Lecture 6. Antigen Structure and Antibody Interactions

Monoclonal Antibodies and Immune Response

  • Fully human monoclonal antibodies can induce immune responses via allotypic and idiotypic determinants.

    • Better tolerated and less severe reactions than mouse monoclonal antibodies.

Overview of Antigens and Immunogens

  • Immunogen: Generates a humoral or cellular immune response.

  • Antigen: Reacts with antigen receptors, regardless of immune response generation ability.

    • May or may not elicit an immune response;

    • Small molecules may react with preformed antibodies but do not stimulate immune responses unless complexed with larger proteins.

Types of Antigens

  • Proteins:

    • Most potent immunogens.

  • Polysaccharides:

    • Second in immunogenicity.

  • Lipids and Nucleic Acids:

    • Generally non-immunogenic unless complexed with proteins or polysaccharides.

  • Activation:

    • Proteins and polysaccharides activate B cells and induce antibody responses.

    • Proteins (peptides presented by MHC) and glycolipids/lipids presented by CD1 induce T cell responses.

Properties of Antigens

  • Foreignness: Must be recognized as non-self.

  • Molecular Size: Optimal immunogenicity occurs with sizes greater than 100 kDa; poor immunogens are usually less than 5-10 kDa.

  • Chemical Complexity and Composition:

    • More complex antigens are generally more immunogenic (e.g., homopolymers lack immunogenicity; copolymers can be immunogenic if sufficiently sized).

  • Susceptibility to Processing and Presentation:

    • Macromolecules must be processable by antigen-presenting cells to stimulate T helper cells; non-processable antigens are poorly immunogenic.

Antigen Structure

  1. Primary Structure: Linear sequence of amino acids (example: Lys-Ala-His-Gly-Lys-Lys-Val-Leu).

  2. Secondary Structure:

    • Alpha helix

    • Beta pleated sheet

  3. Tertiary Structure: Folded polypeptide chain

  4. Quaternary Structure: Multi-polypeptide complex

Biological Factors Affecting Immunogenicity

  • Genotype of Recipient: Variation in immune response due to individual gene expression profiles.

  • Immunogen Dosage:

    • An inadequate amount fails to activate immune response.

    • Too much induces tolerance.

    • Repeated doses enhance the immune response.

  • Route of Administration:

    • Intradermal and subcutaneous are superior to the oral route (which may induce non-responsiveness).

Adjuvants

  • Substances that enhance immunogenicity when mixed with an antigen.

  • Mechanisms:

    • Prolong antigen persistence.

    • Enhance costimulatory signals.

    • Increase local inflammation (macrophage activation and antigen presentation).

    • Stimulate nonspecific lymphocyte proliferation.

Epitopes

  • Defined as discrete sites on an antigen recognized by lymphocyte antigen receptors.

  • Epitopes are immunologically active regions of immunogens.

  • T cell and B cell recognition is fundamentally different and does not involve the same epitopes.

Comparison of Antigen Recognition: T Cells vs. B Cells

Characteristic

B cells

T cells

Interaction with antigen

Binary complex of membrane Ig and Ag

Ternary complex of T cell receptor, Ag, and MHC molecule

Binding of soluble antigen

Yes

No

Involvement of MHC molecules

None required

Required to display processed antigen

Chemical nature of antigens

Protein, polysaccharide, lipid

Mostly proteins (some lipids and glycolipids on MHC-like molecules)

Epitope properties

Accessible, hydrophilic, mobile peptides

Internal linear peptides from antigen processing bound to MHC molecules

B Cell Epitopes

  • Recognize native antigens via accessible and hydrophilic epitopes.

  • Interactions occur through:

    • Linear sequences of amino acids

    • Nonsequential or conformational epitopes.

  • Interaction is mediated by weak noncovalent forces due to shape complementarity.

Antigen Structural Considerations

  • Most antibodies against globular proteins target surface structures dependent on protein conformation.

  • B cell epitopes are commonly found in flexible regions and can be numerous in large proteins.

  • Immunodominant determinants are most accessible to antibody binding.

Antigen Valency

  • Multivalent Antigen: Contains different epitopes or repeated epitopes.

T Cell Epitopes

  • T cells recognize peptides in complex with self-MHC.

  • T cell epitopes are often internal and are amphipathic sequences revealed by antigen processing.

  • Bind MHC molecules via hydrophobic residues and interact with T cell receptors through hydrophilic residues.

  • Immunodominance is linked to interactions with specific MHC types.

Mitogens and Superantigens

  • Mitogens: Activate multiple clones of B or T cells regardless of specificity; known as polyclonal activators.

    • Examples:

    • Lectins like concanavalin A preferentially activate T cells.

    • LPS preferentially activates B cells.

  • Superantigens: Activate large numbers of T helper cells by cross-linking T cell receptors with MHC class II molecules on antigen-presenting cells, providing potent T cell mitogenic activity.

Antigen-Antibody Interactions

  • Mediated by weak forces:

    • Ionic or electrostatic interactions.

    • Hydrogen bonds.

    • Hydrophobic forces.

    • van der Waals forces.

  • Exclusion of water enhances these interactions.

  • Antigen-antibody binding is reversible and does not involve covalent bonds.

Affinity and Avidity of Antibody Binding

  • Affinity: Strength of interaction between epitope and antibody binding site.

  • Avidity: Overall binding strength between multivalent antibodies and multivalent antigens, noting that avidity of IgM is greater than IgG.

Cross-Reactivity

  • Occurs when antibodies against one antigen cross-react with a similar antigen due to shared epitopes.

  • Example: Cross-reactive antibodies from intestinal microbes resembling blood type antigens.

Precipitation Reactions

  • Formed when mixing specific antibodies with antigens in optimal ratios, resulting in a visible precipitate (precipitins).

  • Structures formed are 3-dimensional lattices from cross-linking multivalent soluble antigens with divalent (IgG) or pentameric (IgM) antibodies.

  • No precipitate forms with antibody or antigen excess.

Radial Immunodiffusion

  • A technique involving antigen diffusion in agar, forming a precipitate ring upon sufficient antigen-antibody interaction.

Agglutination Reactions

  • Cross-linking of particulate multivalent antigens by at least divalent antibodies leads to particle agglutination (agglutinins).

  • Excess antibody inhibits agglutination (prozone effect).

  • Effective agglutination requires sufficient links formed at a distance to overcome mutual repulsion (e.g., red blood cells).

  • IgM is a more effective agglutinin compared to IgG.

  • Applications include medical diagnostics (identifying bacteria, blood typing, disease testing).

Enzyme-Linked Immunosorbent Assay (ELISA)

  • Detects specific antibodies (indirect ELISA) or antigens (sandwich ELISA).

  • Indirect ELISA Process:

    1. Antigen is adsorbed to plastic wells.

    2. Excess antigen is washed away; blocking protein added.

    3. Patient serum added; washed again.

    4. Enzyme-conjugated anti-immunoglobulin is introduced; unbound antibody washed away.

    5. Colorless substrate added to generate a color reaction measured by absorbance.

  • Sandwich ELISA: A capture antibody is first adsorbed before adding antigen preparation.

ELISPOT Assays

  • Modified ELISA for measuring individual cell secretions.

  • Wells contain detection antibody, washed and blocked; cell population added to allow molecule secretion.

    • Additional steps include adding detection antibody and substrate.

Immunofluorescence

  • Visualizes antigen-antibody complexes under UV light after attaching fluorescent dyes to antibodies.

  • Direct Method: Primary antibody is tagged with fluorochrome.

  • Indirect Method: Unlabeled primary antibody is visualized with fluorochrome-conjugated anti-immunoglobulin or protein A; benefits include increased sensitivity and simplicity.

Flow Cytometry

  • Analyzes fluid streams of antibody-labeled cells using lasers.

    • Produces histograms and dot plots quantifying cell populations stained with labeled antibodies (Example: Analysis of TCR quantification).

Sensitivity of Immunoassays (Table 20-1)

  • Assays and their sensitivity levels:

    • Precipitation reaction in fluids: 20-200 μg antibody/ml

    • Precipitation in gels: 20-200 μg antibody/ml

    • Agglutination reactions (various forms): ranging down to ~0.00001-0.01 μg antibody/ml

  • Sensitivity can vary depending on antibody affinity, epitope density, and distribution.

Learning Objectives

  1. Define immunogens and antigens, types, and properties.

  2. Explain factors affecting immunogenicity.

  3. Differentiate T cell and B cell epitopes.

  4. Define mitogens and superantigens.

  5. Understand antibody-antigen interaction forces; differentiate avidity and affinity.

  6. Describe precipitation reactions, including various test methods (radial immunodiffusion, agglutination, ELISA, ELISpot, immunofluorescence/flow cytometry).