Immunoglobulins structure and functions, Antigens, and Antibodies

• Describe the structure of an immunoglobulin molecule
  • Glycoprotein, 2 functional parts separated by papain and pepsin, bound by disulphide bond. Cleavage yields 3 fragments ((Fab)2, 1 Fc fragments)
  • Fc region: Effector domain, formed by heavy chains, determines isotype,
    • binds complement (CH2)
    • binds Fc receptor on phagocytes (CH3)
    • contains carboxyl terminal, has carbohydrate side chains
  • Fab region: antigen binding domain, contains variable and hypervariable regions (VL, VH), formed by light and heavy chains, recognizes and binds epitopes on antigen , determines idiotype
• Write short notes on IgM
  • Pentamer or monomer
  • Largest antibody
  • Located on the surface of mature naïve B cells as a monomer(B cell receptor)
  • Circulates as a pentamer with J chain
  • Formed as the first response to antigen contact
  • Pentameric structure allows strong antigen binding while humoral response is initiated
  • Binds and activated complement (highest avidity)
  • Blood group antibodies
  • Immune response (early reaction) antibodies e.g. anti-HbC (Hepatitis B core) IgM antibody
• Write short notes on IgG
  • Monomer, Divalent
  • 4 subclasses: (IgG1 (65%), IgG2, IgG3, IgG4)
  • Most abundant immunoglobulin in serum (70-75%)
  • Delayed formation
    • Ensures long-term immunity
    • Main antibody in subsequent exposure
  • Titer determination for follow-up (high affinity and specificity)
  • Free floating in serum or bound to the surface of lymphocytes
  • Confers transient passive immunity to the child (only immunoglobulin that can cross the placenta)
  • Activates complement (except IgG4)
  • Opsonizes bacteria (IgG1 and IgG3 more effective)
  • Neutralizes viruses and bacterial toxins
  • Rhesus antibodies
  • Immune response (late reaction) antibodies e.g. anti-Hbc IgG
  • Allergy: mediates type II and III hypersensitivity
• Write short notes on IgA
  • Monomer or dimer
  • Most abundant immunoglobulin in the body
  • Low serum concentration
  • Monomer in circulation
  • Dimer when secreted (contains J chain)
  • Crosses epithelial border by transcytosis
  • Found on mucosal surfaces and in bodily fluids like, saliva, mucus, tears and breast milk
  • Prevents binding of pathogens to mucous membranes of host cells
  • Protected from proteases by a secretory component acquired from epithelial cells
  • No complement fixation
  • IgA antibodies to tissue transglutaminase are present in gluten-sensitive enteropathy (coeliac disease)
  • Production by GALT protects against intestinal infections (e.g. giardiasis)
  • Selective IgA deficiency (most common primary immunodeficiency syndrome)
• Write short notes on IgE
  • Monomer
  • Low serum levels
  • Binds to mast cells, cross links upon antigen exposure and release of histamine
  • Binds to basophils
  • Allergy: type I hypersensitivity e.g. anaphylaxis
  • Defense against parasites by stimulating eosinophils
• Write short notes on IgD
  • Monomer
  • Found in blood serum
  • Found on the surface of mature B lymphocytes
  • Function is incompletely understood
• The functions of antibodies are structurally separated. Discuss the statement
  • Opsonization: CH3 Fc portion of IgG
  • Antibody dependent cell-mediated cytotoxicity (ADCC): CH3 Fc portion of IgG
  • Agonism or antagonism on receptors: Fab portion of IgG antibodies
  • Complement fixation: CH2 Fc portion of IgG, IgM
  • Neutralization of antigens: VH and VL Fab portion of IgA, IgG and IgM
• State the mechanisms involved in generation of antibody diversity
  • Somatic recombination (VDJ recombination) at BCRs and TCRs
  • Somatic hypermutation
• Define Immunogenicity
  • The ability of a substance to induce the immune response, either humoral or cell-mediated
• Define Antigenicity
  • The ability of an antigen to combine specifically with the final products of the immune response (Immunological reactivity)
• Define Antigen
  • Any substance which when introduced into the body evokes the immune system and reacts with the products of immune system in a specific and observable manner
    • Satisfies immunogenicity and antigenicity
• Define Immunogen
  • Any substance which induces a detectable immune response (either cell-mediated or humoral)
  • all immunogens are antigens
• Define Hapten
  • Low molecular weight substances, incapable of inducing antibody formation by themselves but can react specifically with antibodies
    • Non-immunogenic but antigenic
    • can become immunogenic when combined with a large protein molecule as a carrier
• Define Epitope
  • Smallest unit of antigenicity, called an antigenic determinant
• Define Paratope antigen binding site on an antibody
• Define Polyclonal antibodies
  • Antibodies produced by different clones of B-cells against different epitopes of the same antigen
• Define Monoclonal antibodies
  • Antibodies produced by the same clones of B-cells against the same epitope on an antigen
• Define Affinity
  • Strength of interaction of a single antigenic epitope with a single paratope of an antibody
• Define Avidity
  • The overall strength of a collection of affinities
• Define Cross-reactivity
  • Antibodies raised against a specific antigen are able to bind to different but chemically similar antigens
• Define Affinity maturation
  • Process by which the affinity of antibodies produces in response to a protein antigen increases with prolonged or repeated exposure to that antigen, occurs via clonal selection and somatic hypermutation
• Define Specificity
  • The degree of selectivity of an antibody for a specific epitope on antigens in the presence of other epitopes

What are the differences between T-cell dependent and T-cell independent antigens

• Briefly describe 5 factors affecting the immunogenicity of a substance
  • Contribution of the immunogen
    • Chemical composition: Proteins > polysaccharides > lipids
    • Size: The larger the molecule, the more immunogenic it is
    • Chemical complexity: increased chemical complexity, increased immunogenicity
    • Conformation and accessibility
    • Physical form: particulate antigens are more immunogenic than soluble antigens, denature antigens are more immunogenic than the native forms
  • Contributions of the biological system
    • Genetic factors: responders vs non-responders
    • Age: young and old have diminished ability to mount an immune response to an immunogen
  • Method of administration
    • Dose: high dose = increased immunogenicity
    • Route: SC route is better than intravenous or intragastric routes
    • Adjuvant: Increase immunogenicity
• What is the diagnostic application of Heterophile antigens
  • Weil-Felix reaction
    • Typhus fever
    • Proteus antigens act as heterophile antigens to antibodies against rickettsial antigens
  • Paul-Bunnell test
    • Infectious mononucleosis
    • Sheep RBC antigens act as heterophile antigens to detect cross reacting antibodies against EBV in patient’s serum
  • Cold agglutination test
    • Primary atypical pneumonia (Mycoplasma)
    • Human O blood group antigen acts as heterophile antigens against mycoplasma antibodies
• Describe how polyclonal antibodies are produced
  • Lab animal is injected with a specific antigen
  • Activation of multiple clones of B cells
  • Each B cell corresponds to a specific epitope on the antibody
  • B cells differentiate into antibody secreting plasma cells
  • High levels of antibodies are produced after a few weeks
  • Whole serum is collected containing antibodies from multiple B-cell clones
  • Serum is fractionated and antibodies are purified
• Describe in detail the steps involved in the production of monoclonal antibodiesHybridoma technology
  • Isolation of B cells
    • Immunization of lab animal with antigen
    • Multiple immunizations for affinity maturation
    • Splenectomy
    • Fragmentation
  • Cell fusion
    • Fusion of B-cells with multiple myeloma cells with HGPRT and Immunoglobulin gene mutations
      • HGPRT mutation: cannot salvage DNA
      • Immunoglobulin gene mutation: no antibody production
    • Done on polyethylene glycol medium by electrophoresis
  • Outcome of fusion
    • 5 products: Unfused B cells, fused B cells, Unfused myeloma cells, myeloma cells, Hybridoma cell
  • Selection of hybridoma
    • Cultured in Hypoxanthine, Aminopterine, Thymidine (HAT) medium
      • Aminopterine: Inhibits de-novo purine synthesis causing myeloma cells to die
      • B-cells die after some time
      • Hybridomas remain alive
  • Isolation of hybridoma of different specificities
    • Limiting dilution method or limited specificity
  • Screening of products
    • ELISA or RIA to detect antibodies of desired specificity
  • Cloning and propagation of the desired hybridomas
  • Monoclonal antibodies are then characterized and stored
• Using specific examples describe the clinical uses of monoclonal antibodies
  • Therapy
    • Infectious disease
      • Palvizumab: RSV prophylaxis
    • Transplantation
      • OKT3, Basiliximab, Daclizumab against CD25/IL-2 alpha chain
    • Auto-immune diseases
      • Infliximab with MTX for refractory RA
    • Cancer
      • Rituximab for B cell lymphoma
      • Trastuzumab (Herceptin) for Breast Ca
  • Diagnostic
    • In-Vitro diagnosis
      • Western blot, Immunodot blot, flow cytometry, ELISA, RIA, Immunofluorescence microscopy
    • Rapid-antigen tests against infectious disease
      • Gonorrhoea, chlamydia Lateral flow
    • Endocrinology diagnoses
  • Interventional therapy and diagnosis
    • Used in targeted therapy or diagnosis as a conjugate
• What are the clinical uses of polyclonal antibodies
  • Lab diagnosis
    • Rheumatoid factor: prone to cross-reactivity in syphillis infections
  • Precipitation reactions
    • to detect and quantify serum proteins, viruses and other antigens
  • Precipitation reaction in industries
    • to detect the presence of bacteria in clinical and food industry cultures

Differentiate between polyclonal and monoclonal antibodies