Antibody Structure and Function

Antibody Structure and Function

Chapter Overview

  • General characteristics of antibodies

  • Structure of immunoglobulins

  • Antibody classes

  • Immunologic memory

  • Antibody specificity and diversity

  • Immunoglobulin genes

  • Monoclonal antibodies

Antibodies

  • Definition: Antibodies are also known as immunoglobulins.

  • Composition: They are glycoproteins found in serum, consisting of 82% to 96% polypeptide and 2% to 14% carbohydrate.

  • Role: Antibodies are a key element of the humoral immune response.

  • Types: There are five major classes of antibodies:

    • IgG

    • IgM

    • IgA

    • IgD

    • IgE

Serum Profiling

  • Antibodies primarily appear in the gamma (γ) globulin band when serum is electrophoresed at pH 8.6.

Tetrapeptide Structure of Immunoglobulins

  • Basic Structure: The fundamental structure of immunoglobulins consists of a tetrapeptide comprising:

    • Two large heavy (H) chains

    • Two small light (L) chains

  • Binding: Chains are held together by noncovalent forces and disulfide interchain bridges.

Heavy and Light Chains

  • Heavy Chains: Each immunoglobulin class is denoted by a Greek letter corresponding to its heavy chain (e.g., Gamma [γ] for IgG).

  • Light Chains: There are two types of light chains:

    • Kappa (κ) light chain

    • Lambda (λ) light chain

  • Molecular Weight: Light chains have a molecular weight of approximately 22,000 daltons and vary by a few amino acids.

  • Presence: Each immunoglobulin class can contain either type of light chain, but only one type exists in a single antibody molecule.

Unique Regions of Immunoglobulins

  • Variable Region: Located at the amino-terminal end; responsible for binding specificity to particular antigens.

  • Constant Regions: Typically consist of three to four regions at the carboxy-terminal end, which account for the biological functions of the antibody.

Hinge Region

  • The hinge region is a segment of the heavy chain between the CH1 and CH2 regions.

  • Flexibility: Provides flexibility, allowing two antigen-binding sites to operate independently.

  • Functions: Assists in the initiation of the complement cascade and the binding to Fc receptors.

Carbohydrate Portion of Immunoglobulins

  • Found in all classes of immunoglobulins, situated between the CH2 domains of the two heavy chains.

  • Functions:

    • Increases solubility of immunoglobulin.

    • Provides protection against degradation.

    • Enhances functional activity of the Fc domains.

Treatment of Immunoglobulins

Treatment with Papain

  • Outcome: Produces two Fab fragments and one Fc fragment.

    • Fab Fragment: Comprises one light chain plus one-half of a heavy chain held together by disulfide bonds. Contains antigen-binding sites.

    • Fc Fragment: Comprises the carboxy-terminal halves of two heavy chains, also held together by disulfide bonds. Responsible for effector functions of immunoglobulins.

Treatment with Pepsin

  • Process: Pepsin cleaves the immunoglobulin above the set of disulfide bonds binding the heavy chains.

  • Outcome: Results in F(ab')2 fragments containing two antigen-binding sites while the Fc' portion is rendered nonfunctional.

Isotypes, Allotypes, and Idiotypes

  • Isotypes: Unique amino acid sequences common to all immunoglobulins of a given class or subclass. Identical across species but differ between species, located in the constant regions of heavy chains.

    • Greek Designations: IgG (γ chain), IgM (μ chain), IgA (α chain), IgD (δ chain), IgE (ε chain).

  • Allotypes: Minor variation of amino acid sequences among individuals of the same species, found in constant regions of certain Ig subclasses.

  • Idiotypes: Variations in the variable regions that contribute to the individual antibody specificity, located in the amino-terminal regions of heavy and light chains.

IgG Immunoglobulin

  • Predominance: Constitutes 70%–75% of total serum immunoglobulin.

  • Half-Life: Longest half-life (23 days) of all immunoglobulins.

  • Structure: Monomer with a molecular weight of 150,000 daltons and a sedimentation coefficient of 7S.

  • Subclasses: Four subclasses:

    • IgG1: 66%

    • IgG2: 23%

    • IgG3: 7%

    • IgG4: 4%

Functions of IgG

  • Main Functions:

    • Binding to complement: Initiates inflammation and foreign cell destruction (IgG3 is the most efficient).

    • Neutralization of toxins and viruses: Key role in immunity.

    • Unique Capability: Only immunoglobulin that can cross the placenta, providing immunity to newborns.

    • Precipitation and agglutination of antigens.

IgM Immunoglobulin

  • Description: Known as a macroglobulin, comprising about 5% to 10% of all serum immunoglobulins.

  • Half-Life: Much shorter half-life of 6 days compared to IgG.

  • Forms: Can exist as:

    • Monomer: On the surface of B cells.

    • Pentamer: Found in blood, held together by a J chain forming disulfide bonds.

    • Structure: Pentamer is star-shaped with 10 antigen-binding sites.

Functions of IgM

  • Key Roles:

    • Complement fixation: Activates the complement system.

    • Agglutination: Clumping of antigens.

    • Neutralization: Effective against bacterial toxins and viruses.

  • Primary Response: Known as the primary response antibody; appears first after antigenic stimulation and is critical during infant maturation. Synthesized only as long as the antigen is present, helping to diagnose acute infection.

IgA Immunoglobulin

  • Description: Accounts for 10% to 15% of circulating immunoglobulins in serum; appears as a monomer with three constant regions, molecular weight of 160,000 daltons.

  • Subclasses: Two subclasses are IgA1 and IgA2.

Subclass Functions

  • IgA1: Acts as an anti-inflammatory agent, downregulating phagocytosis and other immune responses.

  • IgA2: Predominantly located in secretions at mucosal surfaces, including respiratory, urogenital, and intestinal tracts.

Secretory IgA

  • Production: Synthesized mainly by plasma cells in mucosal-associated lymphoid tissue, released as a dimer held together by a J chain and includes a secretory component derived from epithelial cells.

Functions of Secretory IgA

  • Defense Mechanism: Patrols mucosal surfaces acting as a first line of defense against pathogens.

  • Neutralization: Neutralizes toxins produced by microorganisms.

  • Prevention: Prevents bacterial adherence and penetration into the body.

  • Immunity Transfer: Passively transfers immunity to the newborn during breastfeeding.

  • Opsonization: IgA can act as an opsonin.

  • Complement Activation: Aggregation of IgA immune complexes can trigger the alternative pathway of complement activation.

IgD Immunoglobulin

  • Description: Extremely rare in serum, constituting less than 0.001% of total immunoglobulins; a monomer with a molecular weight of 180,000 daltons, featuring an extended hinge region, making it more susceptible to proteolysis.

  • Half-Life: Short half-life range of 1 to 3 days.

Functions of IgD

  • IgD in serum does not perform protective functions like binding complement or engaging phagocytic immune cells.

  • Found on surface of immunocompetent but unstimulated B lymphocytes; may play a key role in B-cell activation and maturation.

IgE Immunoglobulin

  • Prevalence: Represents normally about 0.0005% of total serum immunoglobulins; a monomer with a molecular weight of 190,000 daltons, characterized by four constant domains.

  • Function Against Parasites: The binding of IgE to eosinophils triggers enzyme release to destroy larger antigens, such as parasitic worms that are not easily phagocytized.

IgE Function in Allergic Reactions

  • When two adjacent IgE molecules on a mast cell bind to a specific antigen, it triggers a cascade of cellular events leading to mast cell degranulation and the release of vasoactive amines like histamine and heparin, which mediate allergic responses.

Primary Versus Secondary Antibody Response

  • Comparison: Serum antibody levels vary based on primary (first exposure to immunogen) and secondary (subsequent contact) responses.

  • Lag Phase: There is a lag phase evident in the primary response before antibody production, typically taking 5 to 7 days for antibodies to become detectable.

  • Subsequent Responses: Secondary responses occur rapidly due to memory cells and generate a higher antibody titer that persists longer than the primary response,

  • Graph Representation: Visualized antibody levels in IgG and IgM over time in response to immunogen exposure.

Monoclonal Antibodies

  • Definition: Monoclonal antibodies are used in laboratory testing and therapy, developed based on the principle that each B cell is genetically preprogrammed to synthesize a specific antibody.

  • Production: Derived from a hybridoma, a hybrid cell line created from a single antibody-producing B cell fused with a myeloma cell.

  • Development: This technique was pioneered in 1975 by Georges Köhler and César Milstein.

Hybridoma Production Steps

  1. Immunize a mouse with a specific antigen.

  2. Harvest spleen cells post-immunization.

  3. Combine spleen cells with myeloma cells in the presence of PEG (polyethylene glycol).

  4. Select fused cells exhibiting the desired antibody.

  5. Scale up growth of positive cells in larger quantities.

Summary

  • Basic Unit: The basic structural unit for all immunoglobulins is a tetrapeptide comprising two heavy and two light chains joined by disulfide bonds.

  • Immunoglobulin Classes: The five immunoglobulin classes are IgM, IgG, IgA, IgD, and IgE, differentiated by their heavy chains denoted by Greek letters.

  • Regions of Immunoglobulins: Each immunoglobulin molecule contains constant and variable regions, with the variable region serving as the binding site for specific antigens, while the constant region facilitates various Ig functions such as complement binding and phagocyte interaction.

  • Isotypes vs. Allotypes and Idiotypes: The five types of heavy chains are classified into isotypes. Variations among individuals of the same species are called allotypes, while unique variable portions are defined as idiotypes.

  • IgG Functions: Predominantly present in the serum, IgG mediates complement binding, opsonization, antibody-dependent cellular cytotoxicity (ADCC), and neutralization. It is unique in its ability to cross the placenta.

  • IgM Characteristics: IgM is the largest immunoglobulin due to its pentameric structure, effective in complement fixation and agglutination, and crucial during the early immune response.

  • Role of IgA: Major in secretions and mucosal immunity, IgA functions to prevent pathogen invasion and provides neonatal immunity through breastfeeding.

  • IgD Functionality: Though its role in serum is unclear, IgD acts as a B-cell surface receptor aiding in maturation.

  • IgE Mechanism: Mediates allergic reactions and defends against larger parasites, responding aggressively upon antigen reexposure.

  • Response Timeframes: The primary antibody response presents after initial exposure, with a delay due to activation time; however, memory cells facilitate a faster and more robust secondary response upon subsequent encounters with the same antigen.