Lecture 4 - Antibodies .pdf

Antibodies Dr. Simone Sandiford [email protected] Antibodies are immunoglobulins An antibody is a protein that binds specifically to a particular substance - called its ant...

Antibodies Dr. Simone Sandiford [email protected] Antibodies are immunoglobulins An antibody is a protein that binds specifically to a particular substance - called its antigen Antibodies belong to the class of proteins called immunoglobulins (Igs) Antibodies are the secreted form of the B-cell receptor Male et al. Immunology 9th edition Figure 10.1 Antibodies are secreted from differentiated B cells called  Antigens and immunogens Antigens are molecules that can bind specifically to an antibody or generate peptide fragments that are recognised by T cell receptors Immunogens are substances that can elicit an adaptive immune response All antigens have the potential to elicit specific antibodies, but some need to be attached to an immunogen All immunogens are antigens but not all antigens are immunogenic! Requirements for immunogenicity A substance must possess the following characteristics to be immunogenic Foreignness High molecular weight Chemical complexity Degradability www.microbiologybook.com  Haptens Small organic molecules of simple structure that fail to induce immune responses Immune response can be provoked if hapten is attached to a protein carrier Helbert Immunology for medical students 3rd edition Figure 4.1 Antigen, epitope and paratope Epitope is a part of an antigen that binds to an antibody or an antigen receptor Antibodies are specific for epitopes rather than the whole antigen Paratope is the part of an antibody that recognises and binds to the antigen (antigen binding site) Janeway Immunobiology 9th edition Figure 1.14  The antibody molecule can be cleaved into functionally distinct fragments Proteolytic enzymes cleave antibodies into three fragments: Two identical Fab (antigen binding) fragments One Fc (crystallisable) fragment Janeway Immunobiology 9th edition Figure 4.4 Structure of an antibody Antibodies are composed of two types of protein chains Heavy chains (2) Light chains (2) Antigen binding region varies extensively between antibody molecules and is known as the variable or V region. The variable region of an antibody determines its antigen specificity The constant region or C region is far less variable. The constant region of the heavy chain determines the effector functions Janeway Immunobiology 9th edition Figure 4.1  Immunoglobulins Five different classes of immunoglobulins IgM IgD IgG (4 subclasses; IgG1-4) IgA (2 subclasses; IgA 1,2) IgE Nine different isotypes Different antibody isotypes activate different effector https://www.authorea.com/users/348432/articles/473746- systems isotype-selection-for-antibody-based-cancer-therapy Selected properties of human antibodies Helbert. Immunology for Medical Students 3rd edition Table 4-1  Selected features of human antibodies IgM Predominant antibody early in an immune response 10 potential antigen binding sites Most efficient antibody at agglutinating bacteria and activating complement IgD Involved in B cell activation Serum function unknown IgG Most prevalent antibody in serum Able to cross the placenta to allow maternal protection of newborn Selected features of human antibodies IgE Evolved to protect against parasitic infections Involved in allergic reactions IgA Present in serum and seromucous secretions IgA1 predominates in serum Over 80% of serum IgA is a monomer IgA2 predominate antibody in seromucous secretions – saliva, colostrum, tracheobronchial and genitourinary secretions Dimer  Antibody variations Isotype Variation in constant region of heavy chain that results in different classes of antibodies (IgG, IgM, IgA, IgD, IgE) Allotype Variation between individuals due to minor sequence differences in the heavy or light chains of antibodies Idiotype Variation in the antigen binding region of different antibodies Antibody variations https://slideplayer.com/slide/5870818/  Antigen-antibody interactions Affinity: The strength of the interaction between a single antigen binding site (paratope) and its epitope Valence: The number of different molecules that an antigen or an antibody can combine with at one time Avidity: The strength with which a multivalent antibody binds to a multivalent antigen www.slideplayer.com Antigen-antibody interactions Antibodies form multiple non- covalent bonds with antigens Hydrogen bonds Electrostatic bonds Van der Waals forces Hydrophobic forces Cation-pi interactions Janeway Immunobiology 9th edition Figure 4.9  Specificity, cross reactivity, non reactivity Reactions can show a high level of specificity Reactions can also be cross-reactive, binding to structurally related but different antigens Male et al. Immunology 9th edition Figure 10.11 Antibodies act as adaptor molecules for immune effector systems Male et al. Immunology 9th edition Figure 10.2  Fc receptors An antibody acts as an adapter that links a microbe to a phagocyte Fc receptors (FcRs) are expressed on a number of different cells and bind the Fc region of antibodies Fcγ receptors on macrophages and neutrophils bind to Fc portion of IgG Fcα receptors on myeloid Male et al. Immunology 9th edition Figure 1.13 cells bind to the Fc portion of IgA Fcε receptors on mast cells, basophils and activated Antibody participation in host defences Neutralisation Binds to pathogen and blocks their access to cells Opsonisation Coating of pathogens and foreign particles Complement activation Activation of the classical complement pathway Janeway Immunobiology 9th edition Figure 1.28  Neutralising antibodies and SARS-CoV-2 Figure 2: Neutralizing antibodies bind to spike proteins on the surface of SARS-CoV-2 and prevent the virus from binding and entering the host cell. As each antibody can bind to two spike proteins from different viruses, the start forming virus clusters that can be better recognised and destroyed by phagocytes. https://www.fluidic.com/resources/neutralizing-antibodies Phagocytosis of bacteria is greatly enhanced in the presence of antibodies Male et al. Immunology 9th edition Figure 1.14  Immune response and secretion of antibodies Seroconversion The phase of an infection when antibodies against the infecting agent are first detectable in the blood Primary response First exposure to an antigen Slow, sluggish and short lived Low levels of antibodies Secondary response Janeway Immunobiology 9th edition Figure 1.25 Second exposure to an antigen Schematic profiles of dengue viremia, NS-1, and anti-dengue IgM and IgG in blood during primary and secondary dengue infections over time Pang et al 2017, Journal of Clinical Microbiology Progress and Challenges towards Point of Care Diagnostic Developments for Dengue Antigens activate specific clones of lymphocytes Male et al. Immunology 9th edition Figure 1.16 Postulates of the clonal selection hypothesis Janeway Immunobiology 9th edition – Figure 1.17  Fighting infection by clonal selection https://www.youtube.com/watch?v=HUSDvSknIgI Development of the antibody repertoire by gene recombination Immune system creates billions of different antibodies with a limited number of genes Diversity of antibody repertoire is generated by: Somatic recombination Somatic hypermutation Class switching www.kyowa-kirin.com  V(D)J recombination is responsible for the initial antibody repertoire Somatic recombination – rearrangement of segments of genomic DNA within immunoglobulin during B cell development in the absence of antigen Variable (V) region of an immunoglobulin heavy www.biology.arizona.edu and light chain is encoded by more than one gene segment Heavy chain – three Immunology wars: A billion antibodies https://www.youtube.com/watch?v=Na-Zc- xWCLE  Secondary diversification of the antibody repertoire – somatic hypermutation Secondary phase of diversification occurs in activated B cells Somatic hypermutation is the random mutation that occurs in the V region and diversifies antibody repertoire. Alters affinity of the antibody for antigens Janeway Immunobiology 8th edition Figure 5.2 Occurs at the same time as class switching and both processes involve the enzyme activation induced cytidine deaminase (AID) Somatic hypermutation enables affinity maturation Affinity maturation is caused by somatic hypermutation and results in the increased affinity for an antigen BCRs are selected for progressively higher affinity for an antigen during an immune response Lindsay B. Nicholson Essays Biochem. 2016;60:275-301  Secondary diversification of the antibody repertoire – class switching Class switching involves the C region of the heavy chain only and increases functional diversity of antibodies Antigen specificity is retained Cytokines and T cells play a major role in class switching Isotype switching is greatly affected Janeway Immunobiology 8th edition Figure 5.2 by the tissue environment Partly due to different cytokines released by T cells at different sites Somatic hypermutation and class switching https://www.youtube.com/watch?v=ba68cC8h3Eo  Use of antibodies as diagnostic, therapeutic and research tools Due to their high specificity and selectivity antibodies are of great use Diagnostics Detections of infections, measurements of biological markers, recognition of allergies Therapeutics Treatment of cancer, infectious diseases, autoimmune diseases Research Immunohistochemistry, western blots, flow cytometry Types of antibodies Luborsky, Judith (August 2015) Tumour Antigens Recognised by Antibodies. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0001433.pub2  Production of polyclonal antibodies Antibodies generated in a natural immune response or after immunization are polyclonal http://ruo.mbl.co.jp/bio/e/support/method/antibody-production.html Blood antibody levels after immunization After immunization, IgM levels increase first With repeated immunization IgG levels increase http://ruo.mbl.co.jp/bio/e/support/method/antibody-production.html  Preparation of monoclonal antibodies Production of an unlimited supply of monoclonal antibodies can be achieved by: Hybridoma technology Fusion between immortal myeloma cell and an antibody producing B cell Recombinant technology Synthetic antibody or antibody fragment https://blog.addgene.org/plasmid-based-recombinant- generated by recombinant DNA technology monoclonal-antibodies Monoclonal antibodies https://www.youtube.com/watch?v=M3zllm8QbCM Production of monoclonal antibodies – Hybridoma http://ruo.mbl.co.jp/bio/e/support/method/antibody-production.html https://www.youtube.com/watch?v=uuT08OT3wTc Learning objectives Describe the basic structure of antibodies Functions and properties of antibodies Define: antibody, antigen, epitope, paratope, immunogen, hapten, affinity, avidity, valency, idiotype, isotype, allotype Discuss antigen-antibody interactions Discuss generation of the antibody repertoire Discuss the use of antibodies in therapeutics and diagnostics Discuss the preparation of monoclonal antibodies  Required reading Antibodies–Chapter 10- 9th edition Omit section on Fc receptors and antibody engineering B Cell development and the antibody response – Chapter 9 -9th edition read the section on somatic hypermutation and class switch recombination Additional reading Janeway Immunobiology 9th edition – Chapter 4 – Antigen recognition by B and T cell receptors Helbert – Immunology for medical students 3rd edition – Chapters 4, 5 and 6  Assignment Tutorial assignment has been uploaded to OurVLE Form 5 groups of at least 3 persons per group Each group needs to chose a topic from the list and present on it in tutorial on Thursday 19th September 2024 Groups will be discussing their respective topics briefly (8 minutes maximum). 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