Immunoglobulin Structure and Classes

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

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Structure of immunoglobulins

Classes of immunoglobulins

Subclasses of immunoglobulins

Polyclonal and monoclonal antibodies

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Antibodies

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 and 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

Generalized structure of an immunoglobulin (IgG).

Annotated diagram of immunoglobulin structure

Annotated diagram of immunoglobulin structure.

Heavy and light chains are held together by a combination of non-covalent 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.

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Antibody Labeling and Immobilization Sites

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 10 light chains, 10 heavy chains and 10 antigen-binding.

IgG class

IgG Class

Properties of IgG:

Molecular weight: 150,000

H-chain type (MW): gamma (53,000)

Percent of total immunoglobulin: 75%

Glycosylation (by weight): 3%

Distribution: intra- and extravascular

Function: secondary response

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IgM class

IgM Class

Properties of IgM:

Molecular weight: 900,000

H-chain type (MW): mu (65,000)

Serum concentration: 0.5 to 2 mg/mL

Percent of total immunoglobulin: 10%

Glycosylation (by weight): 12%

Distribution: mostly intravascular

Function: primary response

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IgA class

IgA Class

Properties of IgA:

Molecular weight: 320,000 (secretory)

H-chain type (MW): alpha (55,000)

Serum concentration: 1 to 4 mg/mL

Glycosylation (by weight): 10%

Distribution: intravascular and secretions

Function: protect mucus membranes

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IgD and IgE class

IgD and IgE Class

Properties of IgD:

Molecular weight: 180,000

H-chain type (MW): delta (70,000)

Serum concentration: 0 to 0.4 mg/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 400 ng/mL

Percent of total immunoglobulin: 0.002%

Glycosylation (by weight): 12%

Distribution: basophils and mast cells in saliva and nasal secretions

Function: protect against parasites

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Immunoglobulin IgA Class

Immunoglobulin IgE Class

Immunoglobulin IgG Class

Immunoglobulin IgM Class

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Isotype Control Antibodies

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, IgG3 and IgG4 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.

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Immunoglobulin Structure and Classes

Antibody Isotyping and Characterization

Antibody Purification

Antibody IgG Binding Proteins

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How to Choose and Select Secondary Antibodies

Immunoglobulin ELISA Kits

Isotype Control Antibodies

Antibody Purification Products

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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Introduction to Antibody Production & Purification

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Guide to Primary Antibody Types

How to Choose and Select Secondary Antibodies

Isotype Control Antibodies

Antibody Isotyping and Characterization Products

References

Alberts, B., et al. (1983). Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY.

Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.

Sites, D.P., et al. (1976). Basic & Clinical Immunology. Lange Medical Publication, Los Altos, CA.

For Research Use Only. Not for use in diagnostic procedures.

Immunoglobulin IgG Class

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.

Structure of IgG

Properties of IgG:

Molecular weight: 150,000

H-chain type (MW): gamma (53,000)

Serum concentration: 10 to 16 mg/mL

Percent of total immunoglobulin: 75%

Glycosylation (by weight): 3%

Distribution: intra- and extravascular

Function: secondary response

The role of IgG in the immune response

IgG subclasses

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Antibody Isotyping and Characterization Products

Antibody Purification Products

The role of IgG in the immune response

IgG is the major immunoglobulin in blood, lymph fluid, cerebrospinal fluid and peritoneal fluid and a key
player in the humoral immune response. Serum IgG in healthy humans presents approximately 15% of
total protein beside albumins, enzymes, other globulins and many more.

The Fc portion of IgG, but not F(ab´)2 or Fab fragments, can cross the placenta of a mother and enter
fetal circulation, providing the fetus with postpartum protection. IgG molecules are able to react with
Fcγ receptors that are present on the surface of macrophages, neutrophils and natural killer cells, and
can activate the complement system.

The binding of the Fc portion of IgG to the receptor present on a phagocyte is a critical step in the
opsonization. Phagocytosis of particles coated with IgG antibodies is a vital mechanism that cells use to
cope with microorganisms.

IgG is produced in a delayed response to an infection and can be retained in the body for a long time.
The longevity in serum makes IgG most useful for passive immunization by transfer of this antibody.
Detection of IgG usually indicates a prior infection or vaccination.

IgG subclasses

There are four IgG subclasses described in human, mouse and rat. The subclasses differ in the number of
disulfide bonds and the length and flexibility of the hinge region. Except for their variable regions, all
immunoglobulins within one class share about 90% homology, but only 60% among classes.
Determination of IgG subclasses can be a valuable tool in indicating a potential antibody deficiency.
Selective IgG subclass deficiencies are associated with disease. In cases with prolonged or severe
infections, determination of IgG levels can provide additional insight into the manifestation of disease. It
is important to interpret IgG subclass concentrations in correlation to the donor's age since the immune
system matures during childhood.

Because of its relative abundance and excellent specificity toward antigens, IgG is the principle antibody
used in immunological research and clinical diagnostics.

IgG1

IgG1 comprises 60 to 65% of the total main subclass IgG, and is predominantly responsible for the
thymus-mediated immune response against proteins and polypeptide antigens. IgG1 binds to the Fc-
receptor of phagocytic cells and can activate the complement cascade via binding to C1 complex. IgG1
immune response can already be measured in newborns and reaches its typical concentration in infancy.
A deficiency in IgG1 isotype is typically a sign of a hypogammaglobulinemia.

IgG2

IgG2, the second largest of IgG isotypes, comprises 20 to 25% of the main subclass and is the prevalent
immune response against carbohydrate/polysaccharide antigens. “Adult” concentrations are usually
reached by 6 or 7 years old. Among all IgG isotype deficiencies, a deficiency in IgG2 is the most common
and is associated with recurring airway/respiratory infections in infants.

IgG3

IgG3 comprises around 5 to 10% of total IgG and plays a major role in the immune responses against
protein or polypeptide antigens. The affinity of IgG3 can be higher than that of IgG1.

IgG4

Comprising usually less than 4% of total IgG, IgG4 does not bind to polysaccharides. In the past, testing
for IgG4 has been associated with food allergies, and recent studies have shown that elevated serum
levels of IgG4 are found in patients suffering from sclerosing pancreatitis, cholangitis and interstitial
pneumonia caused by infiltrating IgG4 positive plasma cells. The precise role of IgG4 is still mostly
unknown.
Immunoglobulin IgM Class

Serum IgM exists as a pentamer in mammals and comprises approximately 10% of normal human serum
Ig content. It predominates in primary immune responses to most antigens and is the most efficient
complement-fixing immunoglobulin. IgM is also expressed on the plasma membrane of B lymphocytes
as a monomer. In this form, it is a B cell antigen receptor, with the H chains each containing 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 within the pentamer structure 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, with the additional
constant domain replacing the hinge region. IgM can recognize epitopes on invading microorganisms,
leading to cell agglutination. 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.

Structure of IgM

Properties of IgM:

Molecular weight: 900,000

H-chain type (MW): mu (65,000)

Serum concentration: 0.5 to 2 mg/mL

Percent of total immunoglobulin: 10%

Glycosylation (by weight): 12%

Distribution: mostly intravascular

Function: primary response

The role of IgM in the immune response

Immunoglobulin M is the third most common serum Ig and takes one of two forms:

a pentamer where all heavy chains are identical and all light chains are identical
a monomer (e.g., found on B lymphocytes as B cell receptors)

The large pentameric structure allows for building of bridges between encountered epitopes on
molecules that are too distant as to be connected by smaller IgG antibodies.

IgM is the first antibody built during an immune response. It is responsible for agglutination and cytolytic
reactions since in theory, its pentameric structure gives it 10 free antigen-binding sites as well as it
possesses a high avidity. Due to conformational constraints among the 10 Fab portions, IgM only has a
valence of 5. Additionally, IgM is not as versatile as IgG. However, it is of vital importance in complement
activation and agglutination.

IgM is predominantly found in the lymph fluid and blood and is a very effective neutralizing agent in the
early stages of disease. Elevated levels can be a sign of recent infection or exposure to antigen.

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Introduction to Immunoglobulins

Immunoglobulin Structure and Classes

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Isotype Control Antibodies

Immunoglobulin ELISA Kits

Antibody Isotyping and Characterization Products

Antibody Purification Products

Anti-IgM Secondary Antibodies

Immunoglobulin IgA Class

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 and tears). The principal function of
secretory IgA may be not to destroy antigens but to prevent passage of foreign substances into the
circulatory system.
Structure of IgA

Properties of IgA:

Molecular weight: 320,000 (secretory)

H-chain type (MW): alpha (55,000)

Serum concentration: 1 to 4 mg/mL

Percent of total immunoglobulin: 15%

Glycosylation (by weight): 10%

Distribution: intravascular and secretions

Function: protect mucus membranes

The role of IgA in the immune response

IgA comprises approximately 15% of all immunoglobulins in healthy serum. Two IgA subtypes exist in
humans, IgA1 und IgA2, while mice have only one subclass. They differ in the molecular mass of the
heavy chains and in their concentration in serum.

IgA in serum is mainly monomeric, but in secretions, such as saliva, tears, colostrums, mucus, sweat, and
gastric fluid, IgA is found as a dimer connected by a joining peptide. Most IgA is present in secreted
form. This is believed to be due to its properties in preventing invading pathogens by attaching and
penetrating epithelial surfaces. IgA is a very weak complement-activating antibody; hence, it does not
induce bacterial cell lysis via the complement system. However, secretory IgA works together with
lysozymes (also present in many secreted fluids), which can hydrolyze carbohydrates in bacterial cell
walls thereby enabling the immune system to clear the infection. IgA is predominantly found on
epithelial cell surfaces where it acts as a neutralizing antibody.

IgA deficiency in disease

The most prevalent antibody defect is a selective IgA deficiency (SIgAD). Alterations in IgA1/IgA2 ratio
often go hand in hand with specific disease states, such as recurring infections of the airways or a kidney
disorder called IgA nephropathy.
There are various severe health conditions that can lead to low levels of Immunoglobulin A in the body,
one of which is gonorrhea. The bacteria that cause gonorrhea produce an enzyme that splices IgA
antibodies into Fc and Fab fragments. The Fab part still can recognize the bacteria, but without the Fc
fragment, attachment to phagocyting cells is not possible. When the body does not have sufficient
quantities of IgA, the person may be diagnosed with selective IgA deficiency.

Patients suffering from selective IgA deficiency can have normal levels of the other antibodies, fully
functioning T-cells, phagocytes, and other components of the immune system. Patients who suffer from
a selective IgA deficiency are more prone to autoimmune disorders like rheumatoid arthritis, lupus,
allergies and asthma.

IgA subclasses

IgA1

IgA1 comprises approximately 85% of total IgA concentration in serum. Although IgA1 shows a broad
resistance against several proteases, there are some that can affect/splice on the hinge region. IgA1
shows a good immune response to protein antigens and, to a lesser degree, polysaccharides and
lipopolysaccharides.

IgA2

IgA2, representing only up to 15% of total IgA in serum, plays a crucial role in the mucosa of the airways,
eyes and the gastrointestinal tract to fight against polysaccharide and lipopolysaccaride antigens. It also
shows good resistance to proteolysis and many bacterial proteases, supporting the importance of IgA2
in fighting bacterial infections.

Immunoglobulin IgE Class

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

Properties of IgE:
Molecular weight: 200,000

H-chain type (MW): epsilon (73,000)

Serum concentration: 10 to 400 ng/mL

Percent of total immunoglobulin: 0.002%

Glycosylation (by weight): 12%

Distribution: basophils and mast cells in saliva and nasal secretions

Function: protect against parasites

Properties of IgD:

Molecular weight: 180,000

H-chain type (MW): delta (70,000)

Serum concentration: 0 to 0.4 mg/mL

Percent of total immunoglobulin: 0.2%

Glycosylation (by weight): 13%

Distribution: lymphocyte surface

Function: unknown

Structures of IgD and IgE

The role of IgE in the allergic response

The heavy chain of IgE contains an extra domain, by which it attaches with high affinity to Fc epsilon
Receptor I (FcεRI) found primarily on eosinophils, mast cells and basophils. When antigens such as
pollen, venoms, fungus, spores, dust mites or pet dander bind with the Fab portion of the IgE attached
to the cells, the cells degranulate and release factors like heparin, histamine, proteolytic enzymes,
leukotrienes and cytokines. As a consequence, vasodilatation and increased small vessel permeability
causes fluid to escape from capillaries into the tissues, leading to the characteristic symptoms of an
allergic reaction. Most of these typical allergic reactions like mucus secretion, sneezing, coughing or tear
production are considered beneficial to expel remaining allergens from the body.

Studies have shown that conditions such as asthma, rhinitis, eczema, urticaria, dermatitis and some
parasitic infections (e.g., helminths and tapeworms) lead to increased IgE levels. Binding of eosinophils
with Fc receptors to IgE-coated parasitic helminth worms results in death of the parasite. Low levels of
IgE can occur in a rare inherited disease that affects muscle coordination (ataxia-telangiectasia).

Mouse studies are important in discovering the mechanisms and treatment of allergic responses, and
quantification of IgE levels is an important testing parameter.

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