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Transcript
Antibodies/ Immunoglobulins Professor Md. Akram Hossain Lesson plan Discovery of antibody Definition of Antibody and Immunoglobulin Structure & classification of Ig Distribution/ location of different classes of Ab/ Ig Biological functions of different classes of Ig/Ab Mechanism of Ab action Review questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. What is antibody and immunoglobulin? What are differences between them? Is there any structural difference between Ab & Ig Ig.. What is the basic structure of an Antibody? Who won the noble prize for that? How Ig/ Ab are classified? How many classes are there? Structure, Location & biological Function of IgG/ IgA/ IgM, IgE. IgE. Which Ig is heaviest / biggest of all Ig? Which Ig is found in secretions? What is the peculiarity of its structure? What is the function of secretory component? Which Igs can activate complement? Which Ig can cross placenta & Why? Which Ig is most abundant? Which Ig is found trace amount & why? Which Ig exists in more than one forms? i.e monomer, dimer, pentamer etc How antibodies defend us? Antibody - a disease fighting protein developed by the body in response to the presence of an antigen Historical and Biochemical Evidence for Immunoglobulin structure. Electrophoretic separation of serum proteins yields albumin, α β γ globulin, in that order. γ globulin levels were increased in immunized animals and could be decreased by incubation with specific antigens. Kabat & Tiselius in 1939 showed that γ globulin fraction of serum contain antibody. Porter proposed of a Y-shaped structure in 1962, after discovering Fc & Fab fragment in 1959. Edelman discovered 4 chains of Immunoglobulin. Porter & Edelman Won noble prize in 1972. Albumin Globulin γ β α Electrophoretic mobility of serum proteins Albumin Globulin γ β α Electrophoretic mobility of serum proteins following immunization Albumin Globulin γ β α Electrophoretic mobility of serum proteins of immunized person after removal of antibody by precipitation with Ag. Antibodies … Also called Immunoglobulins Constitute the gamma globulin portion of blood proteins Are soluble proteins secreted by activated B cells and plasma cells in response to an antigen Are capable of binding specifically with that antigen Antibodies are specific glycoprotein configurations produced by B-lymphocytes and plasma cells in response to a specific antigen and capable of reacting with that antigen. Immunoglobulins structurally similar animal proteins that may or may not be endowed with antibody activity. Thus all antibodies are immunoglobulins but all immunoglobulins are not antibodies however, the terms are used interchangeably. All antibodies are Immunoglobulins although not all Igs can function as antibodies The function of an antibody is to bind its antigen as tightly as possible and then direct it towards other components of the Immune System so that it can be destroyed.. destroyed Antibodies can be made against proteins, carbohydrates, lipids and nucleic acids acids.. • but Antibodies to nucleic acids and lipids can be found in autoimmune diseases diseases.. Antibodies to small organic molecules can cause allergies to drugs Structure of antibody / Immunoglobulin Monomer: A flexible YY-shaped molecule with four protein chains: • 2 identical light chains Kappa, or lambda • 2 identical heavy chains α, ε γ, µ, δ or Variable Regions: Two sections at the end of Y’s arms. Contain the antigen binding sites (Fab). Identical on the same antibody, but vary from one antibody to another. Constant Regions: Stem of monomer and lower parts of Y arms. Fc region: Stem of monomer only. Important because they can bind to complement or cells. Basic Antibody Structure Figure 21.12a, b Antigen binding sites Amino terminal } Variable region Light Chain Constant region Heavy Chain Carboxyl terminal Immunoglobulins structure….. The Heavy chains are α, γ, µ, δ or ε The sites and placing of the di di--sulphide bridges and of the sugar groups varies with the class of heavy chain chain.. The biological functions are dependant on the class of Heavy chain e.g ability to cross the placenta or to fix complement complement.. IgM is pentameric and monomeric IgA is monomeric monomeric,, dimeric and secretory IgG, IgD, IgE are monomeric Structure of Immunoglobins • IgG Y shaped • Antigen binding site located on tip of the Y arms • Fab arms connected to Fc stem domain via a flexible hinge • Two identical H chains and L chains • Each chain has a N-terminal VH and VL domain that together form the antigen binding site • Covalent disulfide bridges between H and H-L Constant and Variable regions of Antibodies • C terminus of both H and L chains • invariant and defined as C (constant) region. • Length ~ 330 amino acids in H chains and ~ 110 amino acids in L chains • N terminal segment • Substantially different in different antibodies, thus named V (variable) region • V region ~ 110 amino acids in length • Contains three subregions that show maximal variation between different antibodies • Defined as hypervariable regions • Designated as complementarity determining regions (CDRs) Function of Antibody To recognize and bind with antigen To remove or kill or neutralize in collaboration with other components of immune system Animation of Phagocytosis by Enhanced Attachment (Opsonization) Animation of ADCC lysis by NK cells Animation showing neutralization of an exotoxin. The Fab portion of the antibodies made against epitopes of the binding site of an exotoxin blocks the exotoxin from binding to the host cell membrane. membrane. As a result, the toxin can not enter the cell and cause harm. harm. Animation showing neutralization of a virus. The Fab portion of the antibodies made against epitopes of the virus attachment site blocks the virus from adsorbing to the receptor site on the host cell membrane. membrane. As a result, the virus can not penetrate and replicate. replicate. Animation of Cytolysis of GramGram-Negative Bacteria The Fab portion of IgG or IgM binds to epitopes on the outer membrane of the gram--negative cell wall. gram wall. This activates the complement pathway enabling the membrane attack complex (MAC) to insert into the outer membrane and possibly the cytoplasmic membrane causing the bacterium to lyse lyse.. Antibodies / Immunoglobulins Five classes of Immunoglobulins (Ig) IgG IgM IgA IgD IgE Due to the C region Immunoglobulin Classes I. IgG Structure: Monomer Percentage serum antibodies: 80% Location: Blood, lymph, intestine Half--life in serum: 23 days Half Complement Fixation: Yes Placental Transfer: Yes Known Functions: Enhances phagocytosis, neutralizes toxins and viruses, protects fetus and newborn, Compliment activation 16. IgC Trophoblast Immunoglobulin Classes II. IgM Structure: Pentamer and monomer Percentage serum antibodies: 55-10% Location: Blood, lymph, B cell surface (monomer) Half--life in serum: 5 days Half Complement Fixation: Yes Placental Transfer: No Known Functions: First antibodies produced during an infection. Effective against microbes and agglutinating antigens. Immunoglobulin Classes III. IgA Structure: Monomer, Dimer and Secretory Percentage serum antibodies: 1010-15% Location: Secretions (tears, saliva, intestine, milk), blood and lymph. 40 mg of secretory IgA /kg body weight is secreted through intestine ( Total daily production of IgG 30 mg/kg) Half--life in serum: 6 days Half Complement Fixation: No Placental Transfer: No Known Functions: Localized protection of mucosal surfaces. Provides immunity to infant digestive tract. Immunoglobulin Classes IV. IgD Structure: Monomer Percentage serum antibodies: 0.2% Location: BB-cell surface, blood, and lymph Half--life in serum: 3 days Half Complement Fixation: No Placental Transfer: No Known Functions: In serum function is unknown. On B cell surface, initiate immune response. Immunoglobulin Classes V. IgE Structure: Monomer Percentage serum antibodies: 0.002% Location: Bound to mast cells and basophils throughout body. Blood. Half--life in serum: 2 days Half Complement Fixation: No Placental Transfer: No Known Functions: Allergic reactions. Possibly lysis of worms. Antibody therapy 1890- Von Behring, 1890Baron KitasatoKitasato- ATS 1901 – First noble prize in Medicine IgG 1. Increases in: a) Chronic granulomatous infections b) Infections of all types c) Hyperimmunization d) Liver disease e) Malnutrition (severe) f) Dysproteinemia g) Disease associated with hypersensitivity granulomas, dermatologic disorders, and IgG myeloma h) Rheumatoid arthritis 2. Decreases in: a) Agammaglobulinemia b) Lymphoid aplasia c) Selective IgG, IgA deficiency d) IgA myeloma e) Bence Jones proteinemia f) Chronic lymphoblastic leukemia IgA 1. Increases (in adults) in: a) Waldenström's macroglobulinemia b) Trypanosomiasis c) Actinomycosis d) Carrión's disease (bartonellosis) e) Malaria f) Infectious mononucleosis g) Lupus erythematosus h) Rheumatoid arthritis I) Dysgammaglobulinemia (certain cases) Note: In the newborn, a level of IgM above 20 ng. Note: ng./dl is an indication of in utero stimulation of the immune system and stimulation by the rubella virus, the cytomegalovirus, syphilis, or toxoplasmosis toxoplasmosis.. 2. Decreases in: a) Agammaglobulinemia b) Lymphoproliferative disorders (certain cases) c) Lymphoid aplasia d) IgG and IgA myeloma e) Dysgammaglobulinemia f) Chronic lymphoblastic leukemia IgD 1. Increases in: a) Chronic infections b) IgD myelomas IgE 1. Increases in: a) Atopic skin diseases such as eczema b) Hay fever c) Asthma d) Anaphylactic shock e) IgEIgE-myeloma 2. Decreases in: a) Congenital agammaglobulinemia b) Hypogammaglobulinemia due to faulty metabolism or synthesis of immunoglobulins Antibody Targets Antibodies themselves do not destroy antigen;; they inactivate and tag it for antigen destruction All antibodies form an antigen antigen--antibody (immune) complex Defensive mechanisms used by antibodies are neutralization, agglutination, precipitation, and complement fixation ANTIBODY FORMATION B lymphocytes: formed & mature in bone marrow maturation: produce antibody molecule, surface receptor stimulation: antigen binds receptor lymphocyte activated by immune system multiplication: forms clone of cells, ‘clonal selection’ plasma cells memory cells ANTIBODY FORMATION Time course primary response - delayed, short-lived IgM first, then IgG secondary response - anamnestic response, rapid, strong Role of other cells Macrophages - ‘Antigen Presenting Cell’ (APC) engulf antigen; cut to epitopes epitopes bond to MHC class II on surface T cell with appropriate receptor recognizes MHC + epitope (CD4 receptor on T cell) APC secretes Interleukin 1 (IL-1), activating T cell T cell secretes other IL molecules Il-2 & IL-4 activate T cell, causing proliferation & release or IL-4 & IL-5 Antibody responses •Most antigens are T-dependent (T cells are required), and these must be processed (fragmented) and presented on antigen presenting cells in association with MHC class II protein. This activates Th cells, which can bind to Ag fragment + MHC II on the B cell surface-leading to B cell activation •Each B cell expresses only one Fab type on its surface Immunoglobulin. •The diversity of these Fab regions is generated during development of B cells in the bone marrow by random rearrangement of V, D, and J gene segments. •The role of antigen is to stimulate those B cells with the “correct” receptor (Ab) to induce clonal proliferation and expansion. Monoclonal Antibodies Commercially prepared antibodies are used: To provide passive immunity In research, clinical testing, and treatment of certain cancers Monoclonal antibodies are pure antibody preparations Specific for a single antigenic determinant Produced from descendents of a single cell Monoclonal Antibodies Hybridomas – cell hybrids made from a fusion of a tumor cell and a B cell Have desirable properties of both parent cells – indefinite proliferation as well as the ability to produce a single type of antibody Polyclonal Antibodies •Produced by immunizing an animal (usually a rabbit or goat) with antigen, usually with adjuvant •Because several different antibodies typically exist that can bind to any particular antigen or even a particular epitope, the B cells producing these antibodies will be activated and the resultant immune response will include several different antibodies against the antigen. •These antibodies are derived from several clones of B cells, so the serum that contains them is called a polyclonal antiserum. The Abs will have several different binding strengths (affinities). Problems with Polyclonal Antibodies •Depending on the antigen, the animal may already have had a natural exposure. •Antibodies in a complex mixture like serum may include crossreacting antibodies. •No two antisera are exactly the same, even if the same animal is immunized with the same antigen. Antibody affinity increases with increased numbers of antigen exposures. Thus, antisera cannot be used like a chemical reagent and expected to behave in an absolutely predictable and reliable manner. •Even so, with appropriate controls and characterization, they can be (and still are) very useful. Monoclonal Antibodies •In 1975 Köhler and Milstein developed a method for the production of unlimited amounts of antibody of a predetermined specificity from a single clone of cells. • These cell lines are immortal and can be grown by any lab to purify the antibody. •These antibodies are very much like a chemical reagent. They behave in a predictable way and are reproducible from one lab and one time to the next. •Although these antibodies recognize just one epitope on the antigen of interest, cross-reactions can occur. Also, use of monoclonal antibodies in diagnostic tests often requires a cocktail of more than one Ab, to avoid loss of detection due to a mutation affecting only one epitope. Animation of Phagocytosis by Enhanced Attachment (Opsonization) Animation of Phagocytosis by Enhanced Attachment (Opsonization) Animation of ADCC lysis by NK cells Animation of Cytolysis of GramGram-Negative Bacteria The Fab portion of IgG or IgM binds to epitopes on the outer membrane of the gram--negative cell wall. gram wall. This activates the complement pathway enabling the membrane attack complex (MAC) to insert into the outer membrane and possibly the cytoplasmic membrane causing the bacterium to lyse lyse.. Animation showing neutralization of an exotoxin. The Fab portion of the antibodies made against epitopes of the binding site of an exotoxin blocks the exotoxin from binding to the host cell membrane. membrane. As a result, the toxin can not enter the cell and cause harm. harm. Animation showing neutralization of a virus. The Fab portion of the antibodies made against epitopes of the virus attachment site blocks the virus from adsorbing to the receptor site on the host cell membrane. membrane. As a result, the virus can not penetrate and replicate. replicate.