Premed · Premed · Immunology
Lecture 10: Antibody Structure and Function
Immunology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the basic structure of an immunoglobulin molecule (heavy chains, light chains, domains)
- Explain the structural basis of antigen binding and antibody effector functions
- Define the variable, constant, hypervariable, and framework regions
- Describe how antibody diversity is generated through V(D)J recombination
- List the major effector functions of antibodies
Lecture Content
I. Basic Antibody Structure
Antibodies (immunoglobulins, Ig) are glycoproteins produced by B cells and plasma cells. The basic unit is a Y-shaped molecule composed of four polypeptide chains: two identical heavy (H) chains of approximately 50-70 kDa each and two identical light (L) chains of approximately 25 kDa each, held together by inter-chain disulfide bonds and non-covalent interactions. Each chain has an amino-terminal variable (V) region and a carboxyl-terminal constant (C) region.
Light chains come in two types: kappa (κ), which accounts for approximately 60% of human antibodies, and lambda (λ), which accounts for approximately 40%. Each antibody molecule contains either two κ or two λ chains, never one of each. Light chains have one variable domain (VL) and one constant domain (CL). Heavy chains define five classes (isotypes) based on their constant region: μ (IgM), δ (IgD), γ (IgG), α (IgA), and ε (IgE). Heavy chains have one variable domain (VH) and either 3 or 4 constant domains (CH1, CH2, CH3, and CH4 for IgM and IgE). The hinge region, a flexible segment between CH1 and CH2 found in IgG, IgA, and IgD, allows the two Fab arms to move independently. It is rich in proline and cysteine residues and is susceptible to proteolytic cleavage.
II. Functional Regions of Antibodies
The Fab (fragment antigen-binding) region is composed of the VH and CH1 domains of the heavy chain paired with the VL and CL domains of the light chain. There are two Fab regions per antibody molecule, and each contains an antigen-binding site. The Fc (fragment crystallizable) region is composed of the CH2 and CH3 domains (and CH4 for IgM and IgE) of both heavy chains. The Fc region mediates effector functions including Fc receptor binding on immune cells, complement activation through C1q binding, placental transfer via FcRn, and determination of antibody half-life through FcRn recycling. The F(ab')2 fragment, produced by pepsin digestion, consists of both Fab regions linked by the hinge, while Fab' is a single Fab with a partial hinge produced by reducing F(ab')2.
<image>A detailed structural diagram of an IgG antibody molecule. The Y-shaped molecule is shown with two heavy chains (blue) and two light chains (yellow). Each chain is divided into immunoglobulin domains (loops): VH and VL at the tips of the Y arms (variable regions, with CDR1, CDR2, CDR3 loops highlighted in red within the variable domains), CH1 paired with CL below, a flexible hinge region with disulfide bonds connecting the two heavy chains, and CH2 and CH3 forming the Fc region stem. The Fab region is bracketed (one arm: VH-CH1 + VL-CL), and the Fc region is bracketed (CH2-CH3 of both heavy chains). N-linked carbohydrate on CH2 is shown. An inset magnifies the antigen-binding site showing the six CDR loops (three from VH, three from VL) forming a complementary surface around a bound antigen epitope. Cleavage sites for papain (above the hinge → two Fab + Fc) and pepsin (below the hinge → F(ab')2 + degraded Fc) are marked.</image>
III. Variable Region and Antigen Binding
The variable (V) domains of both heavy and light chains together form the antigen-binding site. Within each V domain, three regions show extreme sequence variability: these are the complementarity-determining regions (CDRs) -- CDR1, CDR2, and CDR3 -- also called hypervariable regions. CDR3 is the most variable, especially VH CDR3, and is the major determinant of antigen specificity. The CDRs form loops that directly contact the antigen. Surrounding the CDRs are four relatively conserved framework regions (FRs), FR1 through FR4, that provide the structural scaffold. The six CDRs (three from VH plus three from VL) together form the paratope, or antigen-binding site.
Antigen-antibody interaction involves exclusively non-covalent forces: hydrogen bonds, electrostatic interactions, van der Waals forces, and hydrophobic interactions. Affinity describes the strength of interaction between one paratope and one epitope, with typical Kd values ranging from 10^-7 to 10^-11 M. Avidity describes the overall strength of interaction considering all binding sites, so IgM with its 10 binding sites has high avidity despite lower affinity per individual site.
IV. Immunoglobulin Domains and the Ig Superfamily
Each V and C domain folds into a characteristic immunoglobulin fold consisting of two anti-parallel β-sheets connected by a disulfide bond, with approximately 110 amino acids per domain. This fold is found in many immune molecules, defining the immunoglobulin superfamily, which includes the TCR, MHC class I and II, CD4, CD8, CD19, CD28, CTLA-4, ICAMs, and Fc receptors, all sharing a common evolutionary origin.
V. Generation of Antibody Diversity -- V(D)J Recombination
The immune system can produce more than 10^11 different antibody specificities through several mechanisms of diversification. Combinatorial diversity of gene segments arises from random selection among the gene segments on each locus: the heavy chain locus on chromosome 14 contains approximately 65 VH segments, 27 DH segments, and 6 JH segments; the κ light chain locus on chromosome 2 has approximately 40 Vκ and 5 Jκ segments; and the λ light chain locus on chromosome 22 has approximately 30 Vλ and 4 Jλ segments.
V(D)J recombination is catalyzed by RAG1 and RAG2 (recombination-activating genes), which recognize recombination signal sequences (RSS) flanking each gene segment. The 12/23 rule dictates that recombination occurs only between segments flanked by RSS with 12-bp and 23-bp spacers, ensuring proper segment joining. The process involves DNA cleavage, hairpin formation, and joining. Junctional diversity further expands the repertoire through imprecise joining at V-D, D-J, and V-J junctions: P-nucleotides (palindromic nucleotides from hairpin opening), N-nucleotides (random nucleotides added by terminal deoxynucleotidyl transferase, TdT), and nucleotide trimming by exonucleases all contribute. This generates enormous diversity at CDR3 in particular. Combinatorial association of heavy and light chains adds another layer of diversity, as any heavy chain can pair with any light chain. Finally, somatic hypermutation (SHM) occurs after antigen encounter in germinal centers, introducing point mutations in V region genes through the action of activation-induced cytidine deaminase (AID). Mutations in the CDRs that improve antigen binding are selected, driving affinity maturation.
<image>A schematic of V(D)J recombination at the immunoglobulin heavy chain locus. The top shows the germline configuration on chromosome 14 with multiple VH segments (V1, V2, V3...V65), DH segments (D1-D27), JH segments (J1-J6), and constant region genes (Cmu, Cdelta, Cgamma, etc.). Step 1: D-J joining -- one D segment joins one J segment with deletion of intervening DNA. Step 2: V-DJ joining -- one V segment joins the DJ segment. Step 3: Transcription and RNA splicing -- the VDJ exon is joined to the Cmu constant region. An inset shows junctional diversity: at the V-D and D-J junctions, P-nucleotides (palindromic) and N-nucleotides (random, added by TdT) create unique sequences. The bottom shows the final mRNA encoding the complete heavy chain (VDJ-Cmu).</image>
VI. Effector Functions of Antibodies
Antibodies perform several effector functions. Neutralization involves antibodies binding to pathogen surface molecules or toxins, blocking their interaction with host cells and thereby preventing viral attachment and entry, toxin activity, and bacterial adhesion. This is primarily mediated by IgG and IgA. Opsonization and phagocytosis occur when IgG coats pathogens and the Fc region binds FcγR on phagocytes, enhancing uptake. FcγRI (CD64) on macrophages, neutrophils, and DCs has high affinity; FcγRIIA (CD32) on macrophages, neutrophils, and platelets has low affinity; and FcγRIIIA (CD16) on NK cells and macrophages mediates ADCC. Complement activation is triggered when IgM and IgG (especially IgG1 and IgG3) activate the classical pathway via C1q binding, leading to opsonization, inflammation, and lysis. ADCC occurs when IgG-coated target cells are killed by NK cells via CD16, or by eosinophils via FcεRI for IgE-coated parasites. Neonatal immunity is provided when maternal IgG crosses the placenta via FcRn, delivering passive immunity to the newborn, while maternal IgA in breast milk protects mucosal surfaces. Mast cell sensitization results when IgE binds FcεRI on mast cells and subsequent antigen cross-linking triggers degranulation, the basis of immediate hypersensitivity.
VII. Antibody Valence and Cross-Reactivity
Valence refers to the number of identical antigen-binding sites per antibody molecule. IgG, IgD, and IgE are bivalent (2 sites); secretory dimeric IgA is tetravalent (4 sites); and pentameric IgM is decavalent (10 sites, though steric constraints limit the effective valence). Cross-reactivity occurs when an antibody raised against one antigen binds a structurally similar but different antigen, which is the basis for heterophile antibodies, autoimmune cross-reactivity through molecular mimicry, and certain diagnostic tests such as the Weil-Felix test.
