This page introduces the nomenclature and criteria used to describe the structure, classes and functional types of immunoglobulins.

Structure of Immunoglobulins

Antibody (or immunoglobulin) molecules are glycoproteins composed of one or more units, each containing four polypeptide chains: two identical heavy chains (H) and two identical light chains (L). The amino terminal ends of the polypeptide chains show considerable variation in amino acid composition and are referred to as the variable (V) regions to distinguish them from the relatively constant (C) regions. Each L chain consists of one variable domain VL and one constant domain CL. The H chains consist of a variable domain, VH, and three constant domains CH1, CH2, CH3. Each heavy chain has about twice the number of amino acids and molecular weight (~50,000) as each light chain (~25,000), resulting in a total immunoglobulin monomer molecular weight of approximately 150,000.

Generalized structure of an immunoglobulin (IgG).

Heavy and light chains are held together by a combination of noncovalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. The V regions of H and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. Each Ig monomer contains two antigen-binding sites and is said to be bivalent.

The hinge region is the area of the H chains between the first and second C region domains and is held together by disulfide bonds. This flexible hinge (found in IgG, IgA and IgD, but not IgM or IgE) region allows the distance between the two antigen-binding sites to vary.

Antibody production and purification guide

The updated Antibody Production and Purification Technical Handbook is an essential resource for any laboratory working with antibodies. The handbook provides an overview of antibody structure and types, as well as technical information on the procedures, reagents and tools used to produce, purify, fragment and label antibodies.

Classes of Immunoglobulins

The five primary classes of immunoglobulins are IgG, IgM, IgA, IgD and IgE. These are distinguished by the type of heavy chain found in the molecule. IgG molecules have heavy chains known as gamma-chains; IgMs have mu-chains; IgAs have alpha-chains; IgEs have epsilon-chains; and IgDs have delta-chains.

Differences in heavy chain polypeptides allow these immunoglobulins to function in different types of immune responses and at particular stages of the immune response. The polypeptide protein sequences responsible for these differences are found primarily in the Fc fragment. While there are five different types of heavy chains, there are only two main types of light chains: kappa (κ) and lambda (λ).

Antibody classes differ in valency as a result of different numbers of Y-like units (monomers) that join to form the complete protein. For example, in humans, functioning IgM antibodies have five Y-shaped units (pentamer) containing a total of ten light chains, ten heavy chains and ten antigen-binding sites (see below).

IgG Immunoglobulins

IgG, a monomer, is the predominant Ig class present in human serum. Produced as part of the secondary immune response to an antigen, this class of immunoglobulin constitutes approximately 75% of total serum Ig. IgG is the only class of Ig that can cross the placenta in humans, and it is largely responsible for protection of the newborn during the first months of life. Because of its relative abundance and excellent specificity toward antigens, IgG is the principle antibody used in immunological research and clinical diagnostics.

Properties of IgG:

  • Molecular weight: 150,000
  • H-chain type (MW): gamma (53,000)
  • Serum concentration: 10 to 16mg/mL
  • Percent of total immunoglobulin: 75%
  • Glycosylation (by weight): 3%
  • Distribution: intra- and extravascular
  • Function: secondary response

IgM Immunoglobulins

Serum IgM exists as a pentamer in mammals, predominates in primary immune responses to most antigens, is the most efficient complement fixing immunoglobulin and comprises approximately 10% of normal human serum Ig content. IgM is also expressed on the plasma mem- brane of the B lymphocytes as a monomer. It is the B cell antigen receptor and the H chains each contain an additional hydrophobic domain for anchoring in the membrane. Monomers of serum IgM are bound together by disulfide bonds and a joining (J) chain.

Each of the five monomers is composed of two light chains (either kappa or lambda) and two heavy chains. Unlike in IgG (and the generalized structure shown above), the heavy chain in IgM monomers is composed of one variable and four constant regions, the additional constant domain replacing the hinge region. IgM can cause cell agglutination as a result of recognition of epitopes on invading microorganisms. This antibody-antigen immune complex is then destroyed by complement fixation or receptor mediated endocytosis by macrophages. IgM is the first immunoglobulin class to be synthesized by the neonate and plays a role in the pathogenesis of some autoimmune diseases.

Properties of IgM:

  • Molecular weight: 900,000
  • H-chain type (MW): mu (65,000)
  • Serum concentration: 0.5 to 2mg/mL
  • Percent of total immunoglobulin: 10%
  • Glycosylation (by weight): 12%
  • Distribution: mostly intravascular
  • Function: primary response

IgA Immunoglobulins

IgA exists in serum in both monomeric and dimeric forms, comprising approximately 15% of the total serum Ig. Secretory IgA, a dimer, provides the primary defense mechanism against some local infections because of its abundance in mucosal secretions (e.g., saliva, tears). The principal function of secretory IgA may not be to destroy antigen but to prevent passage of foreign substances into the circulatory system

Properties of IgA:

  • Molecular weight: 320,000 (secretory)
  • H-chain type (MW): alpha (55,000)
  • Serum concentration: 1 to 4mg/mL
  • Percent of total immunoglobulin: 15%
  • Glycosylation (by weight): 10%
  • Distribution: intravascular and secretions
  • Function: protect mucus membranes

IgD and IgE Immunoglobulins

IgD and IgE are found in serum in much smaller quantities than other Igs. Membrane IgD is a receptor for antigen found mostly on mature B-lymphocytes. IgE primarily defends against parasitic invasion and is responsible for allergic reactions.

Properties of IgD:

  • Molecular weight: 180,000
  • H-chain type (MW): delta (70,000)
  • Serum concentration: 0 to 0.4mg/mL
  • Percent of total immunoglobulin: 0.2%
  • Glycosylation (by weight): 13%
  • Distribution: lymphocyte surface
  • Function: unknown

Properties of IgE:

  • Molecular weight: 200,000
  • H-chain type (MW): epsilon (73,000)
  • Serum concentration: 10 to 400ng/mL
  • Percent of total immunoglobulin: 0.002%
  • Glycosylation (by weight): 12%
  • Distribution: basophils and mast cells in salive and nasal secretions
  • Function: protect against parasites

Subclasses of Immunoglobulins

In addition to the major immunoglobulin classes, several Ig subclasses exist in all members of a particular animal species. Antibodies are classified into subclasses based on minor differences in the heavy chain type of each Ig class.

In humans there are four subclasses of IgG: IgG1, IgG2, IgG3 and IgG4 (numbered in order of decreasing concentration in serum). Variance among different subclasses is less than the variance among different classes.

For example, IgG1 is more closely related to IgG2, 3 or 4 than to IgA, IgM, IgD or IgE. Consequently, antibody-binding proteins (e.g., Protein A or Protein G) and most secondary antibodies used in immunodetection methods cross-react with multiple subclasses but usually not multiple classes of Ig.

Polyclonal and Monoclonal Antibodies

Antibodies (whatever their class or subclass) are produced and purified in two basic forms for use as reagents in immunoassays: polyclonal and monoclonal. Typically, the immunological response to an antigen is heterogeneous, resulting in many different cell lines of B-lymphocytes (precursors of plasma cells) producing antibodies to the same antigen. All of these cells originate from common stem cells, yet each develops the individual capacity to make an antibody that recognizes a particular determinant (epitope) on the same antigen. As a consequence of this heterogeneous response, serum from an immunized animal will contain numerous antigen-specific antibody clones, potentially of several different immuglobulin classes and subclasses comprising generally 2 to 5% of the total immunoglobulin. Because it contains this heterogeneous collection of antigen-binding immunoglobulins, an antibody purified from such a sample is called a polyclonal antibody. Polyclonal antibodies, which are generally purified directly from serum, are especially useful as labeled secondary antibodies in immunoassays.

Because an individual B-lymphocyte produces and secretes only one specific antibody molecule, clones of B-lymphocytes produce monoclonal antibodies. All antibodies secreted by a B-cell clone are identical, providing a source of homogeneous antibody having a single defined specificity. However, while B-lymphocytes can be isolated from suspensions of spleen or lymph node cells excised from immunized animals, they have a limited life span and cannot be cultured directly to produce antibody in useful amounts. Fortunately, this restriction has been overcome with the development of hybridoma technology, wherein isolated B-lymphocytes in suspension are fused with myeloma cells from the same species (usually mouse) to create monoclonal hybrid cell lines that are virtually immortal while still retaining their antibody producing abilities. Such hybridomas may be stored frozen and cultured as needed to produce the specific monoclonal antibody. Monoclonal antibodies are especially useful as primary antibodies in applications that require single epitope specificity and an unchanging supply over many years of use. Hybridoma clones may be grown in cell culture for collection of antibodies from ascites fluid.

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