECRETORY IGA  Synthesized mainly by plasma cells in mucosal- associated lymphoid tissue  Released as a dimer held together by a J chain  Contains a secretory component (SC) derived from epithelial cells  Secretory IgA patrols mucosal surfaces and acts as a first line of defense  Neutralizes toxins produced by microorganisms  Prevents bacteria from adhering to mucosal surfaces and penetrating farther into body  Passively transfers immunity to newborn during breastfeeding  IgA can act as an opsonin.  Aggregation of IgA immune complexes may trigger alternative pathway of complement. IGA FUNCTIONS SECRETORY IGA FUNCTIONS Neutrophils, monocytes, Binding to these sites Both forms of IgA can and macrophages possess triggers a respiratory thus act as opsonins, or specific receptors for burst and degranulation promoters of serum and secretory IgA of the cells involved phagocytosis IGD  Extremely scarce in the serum  Less than 0.001% of total immunoglobulins  A monomer with a molecular weight of 180,000 d and an extended hinge region  More susceptible to proteolysis than other immunoglobulins  Has a short half-life (1 to 3 days)  IgD in serum does not appear to serve a protective function; it does not bind complement, bind to neutrophils or macrophages, or cross the placenta. IGD FUNCTIONS IgD is also found on the surface of immunocompetent but unstimulated B lymphocytes: Appears second (after IgM) May play a role in B-cell activation IGE  Normally 0.0005% of total serum immunoglobulins  A monomer with a molecular weight of 190,000 daltons and a heavy chain with four constant domains  Binding of IgE to eosinophils results in release of enzymes that destroy large antigens like parasitic worms that cannot be easily phagocytized  Does not participate in complement fixation, agglutination, or opsonization Is incapable of crossing the placenta IGE Attaches to basophils, eosinophils and tissue mast cells through high-affinity Fc ε RI receptors Eosinophils play a major part in the destruction of large antigens, such as parasitic worms, that cannot be easily phagocytized IGE FUNCTION: ALLERGIC REACTIONS  Two adjacent IgE molecules on a mast cell bind a specific antigen.  Cascade of cellular events results in degranulation of mast cells and release of vasoactive amines (such as histamine and heparin).   Based on clonal selection and Ig genetics ANTIBODY  1950s: Jerne and Burnet’s theory of clonal selection for antibody formation: SPECIFICITY AND Lymphocytes are genetically preprogrammed to produce one type of DIVERSITY immunoglobulin. A specific antigen finds the particular cells capable of responding to it, causing them to proliferate. Would require a large number of genes.  1965: Dryer and Bennett: Constant and variable portions of immunoglobulin chains are coded for by separate genes.  1987: Susumu Tonegawa: Specific Ig gene segments are selected and joined together during B-cell maturation. CLONAL SELECTION  Each B cell has a BCR specific for a particular antigen.  When antigen enters the body, it binds only to the B cells that possess BCRs specific for it.  Only those B cells proliferate and differentiate into antibody-producing plasma cells. IMMUNOGLOBULIN GENES  Chromosomes contain building blocks from which genes can be assembled  Human immunoglobulin genes are found in three unlinked clusters:  H chain genes on chromosome 14  κ chain genes on chromosome 2  λ chain genes on chromosome 22  Rearrangement of genes is needed to produce functional antibody molecules. IMMUNOGLOBULIN GENES (CONTINUED_1) More than one gene controls synthesis of a particular immunoglobulin H chains Variable-region genes:VH, D, and J Constant-region genes: set of C genes L chains:VL, J, and C (lack a D region) Through random selection during B-cell maturation, individual segments are joined together, committing that B lymphocyte to making antibody of a single specificity. IMMUNOGLOBULIN GENES (CONTINUED_2)  Heavy-chain gene rearrangement IMMUNOGLOBULIN GENES (CONTINUED_3)  Light-chain gene rearrangement ADDITIONAL SOURCES OF ANTIBODY DIVERSITY Different heavy chains Somatic Immunoglobulin class Junctional diversity can combine with hypermutation switching different light chains. Joining of V, J, and D Genetic mutations segments does not during the course of always occur at a the immune fixed position. response that result in stronger binding of antigen (affinity maturation) MONOCLONAL ANTIBODIES Used for laboratory testing and therapy Developed based on knowledge that each B cell is genetically preprogrammed to synthesize a very specific antibody Derived from a hybrid cell line (“hybridoma”) generated from a single antibody-producing B cell and a myeloma cell Technique developed in 1975 by Georges Kohler and Cesar Milstein HYBRIDOMA PRODUCTION  Immunize a mouse with a specific antigen.  Harvest spleen cells.  Combine spleen cells with myeloma cells in the presence of PEG.  Select fused cells and screen for presence of desired antibody.  Grow positive cells in larger quantities. SUMMARY The basic structural unit for all immunoglobulins is a tetrapeptide composed of two heavy chains and two light chains joined together by disulfide bonds. The five classes of immunoglobulins are IgM, IgG, IgA, IgD, and IgE. Kappa and lambda light chains are found in all types of immunoglobulins, but the heavy chains differ for each immunoglobulin class and are denoted by Greek letters. SUMMARY Each Variable region determines the specificity of that molecule for immunoglobulin a particular antigen and contains the antigen-binding sites. molecule has constant and variable regions. Constant region is responsible for Ig functions, such as binding to complement and binding to phagocytic cells. The five different types of heavy chains are called isotypes (g, a, m, d, e). SUMMARY Minor variations in a particular type of heavy chain are called allotypes and can differ among individuals of the same species. The variable portion of heavy and light chains unique to a particular immunoglobulin molecule is known as the idiotype. IgG is the predominant immunoglobulin in the serum. Its functions include binding of complement, opsonization, ADCC, and neutralization of toxins and viruses.  SUMMARY IgM is the largest Ig because it is a pentamer in structure. It excels at complement fixation and agglutination of antigens. It is the primary response antibody: the first to appear during an immune response and during development of the infant. IgA is the major Ig in the body’s secretions, where it occurs as a dimer. It patrols mucosal surfaces and prevents pathogens from further invading the body. It is also present in breast milk and transfers immunity to the newborn infant. IgD has no known function in the serum but can serve as a surface receptor for antigen on the surface of B cells and plays a role in B-cell maturation. IgE binds to mast cells and basophils to initiate an inflammatory reaction that mediates allergic reactions and defense against large parasites. SUMMARY The primary antibody response is the host’s response after the first exposure to an antigen. It takes 5 to 7 days before antibody can be detected due to the time needed to activate the lymphocytes. The secondary response to antigen is caused by the action of memory cells after a repeated exposure to the same antigen. It occurs more rapidly and produces a higher antibody titer that persists for a longer time. SUMMARY Clonal selection is based on the fact that lymphocytes are genetically programmed to respond to a specific antigen. Upon entering the body, the antigen binds to a receptor on the correctly programmed B lymphocyte, causing it to divide and differentiate into antibody-producing plasma cells. Several genes code for a particular immunoglobulin; through a random selection process, these individual segments are joined to make antibody of a single specificity. SUMMARY Monoclonal antibodies are made by hybridoma cells, which are generated by fusing an antibody- producing B cell with a malignant myeloma cell that contributes the property of immortality. Monoclonal antibodies are used as laboratory reagents and as therapies for various diseases. POSTAMBLE  READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES.  USE THE UNIT OBJECTIVES AS A STUDY GUIDE  All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives