Topic 3 Antibodies Part A lecture notes.pdf

Foundations in Pharmacology PHRM2005 Dr Ricky Lareu Antibodies Part A 1 Key Concepts Blood components Major plasma/serum proteins Structure and function of immunoglobulins Polyclonal and monoclonal antibodies 2 Learning Outcomes You need to be able to describe the following: Part A Levels of protein...

Foundations in Pharmacology PHRM2005 Dr Ricky Lareu Antibodies Part A 1 Key Concepts Blood components Major plasma/serum proteins Structure and function of immunoglobulins Polyclonal and monoclonal antibodies 2 Learning Outcomes You need to be able to describe the following: Part A Levels of protein structure. Constituents of blood. Major constituents of plasma/serum. Electrophoresis of serum proteins. Major properties and function of albumin and immunoglobulins. Basic structural components of immunoglobulins and their functional properties. Structure and function of the various types of immunoglobulins. Antibody function. Part B The difference between polyclonal and monoclonal antibodies. How polyclonal and monoclonal antibodies are produced. Examples of how monoclonal antibodies can be used i.e. therapy, diagnosis etc. 3 Review: There Are Four Levels of Protein Structure Proteins give the very shape to our bodies, there are over 50,000 proteins made in a cell. Levels: Primary structure - amino acid linear sequence Secondary structure - regions of regularly repeating conformations of the peptide chain, such as -helices and β-sheets, U-turns and loops Tertiary structure - describes the shape of the fully folded polypeptide chain (minimum level of functionality) Quaternary structure - arrangement of two or more polypeptide chains into a multi-unit molecule (only some proteins) 4 Composition of Blood Formed elements: 45% Red Blood Cells (Erythrocytes) – most abundant cells in blood; produced in bone marrow. Contain protein called hemoglobin carrying oxygen to cells. White Blood Cells (Leukocytes) –part of immune system. Destroy infectious agents. Platelets (Thrombocytes) – Hold clotting factors carried in plasma; they clot together in a process called coagulation to seal a wound and prevent loss of blood. Plasma – 55% 5 Composition of Plasma Yellowish liquid portion of blood, 55% Colloid – a liquid containing suspended substances that don’t settle out i.e. proteins Consists of 91.5% water, 7% proteins and 1.5% other including electrolytes, nutrients, gases, regulatory substances, waste products Protein fraction – 70-80% of plasms proteins synthesized in the liver: Albumin – Important in regulation of water movement between tissues and blood, 54% Globulins – Immune system or transport molecules, 38% Fibrinogen – Responsible for formation of blood clots, 7% All others – e.g. hormones, enzymes, 1% If allowed to clot = serum Does not include cells, platelets, fibrinogen Contains immunoglobulins 19-6 Electrophoresis of Serum Proteins Electrophoresis – the separation of charged particles (e.g. proteins) based on migration speed through a matrix (e.g. agarose) or buffer, in a constant electrical field. Proteins and peptides will migrate: ▪ Net charge – amino acid composition ▪ Size (molecular weight) – only in a matrix – large protein will migrate slower Electrophoresis of serum proteins – cellulose acetate zone electrophoresis: Slightly alkaline buffer (~pH 8.4) proteins will have a net negative charge and migrate to anode (positive electrode) – albumin has the highest negative charge. 7 Albumin Major protein in human plasma, 54% Single chain, 580 amino acids with 50% a-helix, MW 66 000 Da Synthesized by the liver, ½ life in circulation (19 days) Functions Colloid osmotic pressure of blood is 80% due to albumin ▪ relatively low molecular weight & regulates water distribution Transport of fatty acids & calcium / copper / drugs ▪ ▪ Liver to tissues, drug binding and transport e.g. Sulfonamides, penicillin G, aspirin Lower levels of albumin can result due to variety of diseases, particularly those with liver disease, and protein malnutrition e.g. kwashiorkor Immunoglobulins Antigen Binding Sites Light chain – binds to antigen/immunogen 2 types – κ chain, λ chain An Ig always has 2 of the same Heavy chain – provides Ig biological property 5 classes or isotypes: μ IgM δ IgD γ IgG α IgA ε IgE Amount of protein Produced by B cells: activated and differentiated into plasma cells Multiple polypeptide chains (at least 4) ▪ 2 identical heavy (green) & 2 identical light (yellow) Linked by disulphide bonds Intrachain disulphide bonds produce folding - domains Domains determine functional properties Variable carbohydrate component - albumin globulins g b 2 Mobility 1 9 + Ig Regions Fab = fragment antigen binding Fc = fragment crystallizable Complement binding site Antigen binding sites Attach to Fc receptor on cells Hinge region allows for Fab components flexibility to bind to differentially-spaced antigens 10 IgG IgE IgD General structures of the five major classes of secreted antibody. Disulfide bonds are indicated by thick black lines. Note that the IgG, IgA, and IgD heavy chains (blue, orange, and green, respectively) contain four domains and a hinge region, whereas the IgM and IgE heavy chains (purple and yellow, respectively) contain five domains but no hinge region. Note: J Chain needed for multiple forms [Dimer or greater] IgA IgM 11 IgG Predominant Ig in blood, lymph, etc. MW = 150,000 Da longest half-life of all Ig’s causes agglutination / precipitation of antigen: – neutralizes toxins – immobilizes bacteria – neutralizes viruses only class of Ig able to cross the placenta – & passed to newborns via colostrum is an opsonizing Ab → promotes phagocytosis mediates antibody-dependent cell-mediated cytotoxicity (ADCC) – killing via NK cells activates the complement system 12 IgM First Ig produced in immune response exists as a pentameric molecule MW = 900,000 most efficient Ig for activating complement system most efficient Ig for agglutinating antigen 13 Antigen-induced conformational changes in IgM Planar or ‘Starfish’ conformation found in solution. Does not fix complement Staple or ‘crab’ conformation of IgM Conformation change induced by binding to antigen. Efficient at fixing complement 14 IgA major Ig in external secretions: – e.g. saliva, tears, nasal fluids, sweat, colostrum, secretions of respiratory and GI tracts in serum IgA is a monomer in secretions it exists as a dimer MW (monomer) = 165,000 (monomer) important in the primary defense against local respiratory or gastrointestinal infections; prevents attachment to epithelial surfaces possesses bactericidal activity efficient antiviral Ab efficient agglutinating Ab dimers also have ‘secretory component’ 15 Secretion of IgA Secretory IgA is formed during transport through mucous membrane epithelial cells. Dimeric IgA binds to a poly-Ig receptor on the basolateral membrane of an epithelial cell and is internalized by receptor mediated endocytosis. After transport of the receptor-IgA complex to the luminal surface, the poly-Ig receptor is enzymatically cleaved releasing the secretory compound bound to the dimeric IgA. 16 IgD co-expressed with IgM on surface of mature B cells present in serum in very low amounts MW = 180,000 function not fully elucidated, but thought to involve elimination of selfreactive B cells and acts as a receptor for antigen 17 IgE lowest concentration in serum, and shortest half-life of all Ig’s MW = 200,000 Fc portion binds with very high affinity to receptors on mast cells and basophils – hypersensitivity reaction (allergy) plays a role in protection against parasites – eosinophil binding 18 Antibody Binding Characteristics Antibody affinity Is a quantitative measure of binding strength Combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag Antibody avidity Incorporates affinity of multiple binding sites True strength of the Ab-Ag interaction within biological systems The interaction at one site will increase the possibility of reaction at a second site High avidity can compensate for low affinity ▪ e.g. secreted pentameric IgM has a higher avidity than IgG 19 Why do Antibodies Need an Fc Region? The (Fab)2 fragment can: Detect antigen Precipitate antigen Block the active sites of toxins or pathogen-associated molecules Block interactions between host and pathogen-associated molecules The Fc component can: Initiate inflammatory and effector functions associated with cells Initiate inflammatory and effector functions of complement Cause the trafficking of antigens into the antigen processing pathways 20 Functions of Antibodies Antibodies can participate in host defense in three main ways. 1) Antibodies neutralize bacterial toxins and complex to virus particles and bacterial cells. 2) Antibodies coating an antigen render it recognizable as foreign by phagocytes (macrophages and neutrophils), which then ingest and destroy it; this is called opsonization. 3) Antibodies activate the complement system by coating a bacterial cell. Ag processing pathways 21 Summary of Ig Properties 22