Antigens and Antibodies: Comprehensive Notes

Nature of antigens and antibodies

Antigens are substances that are found on the surface of cells, and antibodies are substances that attach to the surface of our cells. In simple terms, antigens are the substances that confer immunogenicity to a cell and provoke a reaction with antibodies. Antigens are composed of proteins and polysaccharides, whereas antibodies are glycoproteins.

Epitopes and antigen recognition

Epitopes (also called antigenic determinants) are the binding sites on antigens (and on antibodies) that are recognized by immune receptors. Key points about epitopes:

  • They are the active regions of an immunogen that engage with receptors, especially the T cell receptor, which leads to antigen presentation and T cell–driven antibody production by B cells.

  • They are also known as determinant sites that are recognized for antibody production and antigen presentation by T cells or B cells.

Factors influencing immunogenicity

Immunogenicity refers to the ability of a substance (an immunogen) to elicit an immune response. There are five factors that influence immunogenicity:
1) Foreignness
2) Size
3) Chemical composition and complexity
4) Route, dosage, and timing
5) Adjuvants

Foreignness

The more foreign a molecule is to an individual’s genetic makeup, the higher its potential to provoke an immune reaction. Examples and terminology used in the lecture include:

  • Autoantigens: antigens derived from the same individual; these typically provoke the least immune reaction and are linked to autoimmune disorders when misregulated.

  • Alloantigens: antigens from the same species but of a different individual (e.g., human-to-human variants with different blood group or HLA antigens).

  • Hetero antigens: antigens from other species (e.g., animals, plants used in diagnostics or research). These generally carry a higher risk of immune recognition but can vary in cross-reactivity.

  • Heterophile antigens: antigens taken from completely different organisms and sometimes used in diagnostic antisera (often plant-derived antisera in blood typing).
    A common practical example discussed is blood typing and the Rh system: some populations are more often Rh negative or positive, illustrating how foreignness can influence immune reactions.

Autoantigens are produced by the body itself and tend to provoke the least reaction, whereas heteroantigens (from other species) and heterophile antigens can provoke stronger responses and are used in diagnostic settings.

Size

Generally, larger molecules are more immunogenic than smaller ones.

Chemical composition and complexity

Proteins are typically the most immunogenic macromolecules because they can adopt multiple structural forms. The macromolecules differ in the structures they can form:

  • Proteins can adopt four levels of structure: primary, secondary, tertiary, and quaternary.

  • Polysaccharides can form three levels: monomer, dimer, and polymer.

  • Lipids and nucleic acids have different structural categories. In this context, proteins are among the most immunogenic due to their higher structural complexity.
    A practical note from the lecture: the minimum size for a potent immunogen is greater than or equal to
    M≥ 10,000 DaM \ge \ 10{,}000 \ \mathrm{Da}

Route, dosage, and timing

Different routes of administration have different onset times and implications for dosing:

  • Oral: onset around \( \approx 1 \\ hour \).

  • Intramuscular and intradermal: onset around \( \approx 30 \) minutes.

  • Intravenous and intraperitoneal: onset within less than \( 15 \) minutes.
    Examples:

  • Oral: drugs like paracetamol; tablets (e.g., a generic oral medication).

  • Intramuscular: vaccines.

  • Intradermal: allergy tests (e.g., tuberculin skin test).

  • Intraperitoneal: insulin injections (used as an example).

Dosage considerations:

  • Oral and intramuscular routes generally require larger dosages due to metabolism and first-pass effects.

  • Intravenous and intraperitoneal routes deliver more directly into circulation and carry a higher risk of overdose because they bypass initial metabolism. These routes often require closer monitoring.

  • Metabolism differences:

    • Oral (and intramuscular) drugs undergo intestinal absorption and liver metabolism, reducing bioavailability before systemic circulation.

    • Intravenous/intraperitoneal drugs go directly into circulation and act faster, with little or no initial metabolism before reaching the target organ.

Adjuvants

Adjuvants are substances added to vaccines to enhance the immune response, effectively acting as catalysts to augment immunogenicity. The lecture emphasizes memorization of adjuvant examples (specific examples were not enumerated in the transcript).

Types of antigens: T-independent vs T-dependent

Antigens are categorized based on whether they require antigen presentation to induce an immune response:

  • T-independent antigens: do not require antigen presentation and can directly react with B cells to elicit an antibody response.

  • T-dependent antigens: require stimulation by T cells (antigen presentation) to drive B cell antibody production.
    Examples and notes from the transcript include the notion that T-independent antigens tend to be associated with first-time (initial) infections, while T-dependent antigens are often related to viral infections requiring T cell help.

Antibodies: structure, function, and diversity

Antibodies are glycoproteins that neutralize toxins, promote phagocytosis, and stimulate chemotaxis via opsonization.

Basic structure (monomer)

  • Each antibody monomer contains two types of polypeptide chains: heavy chains and light chains. Each monomer typically has two heavy chains and two light chains connected by disulfide bonds.

  • Disulfide bonds:

    • One disulfide bond links each light chain to its corresponding heavy chain (light-heavy linkage).

    • Two disulfide bonds connect the two heavy chains (heavy-heavy linkage), forming the hinge region.

  • The antibody monomer comprises two regions with distinct roles:

    • Fab (Fragment antigen-binding) region: contains the variable regions responsible for binding the antigen. The Fab is formed by the variable domains of both the heavy and light chains (VH and VL).

    • Fc (Fragment crystallizable) region: contains the constant regions that determine the antibody class and effector functions.

  • The Fc region is defined by the constant domains in the heavy chain (CD regions), typically starting at the second constant region (C2) through C3, and, for many isotypes, C4 as well. The Fab region uses the variable domains and the first constant region (C_1) of the heavy chain and the variable region of the light chain.

  • The Fab region harbors four variable segments in total that determine binding specificity (two per binding site, corresponding to one heavy-chain variable domain and one light-chain variable domain per site).

  • The heavy chain variable region generally includes one variable domain, and the light chain variable region includes one variable domain. A monomer has two binding sites, hence two Fab regions per monomer.

  • The constant Fc region contains the code for immunoglobulin identity (e.g., IgG, IgA, IgM, IgD, IgE), which is determined by the heavy chain constant regions (C2 to C3, and sometimes C_4 depending on isotype).

  • The heavy chain constant region coding determines the immunoglobulin class:

    • IgG, IgA, IgE, IgD, IgM (heavy chains γ, α, ε, δ, μ respectively).

  • The variable domain of the heavy chain comprises about 110 amino acids. Therefore, a single heavy chain variable domain contains roughly 110 amino acids, and a full heavy chain variable region plus its constant domains contribute to the monomer’s size. The transcript notes, for context, that a heavy chain variable region is about 110 amino acids, yielding a total of around 220 amino acids for the heavy-chain variable region per monomer (two heavy chains).

Light chain

  • Light chains come in two isotypes: κ (kappa) and λ (lambda).

  • Light chains possess a constant region and a variable region. The transcript notes the distribution as approximately 65% κ and 35% λ (roughly a 2:1 ratio).

  • A notable clinical marker related to light chains is the Bence Jones protein, which are light chains found in the urine of many multiple myeloma patients. These light chains precipitate at
    600˘0b0extC60^{\u00b0} ext{C} and dissolve at
    800˘0b0extC.80^{\u00b0} ext{C}.

  • The light chain has two domains: a variable region and a constant region. The overall light chain contributes to the Fab region alongside the heavy chain’s variable region to form the antigen-binding site.

Heavy chains and domains

  • Each heavy chain contains one variable region and a constant region. The Fc portion contains the code for the identity of the immunoglobulin (isotype) through the constant region, typically C2 to C3 (and C_4 for certain isotypes like IgM and IgE).

  • The heavy chain isotype is determined by the constant region:

    • γ (IgG), α (IgA), ε (IgE), δ (IgD), μ (IgM).

  • The number of domains per chain:

    • Normally, each heavy chain has one variable domain and three constant domains (C1, C2, C3). IgM and IgE are noted to have a fourth constant domain (C4) in some configurations, increasing the potential domain count per heavy chain.

    • Therefore, a typical monomer has eight domains in total from the heavy chain (4 per chain × 2 chains) plus four domains from the two light chains (2 per chain × 2 chains) for a total of 12 domains per monomer in the simplified accounting, with exceptions noted for IgM/IgE.

  • Light chains contribute two domains each: one variable (VL) and one constant (CL).

Hinge region, disulfide bonds, and domains

  • The hinge region is rich in proline and provides flexibility to the Fab arms due to the disulfide bonding pattern.

  • Fragmentation and domains:

    • Papain digestion yields three fragments: two Fab fragments (antigen-binding) and one Fc fragment. Papain cleaves above the hinge region (between C1 and C2).

    • Pepsin digestion yields two fragments, cutting below the hinge region, producing one large fragment containing the Fc region (F_c) and one smaller fragment comprising the Fab portions (F(ab')2 in typical descriptions).

Fragmentation of antibodies by size types

  • Monomer antibodies include IgG, IgD, IgE, and IgA1; IgA2 forms a dimer when secreted and functions as a secretory antibody in mucosal tissues.

  • Dimers: IgA2 is primarily found as a dimer (secretory IgA) in mucosal secretions.

  • Polymers: IgM exists as a pentamer (and is the largest antibody form). A polymer is defined as more than two monomers; IgM is typically a pentamer in circulation.

Types of immunoglobulins by class and their subclasses

  • IgG: the most abundant immunoglobulin in the human body; has four subclasses (IgG1, IgG2, IgG3, IgG4).

    • IgG1 is known for transplacental transfer (fetal immunity).

    • IgG3 is a particularly effective complement activator due to its multiple disulfide bonds (noted as having 15 disulfide bonds in the transcript).

  • IgM: the largest immunoglobulin; has a star-like or snowflake appearance; the first immunoglobulin produced during infection, detectable around two months after birth and stabilizing by three to five years depending on development.

  • IgA: two forms—IgA1 (usually a monomer in serum) and IgA2 (predominant as a dimer in secretions). Secretory IgA is found in body fluids such as breast milk, saliva, tears, and mucous secretions, contributing to mucosal immunity and providing antimicrobial action at epithelial surfaces.

  • IgD: function not fully understood; implicated in immunoregulation and control of IgM and IgG secretion during infection.

  • IgE: the least abundant immunoglobulin; historically associated with allergic reactions and parasitic infections; linked with eosinophils and basophils in allergic processes and capable of mediating antiparasitic responses.

Functional themes and clinical notes

  • Immunoglobulin functions: neutralization of toxins, promotion of phagocytosis, and facilitation of chemotaxis through opsonization.

  • Theories of antibody diversity:

    • Side-chain theory (Paul Ehrlich): proposed one antigen binds to one specific antibody.

    • Template theory (Horowitz): binding sites are shaped by the antigen and binding is guided by a-template fit.

    • Clonal selection theory (Burnet and others): most widely accepted; individual lymphocytes are programmed to produce a single type of immunoglobulin, and exposure to a specific antigen selects those lymphocytes to proliferate.

  • The lecture emphasizes that while the template theory contributed to understanding, the clonal selection theory best explains how lymphocytes respond to antigens by expanding specific clones.

Summary of practical biology terms and markers

  • Benz Jones proteins: light chain fragments used as a marker in multiple myeloma; they precipitate at about
    60∘C60^{\circ}\text{C} and dissolve at about
    80∘C80^{\circ}\text{C}.

  • The antibody’s variable region is the source of antigen-binding diversity, while the constant region determines effector functions and isotype.

  • The heavy chain variable region is about 110 amino acids in length; this contributes to the diversity of antigen recognition.

Monoclonal antibodies and further reading

The lecturer notes an external resource for monoclonal antibody production (Stevens) and suggests reading the module for an in-depth discussion on how monoclonal antibodies are produced. The content here provides foundational understanding of antigen–antibody structure, function, and diversity needed for that advanced topic.