Antibody Structure and Function

• Antibodies, also known as immunoglobulins, are critical for adaptive immunity and have applications in diagnostics, therapeutics, and vaccine design.

Basic Composition

• Antibodies consist of two identical heavy chains (50–70 kDa each) and two identical light chains (25 kDa each), linked by disulfide bonds, forming a symmetrical Y-shape.
• Each chain contains constant regions (C) and variable regions (V).
• Variable regions form the antigen-binding sites (paratopes).
• Constant regions determine effector functions.

Domains

• Fab (Fragment antigen-binding): Two arms of the Y, composed of variable (VH, VL) and constant (CH1, CL) domains, bind antigens with high specificity.
• Fc (Fragment crystallizable): The stem of the Y, formed by the constant domains (CH2, CH3) of the heavy chains; interacts with immune cells (macrophages) and complement proteins to trigger immune responses.
• Hinge Region: Flexible segment between Fab and Fc, allowing antibodies to bind antigens at varying distances and angles.

Classes (Isotypes)

• Determined by heavy-chain constant regions:
  • IgG (γ chain): Most abundant in serum, crosses the placenta for passive immunity.
  • IgM (μ chain): Pentamer in serum, first responder in primary immune responses.
  • IgA (α chain): Dimer in mucosal secretions (e.g., saliva, breast milk).
  • IgE (ε chain): Binds mast cells/basophils and mediates allergic reactions.
  • IgD (δ chain): Expressed on B-cell surfaces and plays a role in immune activation.

Functions

• Antigen Binding: Variable regions recognize and bind specific epitopes on antigens (e.g., pathogens, toxins), neutralizing their activity.
• Effector Functions (via Fc Region):
  • Opsonization: Coating pathogens to enhance phagocytosis by macrophages.
  • Complement Activation: IgM and IgG trigger the complement cascade, leading to pathogen lysis.
  • Antibody-Dependent Cellular Cytotoxicity (ADCC): Fc binds to NK cells, inducing target cell destruction.
• Immune Regulation:
  • Neutralization: Blocking pathogen entry into host cells (e.g., viruses).
  • Agglutination: Cross-linking pathogens to immobilize them.

Antibody Types and Functions

• IgG:
  • Primary antibody in blood serum (70-75% of total antibodies).
  • Neutralizes pathogens (viruses/bacteria) through opsonization and antibody-dependent cellular cytotoxicity (ADCC).
  • Crosses the placenta to provide passive immunity to newborns.
  • Activates the complement system to enhance pathogen destruction.
• IgM:
  • First antibody produced during primary immune responses.
  • Pentameric structure with 10 antigen-binding sites, enabling strong agglutination of pathogens.
  • Efficiently activates the complement system.
  • Found in blood and lymph, acting as a rapid defense against bloodstream infections.
• IgA:
  • Dominant antibody in mucosal secretions (saliva, tears, breast milk).
  • Forms dimers to neutralize pathogens at mucosal surfaces, preventing attachment to host cells.
  • Protects newborns via IgA-rich colostrum.
  • Resists enzymatic degradation in harsh mucosal environments.
• IgE:
  • Binds to mast cells and basophils, triggering allergic reactions (e.g., histamine release).
  • Defends against parasitic infections (e.g., helminths).
  • Present in trace amounts in serum.
• IgD:
  • Primarily expressed as a B-cell receptor (BCR) on immature B cells.
  • Role in B-cell activation and differentiation into antibody-producing plasma cells.
  • Minor presence in serum and its exact pathogen-related function remains unclear.

Antibodies Against Viral and Bacterial Infections

• Against Viral Infections
  • Neutralization: Block viral entry into host cells by binding to surface proteins (e.g., spike proteins of coronaviruses). Example: Anti-HIV antibodies neutralize viral particles in blood.
  • Complement Activation: IgM and IgG trigger the complement cascade, leading to lysis of enveloped viruses.
  • Opsonization: Coat viruses to enhance phagocytosis by macrophages and neutrophils via Fc receptor binding.
  • Antibody-Dependent Cellular Cytotoxicity (ADCC): Fc regions recruit NK cells to destroy virus-infected host cells.
  • Mucosal Protection (IgA): Secretory IgA neutralizes viruses in mucosal surfaces (e.g., respiratory/gastrointestinal tracts).
• Against Bacterial Infections
  • Neutralization of Toxins: Bind to bacterial toxins (e.g., tetanus toxin) to prevent host cell damage.
  • Opsonization: IgG coats bacteria (e.g., Streptococcus pneumoniae), promoting phagocytosis.
  • Agglutination: IgM cross-links bacterial cells into clumps, immobilizing them for clearance.
  • Complement-Mediated Lysis: IgM/IgG activate complement to lyse gram-negative bacteria (e.g., E. coli).
• Specialized Roles by Antibody Class:
  • IgG: Dominant in serum, neutralizes pathogens, crosses placenta for infant immunity.
  • IgM: First responder in primary infections, potent complement activator.
  • IgA: Protects mucosal surfaces and is critical in breast milk for infant gut immunity.

Therapeutic Applications

• Passive Immunity: Administered antibodies (e.g., convalescent plasma) neutralize pathogens during acute infections.
• Vaccine Support: Antibodies generated post-vaccination provide long-term protection via memory B cells.

Antibody Antigen-Binding Site Diversity

• Antigen-binding sites exhibit diversity in shape and physical properties, enabling specific interactions with various antigens.
• Structural Diversity:
  • The antigen-binding site is formed by variable domains (VH and VL) of heavy and light chains, with six complementarity-determining regions (CDRs) creating a unique 3D surface.
  • Shapes range from long, shallow grooves (for small molecules like haptens) to wide, open crevices or protruding surfaces (for larger protein epitopes).
  • CDR loop length and sequence directly determine binding site morphology, enabling adaptation to diverse antigen geometries.
• Physical Properties:
  • Binding sites feature residues (e.g., Tyr, Trp) with flexible side chains and amphipathic properties, allowing hydrophobic, electrostatic, and hydrogen-bond interactions.
  • Flexible CDR loops enable conformational adjustments to improve antigen fit (induced-fit model).
  • Hydrophobic pockets enhance binding to non-polar epitopes, while charged residues (e.g., Asp, Lys) mediate ionic interactions.
• Functional Implications:
  • Structural flexibility allows binding to structurally similar antigens (e.g., viral variants).
  • Precise molecular complementarity between CDRs and epitopes ensures high specificity, with mismatches as small as 1-2 Å disrupting binding.
  • Variations in heavy/light chain packing angles modulate binding site conformation, enabling recognition of different antigen classes (e.g., proteins vs. polysaccharides).

Generation of Immunoglobulin Diversity

• Immunoglobulin diversity in B cells before antigen exposure occurs through three primary mechanisms during early B-cell development in the bone marrow:
• V(D)J Recombination:
  • Heavy and light chain genes undergo random rearrangement of variable (V), diversity (D, heavy chain only), and joining (J) gene segments.
  • For heavy chains: ~45 VH, ~23 DH, and ~6 JH segments recombine.
  • For light chains (κ/λ): ~40 Vκ/30 Vλ and ~5 Jκ/4 Jλ segments recombine.
  • This generates ~106 unique combinations for heavy chains and ~103 for light chains.
• Junctional Diversification:
  • Random nucleotide additions (N-region) by terminal deoxynucleotidyl transferase (TdT) at V-D-J junctions.
  • Exonucleases remove nucleotides at recombination junctions, creating frameshifts and novel amino acid sequences.
• Combinatorial Pairing:
  • Random association of independently rearranged heavy and light chains amplifies diversity (e.g., 106 heavy × 103 light ≈ 109 combinations).
• Key Features:
  • Mediated by RAG1/RAG2 recombinases that recognize recombination signal sequences (RSS).
  • Occurs without antigen stimulation during B-cell maturation in the bone marrow.
  • Generates a naïve B-cell repertoire with ~1011 unique antigen-binding specificities.

Somatic Hypermutation (SHM)

• B cells generate diverse immunoglobulin (Ig) repertoires through two sequential mechanisms: V(D)J recombination during early development and somatic hypermutation (SHM) after antigen exposure.
• V(D)J Recombination Mechanism
  • Gene segment rearrangement for heavy and light chains, generating combinatorial diversity.
  • RAG1/RAG2 recombinases mediate DNA cleavage at recombination signal sequences (RSS) flanking V, D, and J segments.
  • Junctional diversification through nucleotide additions via terminal deoxynucleotidyl transferase (TdT) and imprecise joining, creating frameshifts and novel CDR3 sequences.
  • Outcome: Produces a primary antibody repertoire with broad antigen-binding potential before antigen exposure.
• Somatic Hypermutation (SHM)
  • Triggered by antigen binding to B-cell receptors (BCRs) in germinal centers of lymphoid tissues.
  • Activation-induced cytidine deaminase (AID) deaminates cytosine to uracil in variable region DNA, creating U mismatches.
  • Error-prone repair by DNA polymerases introduces point mutations.
  • Mutation hotspots preferentially target complementarity-determining regions (CDRs) in variable domains, enhancing antigen-binding affinity.
  • Results in affinity maturation, increasing antibody-antigen binding strength through Darwinian selection, and clonal diversification, generating ~103 variants per B cell clone.
• Synergy of Both Processes
  • V(D)J recombination establishes a baseline diversity of ~1011 unique BCRs.
  • SHM fine-tunes antigen specificity post-antigen exposure, increasing affinity by up to 10,000-fold.
• Enzymes Involved
  • V(D)J recombination: RAG1/RAG2 cleave DNA at RSS sites.
  • SHM: AID deaminates cytosine in Ig genes.

Human IgG Subclasses

• The four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) differ in structure, biological functions, and clinical roles:
• Structural Differences
  • Hinge Region: IgG1/IgG3 have longer, more flexible hinge regions, while IgG2/IgG4 have shorter, less flexible ones.
  • Fc Region: IgG1 and IgG3 bind strongly to Fcγ receptors and activate complement, while IgG2 and IgG4 have weaker binding.
• Serum Abundance: IgG1 (~60-70%) > IgG2 (~20-25%) > IgG3 (5-10%) > IgG4 (1-5%).
  • IgG1 is the dominant response to protein antigens (e.g., viral/bacterial proteins).
  • IgG2 specializes in polysaccharide antigens (e.g., bacterial capsules) and is associated with immunity to encapsulated bacteria.
  • IgG3 is the most potent complement activator but has a short serum half-life.
  • IgG4 has an anti-inflammatory role, forms bispecific antibodies, and competes with IgE in allergy responses.
• Clinical Relevance
  • IgG1/IgG3 deficiency increases susceptibility to bacterial/viral infections; IgG2 deficiency increases the risk of encapsulated bacterial infections. IgG4-related disease is associated with fibroinflammatory disorders.
• Therapeutic Applications:
  • IgG1 is preferred for monoclonal antibodies requiring ADCC (e.g., trastuzumab, rituximab). , IgG4 is used for blocking antibodies (e.g., nivolumab) to minimize immune activation.