Chapter Notes: Antibodies—Immunoglobulins

Table of Contents
1. Learning Objectives
2. Introduction
3. Physicochemical properties of antibodies
4. Antibody structure
5. Immunoglobulin classes (isotypes): overview and properties
6. Immunoglobulin G (IgG)
7. Immunoglobulin A (IgA)
8. Immunoglobulin M (IgM)
9. Immunoglobulin D (IgD)
10. Immunoglobulin E (IgE)
11. Role of different immunoglobulin classes (summary)
12. Antigenic determinants on immunoglobulins
13. Abnormal immunoglobulins and clinical disorders
14. Additional notes and historical terminology
15. Key points
16. Further reading
View more

Learning Objectives

• Define antibody and immunoglobulin and describe their relationship.
• Draw and label the basic structure of an immunoglobulin molecule (four-chain Y-shaped monomer) and identify Fab and Fc regions.
• Explain the structure and principal functions of IgG, IgA and IgM.
• Describe the properties of the five major immunoglobulin isotypes: IgG, IgA, IgM, IgD and IgE.
• Identify antigenic determinants on immunoglobulins (isotypes, allotypes, idiotypes) and summarise common pathological variants (for example, monoclonal gammopathies, cryoglobulinaemia).

Introduction

Towards the end of the 19th century, Emil von Behring and Shibasaburō Kitasato demonstrated that serum from an immunized animal contained specific neutralizing substances (antitoxins). This was the first clear evidence of the activity of the proteins now called antibodies or immunoglobulins.

Antibody (biological/functional term) and immunoglobulin (structural/chemical term) refer to closely related concepts: an immunoglobulin is a serum glycoprotein molecule of a defined structure, while an antibody is an immunoglobulin that is functionally capable of recognising and binding a specific antigen. Thus, all antibodies are immunoglobulins, but not every immunoglobulin necessarily acts as a specific antibody.

Physicochemical properties of antibodies

Electrophoretic mobility

Serum proteins can be separated by electrophoresis into albumin and several globulin fractions: alpha-1, alpha-2, beta and gamma globulins. Antibody activity is largely associated with the gamma globulin fraction; historically the term gammaglobulin became synonymous with antibody.

Sedimentation coefficient and molecular weight

Most monomeric antibodies (for example IgG monomers) sediment at about 7S and have molecular weights around 150,000-180,000 Da. Larger polymeric antibodies such as pentameric IgM sediment at about 19S and have molecular weights near 900,000-1,000,000 Da; such high-molecular-weight antibodies were sometimes called macroglobulins.

Physicochemical and antigenic classes

On the basis of physicochemical and antigenic structure, immunoglobulins are divided into five major classes (isotypes): IgG, IgA, IgM, IgD and IgE. Within some classes there are subclasses that differ slightly in structure and function (for example IgG1-IgG4, IgA1 and IgA2).

Antibody structure

Overall organisation

An immunoglobulin monomer is a Y-shaped molecule composed of four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains. Chains are linked by interchain disulfide bonds. Each light chain is attached to a heavy chain by a disulfide bond; the two heavy chains are joined to each other by one or more disulfide bonds depending on the class.

The two arms of the Y each carry an antigen-binding site; these are formed by the paired variable regions of the heavy and light chains (VH and VL). The stem of the Y is the Fc (fragment crystallisable) region, which mediates effector functions such as complement binding and binding to Fc receptors on cells.

Papain digestion

Treatment of an IgG molecule with the proteolytic enzyme papain cleaves the molecule above the hinge region to produce three fragments: two identical Fab fragments (fragment antigen-binding) that retain antigen-binding ability, and one Fc fragment (fragment crystallisable) that does not bind antigen but mediates biological effector functions.

Pepsin digestion

Digestion with pepsin cleaves below the hinge region and yields a bivalent F(ab')₂ fragment (two Fab-like units held together) and smaller degraded Fc fragments.

Light and heavy chains

Light chains are smaller (approximately 25 kDa) and heavy chains larger (approximately 50 kDa per chain in IgG-like molecules). There are two types of light chains in humans, designated kappa (κ) and lambda (λ). Any given antibody molecule contains either κ or λ light chains but not both; the κ:λ ratio in human serum is roughly 2:1.

Heavy chains determine the immunoglobulin class and are named by Greek letters: γ (gamma) for IgG, α (alpha) for IgA, μ (mu) for IgM, δ (delta) for IgD and ε (epsilon) for IgE. Differences in heavy-chain constant regions account for the distinct properties of each class (distribution, half-life, complement activation, binding to Fc receptors, etc.).

Variable and constant regions

Each chain contains a variable (V) region at the amino terminus and one or more constant (C) regions at the carboxy terminus. The variable regions of one heavy and one light chain fold together to form the antigen-binding site. Within each variable region there are three hypervariable loops called complementarity-determining regions (CDRs) that make most of the direct contacts with antigen; these determine the antibody's specificity.

Heavy chains have several constant domains designated CH1, CH2, CH3 (and CH4 in some classes). The hinge region between CH1 and CH2 provides flexibility to the Fab arms and is a target for enzymatic cleavage (papain, pepsin).

Fc fragment and its functions

The Fc portion is formed by the carboxy-terminal domains of the heavy chains and is responsible for the biologic activities of antibodies that do not involve antigen binding. Important functions of Fc include:

• Binding complement: Fc regions (for example the CH2 of IgG) bind C1q and initiate the classical complement pathway.
• Binding to cellular Fc receptors (FcRs): This mediates opsonisation, antibody-dependent cell-mediated cytotoxicity and other cell interactions.
• Placental transfer: Fc of IgG determines transplacental passage of maternal IgG to the fetus.
• Determining catabolic rate: Fc structure influences antibody half-life.

Immunoglobulin domains

Each chain is composed of independently folded domains stabilised by intrachain disulfide bonds. These domains adopt the characteristic immunoglobulin fold found throughout Ig molecules and many other proteins. Different domains have specific roles: VH and VL form antigen-binding sites; CH2 is important for complement binding; CH3 mediates interactions with cells.

Immunoglobulin classes (isotypes): overview and properties

Human serum contains five major immunoglobulin classes in descending order of concentration: IgG, IgA, IgM, IgD and IgE. The table below summarises their major physical, physiological and biologic properties.

1. Immunoglobulin G (IgG)

• IgG is the major serum immunoglobulin (approximately 75-85% of total serum Ig).
• Monomeric, 7S, MW ~150 kDa; relatively low carbohydrate content.
• Normal serum concentration is roughly 8-16 mg/ml (typical average ~12 mg/ml); half-life ≈ 23 days (longest of the isotypes).
• Distributed nearly equally between intra- and extravascular compartments-suitable for passive immunization via serum transfer.
• Four human subclasses exist (IgG1, IgG2, IgG3, IgG4) present in approximate proportions 65%, 23%, 8%, 4% respectively; subclasses differ in Fc-mediated functions and placental transfer (most cross placenta; IgG2 less efficiently).
• Functions:
  • Maternal transfer: IgG crosses the placenta and provides neonatal protection for the first months of life (subclass differences exist).
  • Opsonisation: IgG coats microbes and enhances phagocytosis by cells with Fcγ receptors.
  • Complement activation via classical pathway, neutralisation of toxins/viruses, immobilisation of motile bacteria by binding flagella/cilia.
  • Can mediate antibody-dependent cellular cytotoxicity (ADCC) by NK cells.
  • Passively administered IgG can suppress endogenous antibody production (used clinically to prevent Rh immunisation by anti-D immunoglobulin).

2. Immunoglobulin A (IgA)

• IgA constitutes about 10-13% of serum immunoglobulins; serum concentration typically 0.6-4.2 mg/ml (average ~1.2 mg/ml); half-life ≈ 6-8 days.
• Two major forms:
  • Serum IgA: largely monomeric (7S).
  • Secretory IgA (SIgA): usually a dimer joined by a J chain and associated with a secretory component derived from epithelial poly-Ig receptor. SIgA is found in mucus, saliva, tears, colostrum and other secretions and is resistant to proteolytic degradation.
• Two human subclasses: IgA1 (dominant in serum) and IgA2 (more prominent in secretions and more resistant to certain bacterial IgA1-specific proteases).
• Functions:
  • Local mucosal immunity: SIgA prevents microbial attachment to mucosal surfaces and neutralises pathogens and toxins in secretions (an "immune barrier").
  • Inhibition of microbial entry into tissues by preventing adherence.
  • Present in breast milk: contributes to neonatal protection of the gut and mucosa.
  • Can agglutinate organisms and impede viral entry into cells; may activate the alternative complement pathway in some contexts and promote phagocytosis.

3. Immunoglobulin M (IgM)

• IgM represents ~5-10% of serum immunoglobulins. It is a pentamer in the circulation (five monomeric units joined by J chain), sedimentation ~19S, MW ~900,000-1,000,000 Da.
• Normal serum level ≈ 1.2 mg/ml; half-life ≈ 5 days.
• IgM is the first immunoglobulin class produced in the primary immune response and is the first class expressed by developing B cells (co-expressed with IgD on naïve mature B cells as membrane forms).
• Functional properties:
  • High valency: pentameric IgM has 10 antigen-binding sites (theoretical valency 10), making it effective at agglutination and precipitation; effective valency against large antigens is often about 5 due to steric factors.
  • Powerful activator of the classical complement pathway-excellent at complement fixation.
  • Mostly intravascular because of its large size.
  • Clinical utility: presence of specific IgM in a neonate indicates intrauterine infection (IgM does not cross the placenta). IgM is an indicator of recent or acute infection; IgM is relatively short-lived.

4. Immunoglobulin D (IgD)

• IgD is present in very low concentrations in serum (≈ 0.03 mg/ml) and has a monomeric structure (MW ~180 kDa; sedimentation ~7S).
• Half-life approximately 2-8 days (often cited ≈ 3 days).
• IgD is expressed on the surface of mature, naive B cells (usually co-expressed with IgM) where it functions as an antigen receptor and participates in B-cell activation. The precise effector role of circulating IgD remains less well defined than that of other isotypes.

5. Immunoglobulin E (IgE)

• IgE is present in extremely low concentrations in serum (typical serum level ≈ 0.00004 mg/ml). Sedimentation coefficient ≈ 8S and MW ≈ 190,000 Da; half-life in serum 1-5 days, though bound IgE on mast cells is much more persistent.
• IgE does not efficiently activate complement and does not agglutinate antigens.
• Unique properties and functions:
  • IgE binds with high affinity by its Fc portion to Fcε receptors on mast cells and basophils. Cross-linking of bound IgE by allergen triggers degranulation, releasing histamine and other mediators that cause allergic symptoms (type I hypersensitivity, atopic reactions, bronchial asthma, anaphylaxis).
  • IgE is important in immunity to helminthic parasites; elevated IgE levels are seen in parasitic infections and in atopic individuals.
  • IgE is largely tissue-associated (extravascular), concentrated in mucosal linings and skin.

Role of different immunoglobulin classes (summary)

• IgG: Protects the body fluids and tissues; systemic defence; crosses placenta.
• IgA: Protects mucosal surfaces and secretions.
• IgM: Protects the bloodstream; first responder in primary immune response; excellent at complement activation.
• IgE: Mediates type I hypersensitivity; defence against helminths.
• IgD: Functions primarily as a B-cell receptor; circulating role less clear.

Antigenic determinants on immunoglobulins

Because immunoglobulins are proteins with variable amino acid sequences, they themselves can act as antigens and carry distinct antigenic determinants. Three major categories of antigenic determinants on Igs are recognised:

1. Isotypes

Isotypic determinants are structures shared by all individuals of a species and correspond to the constant regions that define immunoglobulin classes and subclasses (for example γ, α, μ, δ, ε on heavy chains and κ or λ on light chains). Isotype differences distinguish IgG, IgA, IgM, IgD and IgE.

2. Allotypes

Allotypic determinants are genetically determined polymorphic variants of immunoglobulin constant regions that differ between individuals of the same species. They are allelic differences in immunoglobulin genes; examples include the Gm system (gamma chain markers), Am markers (alpha chains) and Km markers (kappa light chains). Allotypic markers have applications in paternity testing and population genetics.

3. Idiotypes

Idiotypic determinants are antigenic specificities located in the variable regions (particularly the hypervariable CDRs) of an individual antibody molecule. The set of idiotopes on an antibody is called its idiotype; these are unique to antibodies that recognise a particular epitope.

Abnormal immunoglobulins and clinical disorders

Multiple myeloma

Multiple myeloma is a neoplasm of plasma cells in which a single clone of plasma cells proliferates and secretes a large amount of a single immunoglobulin (a monoclonal protein, or M protein). The secreted M protein can be of various isotypes (commonly IgG or IgA) and results in an abnormal serum protein spike on electrophoresis.

Waldenström's macroglobulinaemia

A lymphoplasmacytic neoplasm characterised by production of large amounts of monoclonal IgM, leading to hyperviscosity of blood and related complications.

Bence-Jones proteins

Excess free immunoglobulin light chains produced by some myeloma clones are excreted in urine and are historically called Bence-Jones proteins. These light chains may cause renal damage. In an individual patient the free light chains are of a single type-either κ or λ but not both.

Heavy chain disease

A rare lymphoid neoplasia in which truncated heavy chains (often lacking associated light chains) are produced in excess; clinical features vary with the type of heavy chain involved.

Cryoglobulinaemia

Cryoglobulins are immunoglobulins that precipitate at low temperatures and redissolve on warming. They may be monoclonal or mixed (for example IgM with rheumatoid factor activity). Cryoglobulinaemia can occur in myeloma, macroglobulinaemia and autoimmune diseases and may cause vascular occlusion and tissue ischaemia in cold-exposed areas.

Additional notes and historical terminology

The generic term immunoglobulin was endorsed internationally to denote the family of antibody proteins and related variants (including abnormal proteins found in myeloma and macroglobulinaemia). Immunoglobulins make up approximately 20-25% of the total serum protein fraction.

Key points

• An antibody or immunoglobulin (Ig) is a glycoprotein produced in response to an antigen and capable of recognising and binding that antigen.
• An antibody molecule consists of two identical light chains and two identical heavy chains linked by disulfide bonds; each chain has variable and constant regions.
• The heavy-chain isotype defines the antibody class (IgG, IgA, IgM, IgD, IgE) and determines many effector functions.
• Each variable domain contains three complementarity-determining regions (CDRs) that form the antigen-binding site and determine specificity.
• Immunoglobulins carry antigenic markers of three types: isotypes (class-specific), allotypes (individual genetic variants) and idiotypes (unique antigen-binding region determinants).
• Abnormal immunoglobulins occur in disorders such as multiple myeloma, Waldenström's macroglobulinaemia, heavy chain disease and cryoglobulinaemia and have diagnostic and pathophysiological importance.

Further reading

• Burton DR. Antibody: the flexible adapter molecule. Trends Biochem Sci. 1990;15:64-69.
• Goodman JW. Immunoglobulin Structure and Function. In: Basic and Clinical Immunology. 7th ed. (chapter and year as referenced in teaching texts).
• Roitt IM, Delves PJ. Encyclopedia of Immunology. 1992.