The document summarizes the structure, function, and types of immunoglobulins (antibodies). It discusses the basic four-chain antibody structure consisting of two heavy and two light chains. The five major classes of antibodies - IgG, IgM, IgA, IgE, and IgD - are described along with their structures and functions including antigen binding, opsonization, complement activation, antibody-dependent cytotoxicity, and transcytosis. Electrophoretic studies in the 1930s first identified immunoglobulins in the gamma globulin fraction of serum.

1. IMMUNOGLOBULIN
STRUCTURE, FUNCTION AND TYPES
Aman Kumar Jaiswar, 422MB00173, I M.Sc. Applied Microbiology
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2. INTRODUCTION
◦ Antibodies are antigen binding proteins present on B-cell membrane
and secreted by plasma cells.
◦ Antibodies circulate in the blood, where they participate in humoral
immunity.
◦ They act by searching out and neutralizing antigens or marking them
for elimination.
◦ All antibodies share structural features, bind to antigen, and
participate in a limited number of effector functions.
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3. ELECTROPHORETIC STUDY OF
IMMUNE SERA
◦ Carried out by A. Tiselius and E. A. Kabat in 1939.
◦ They immunized rabbits with the protein ovalbumin and then divided the immunized rabbits’ serum
into two aliquots.
◦ Electrophoresis of one serum aliquot revealed four peaks corresponding to albumin and the alpha (α),
beta (β), and gamma (γ) globulins.
◦ The other serum aliquot was reacted with ovalbumin, and the precipitate that formed was removed.
◦ The remaining serum proteins, which did not react with the antigen, were then electrophoresed.
◦ On comparison, significant drop in γ-globulin peak in the aliquot that had been reacted in the antigen.
◦ Thus, the γ -globulin fraction was identified as containing serum antibodies, which were called
immunoglobulins to distinguish them from any other proteins in the same fraction.
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4. Blue line: electrophoretic pattern
of untreated antiserum
Black line: pattern of antiserum
that was incubated with OVA
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5. BASIC STRUCTURE
OF AN ANTIBODY
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6.  4 peptide chains, 2 identical light (L) chains and 2
identical heavy (H) chains.
 Light chains have a molecular weight of about
25,000 Da whereas heavy chains have a molecular
weight of 50,000 Da and more.
 Each light chain is bound to the heavy chain by
disulphide bond to form a heterodimer (H-L).
 The two heterodimers are also linked together by
disulphide bonds to form the basic four chain
structure (H-L)2.
 The first 110 amino acids in each chain vary
greatly among antibodies of different specificities.
This region is called the variable region (V), where
VL is variable light chain and VH is variable heavy
chain.
 The regions of relatively constant sequence
beyond the variable regions are called C regions,
CL on the light chain and CH on the heavy chain.
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7. CHEMICAL / ENZYMATIC ANALYSIS
OF ANTIBODY MOLECULE
◦ When IgG is briefly digested with the enzyme papain, three fragments are produced- two
identical and one non-identical.
◦ The two identical fragments of molecular weight 45,000 Da have antigen-binding activity
and are called Fab fragments (Fragment, antigen-binding).
◦ The other fragment of 50,000 Da molecular weight has no antigen-binding activity and is
called Fc fragment (Fragment, crystallizable) because it was found to crystallize during
cold storage.
◦ Similarly, pepsin digestion produces a single fragment called F(ab’)2 which binds antigen.
◦ Mercaptoethanol irreversibly cleaves disulfide bonds and separates the molecule into 4
subunits, 2 heavy and 2 light chains.
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8. 8

9. TYPES OF LIGHT AND HEAVY CHAINS
LIGHT CHAIN
◦ Light chains have a 100-110 long variable region
(VL) and a constant region (CL).
◦ The constant region has 2 basic amino acid
sequences and hence 2 types are recognized –
kappa (κ) and lambda (λ).
◦ In humans, 60% light chains are kappa and 40%
are lambda, in mice 95% are kappa and 5% are
lambda.
◦ Lambda light chains have 3 subtypes – λ1, λ2,
and λ3, and have minor differences in amino acid
sequences.
HEAVY CHAIN
◦ Similar to light chains, heavy chains also have a
100-110 long variable region (VH) and a constant
region (CH).
◦ The constant chain has 5 basic sequences called
mu (μ), delta (δ), gamma (γ), epsilon (ε) and
alpha (α).
◦ δ, γ and α chains have a length of 330 amino
acids, μ and ε are 440 amino acids long.
◦ The heavy chains determine the class of the
antibody - IgM(μ), IgD(δ), IgG(γ), IgE(ε) and
IgA(α)
◦ In humans, α chains have 2 subtypes – α1 and α2,
and γ chains have 4 subtypes – γ1, γ2, γ3 and γ4.
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10. OTHER CHARACTERISTICS
◦ Both heavy and light chains are folded into domains, homologous units of 110 amino acids
with an intrachain disulphide bond that forms a loop of about 60 amino acids.
◦ Light chains have 1 constant (CL) and 1 variable (VL) domain. Heavy chains have 1 variable
(VH) and 3-4 constant domains (CH1, CH2, CH3 and CH4).
◦ The loops present in VL and VH chains show maximum variability and are called
hypervariable regions or complementarity determining regions (CDRs) and are the antigen
binding sites.
◦ The remainder of VL and VH show less variability and are called framework regions (FRs).
◦ δ, γ and α have an extended peptide sequence between CH1 and CH2 called hinge region
which is rich in proline and gives IgD, IgG and IgA flexibility.
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12. FUNCTIONS
Apart from antigen-binding, which is carried out by variable regions of the antibody,
there are several other effector functions that are carried out by the constant region.
These include opsonization, complement activation, antibody-dependent cell-mediated
cytotoxicity and transcytosis.
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13. FUNCTIONS
OPSONIZATION
◦ It is the promotion of phagocytosis of
antigens by macrophages and neutrophils.
◦ Macrophages and neutrophils contain Fc
receptors that bind to the Fc region of
immunoglobulins.
◦ When Fc receptors bind to the Fc regions in
an antigen-antibody complex, it initiates
binding of the complex to the phagocyte
membrane as well as phagocytosis.
◦ The complex is then destructed using
enzymes or oxidizing agents.
COMPLEMENT ACTIVATION
◦ IgM and IgG can activate a collection of
serum glycoproteins called complements
that can perforate cell membranes.
◦ C3b binds non-specifically to antigen-
antibody complexes which also acts as a
receptor for macrophages and promotes
phagocytosis.
◦ The collaboration between antibody and the
complement system is important for the
inactivation and removal of antigens and
the killing of pathogens.
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OPSONIZATION COMPLEMENT ACTIVATION

15. FUNCTIONS
ANTIBODY-DEPENDENT CELL-
MEDIATED CYTOXICITY
◦ Some other cells such as Natural Killer
(NK) cells also have Fc receptors and
hence can bind to antigen-antibody
complexes.
◦ This leads to cytotoxic activities of the
effector cell towards target cell by a
process called antibody-dependent cell-
mediated cytotoxicity.
◦ Here, antibody acts as a receptor that
enables the attacking cell to recognize
and kill the target cell.
TRANSCYTOSIS
◦ The delivery of antibodies to mucosal
surfaces as well as breast milk across
epithelial layers is called transcytosis.
◦ It greatly depends on the properties of
the constant region.
◦ Examples for immunoglobulins capable
of transcytosis include IgA, IgM and
IgG.
◦ IgG can also cross maternal placenta to
confer passive immunity to the fetus.
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ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY (ADCC)

17. ANTIBODY CLASSES
There are five major classes of antibodies that differ in the
amino acid sequences in their heavy chain constant regions.
These confer class-specific structural and functional properties.
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18. Immunoglobulin G (IgG)
◦ It is the most abundant in serum, and constitutes about 80% of total
serum immunoglobulin.
◦ IgG molecule consists of 2 γ heavy chains and 2 κ/λ light chains.
◦ There are 4 subclasses distinguished by differences in γ chain
sequences – IgG1, IgG2, IgG3 and IgG4.
◦ The major differences between these subclasses are the size of the
hinge region and number and position of interchain disulphide bonds
between the heavy chains.
◦ These differences affect the biological properties as given below:
◦ 1. IgG1, IgG3 and IgG4 are involved in transplacental movement and
passive immunization.
◦ 2. IgG3 is the most effective complement activator followed by IgG1
and IgG2. IgG4 does not activate complement.
◦ 3. IgG1 and IgG3 have high affinity to Fc receptors and thus mediate
opsonization compared to IgG4 and IgG2 that have low affinity.
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19. Immunoglobulin M (IgM)
◦ IgM accounts for 5-10% of total serum immunoglobulin with an average
serum concentration of 1.5 mg/ml.
◦ It had a molecular weight of about 1,80,000 Da.
◦ It is secreted as a pentamer with 5 monomer units held together with
disulphide bonds that link their heavy chains.
◦ Each pentamer contains an additional Fc-linked polypeptide called J
(joining) chain, which is bound to 2 of the 10 μ chains by disulphide
bonds.
◦ J chain is required for pentamer formation and is added just before the
secretion of the IgM molecule.
◦ It is the first immunoglobulin produced in response to an antigen and
the first immunoglobulin produced by a neonate.
◦ It is a good agglutinin due to its large size and is also more efficient than
IgG in activating complement.
◦ It plays an important accessory role as secretory immunoglobulin and is
present is mucosal tissue.
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20. Immunoglobulin A (IgA)
◦ IgA constitutes about 10-15% of total immunoglobulin in serum, and is
predominant in external secretions such as breast milk, saliva, tears and
mucus.
◦ It exists primarily as a monomer, but polymeric forms are also seen, all
containing a J-chain.
◦ Secretory IgA consists of a dimer, a J-chain and a polypeptide chain called
secretory component, which is receptor-derived from mucosal epithelial
cells.
◦ Secretory component has a molecular weight of 70,000 Da and contains 5
domains that bind to Fc region of the IgA by disulphide bonds.
◦ It is the most secreted antibody, and about 5-15g of IgA is secreted into
mucous secretions daily.
◦ Its functions include cross-linking of large antigens with multiple epitopes.
◦ It also prevents attachment of pathogens to mucosal membranes, thus
inhibiting infection and colonization.
◦ It is present in breast milk and provides passive immunity to the neonate.
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21. Immunoglobulin E (IgE)
◦ It has an extremely low average serum concentration of
0.3 μg/ml.
◦ IgE mediates the immediate hypersensitivity reactions that
are responsible for the symptoms of hay fever, asthma,
hives and anaphylactic shock.
◦ It binds to Fc receptors on the membranes of basophils
and mast cells, which induces them to translocate their
granules to extracellular membrane and release their
contents. This process is called degranulation.
◦ As a result, a variety of pharmacologically active
compounds are released that give rise to allergic
manifestations.
◦ Mast cell degranulation also facilitates the buildup of cells
necessary for antiparasitic defense.
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22. Immunoglobulin D (IgD)
◦ IgD was first discovered when the myeloma
protein of a myeloma patient failed to react with
then known IgA, IgM and IgG antisera.
◦ When rabbits were immunized with this protein,
the resulting antisera was used to identify a new
class of antibody.
◦ This new class, called IgD, has a serum
concentration of 30 μg/ml and constitutes about
0.2% of the total immunoglobulin in the serum.
◦ Along with IgM, it is the major membrane bound
immunoglobulin expressed by mature B cells.
◦ No biological effector function has been
identified for IgD so far.
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