antibody

1. Antibody

2. • Abs are Antigen receptors of the adaptive immune system.
• On stimulation with antigen, B cells secrete antibodies with antigen-binding sites identical to those
on the B-cell membrane antigen receptor.
• Abs are soluble form of the receptor
• Secreted antibodies and their membrane-bound receptor forms belong to the immunoglobulin
family of proteins.
• This large family of proteins, which includes both B- and T-cell receptors, adhesion molecules, some
tyrosine kinases, and other immune receptors, is characterized by the presence of one or more
immunoglobulin domains, thus, Each of these proteins is classified as a member of the
immunoglobulin superfamily
• The immunoglobulin domain is generated when a polypeptide chain folds into an organized series of
antiparallel beta pleated strands.
• In antibody molecules, most immunoglobulin domains contain approximately 110 amino acids, and
each beta sheet contains three to five strands
• the immunoglobulin fold is superbly adapted to provide a single scaffold onto which multiple different
binding sites can be built, as the antigen-binding sites can simply be built into these loosely folded
regions of the antigen-binding domains.

3. Diagram of the immunoglobulin fold structure
of the antibody light chain variable (VL) and
constant (CL) region domains.

5. • All antibodies share a common structure of four
polypeptide chains, consisting of two identical light (L)
chains and two identical heavy (H) chains.
• Each light chain is bound to its partner heavy chain by
• a disulfide bond between corresponding cysteine residues, as
well as by
• noncovalent interactions between the VH and VL domains and
the CH1 and CL domains.
• These bonds enable the formation of a closely
associated heterodimer (H-L).
• Multiple disulfide bridges link the two heavy chains
together about halfway down their length.
• C-terminal parts of the two heavy chains also
participate in noncovalent bonding interactions
between corresponding domains.
• the antibody molecule forms a Y shape with two
identical antigen-binding regions at the tips of the Y.
• Each antigen-binding region is made up of amino acids
derived from both the heavy- and the light chain
amino-terminal domains.
• The heavy and light chains both contribute two
domains to each arm of the Y, with the non–antigen-
binding domain of each chain serving to extend the
antigen-binding arm.
• The base of the Y consists of the C-terminal domains
of the antibody heavy chain.

6. • overall structure of the antibody molecule consists
of three relatively compact regions, joined by a
more flexible hinge region.
• The hinge region is particularly susceptible to
proteolytic cleavage by the enzyme papain.
• Papain cleavage resolves the antibody molecule
into two identical fragments that retain the
antigen-binding specificity of the original antibody
(shown as Fab regions), and the remaining region
of the molecule, which consists of the non
antigen-binding portion. This latter region, which
is identical for all antibodies of a given class,
crystallizes easily and was thus called the Fc
region (fragment crystallizable).
• The Fab regions bind to the antigen, and the Fc
region of the antigen-coupled antibody binds to Fc
receptors on phagocytic or cytolytic cells, or to
immune effector molecules.

7. Prototype structure of IgG, showing chain
structure and interchain disulfide bonds.
• The fragments produced by
enzymatic digestion with pepsin or
papain or by cleavage of the
disulfide bonds with
mercaptoethanol are indicated.
• Light (L) chains are in light blue,
and heavy (H) chains are in dark
blue.

8. • The N-terminal half of light chains is
referred to as the variable, or VL, region of
the light chain, and the less variable part of
the sequence is termed the constant, or
CL, region.
• The two major light chain constant region
sequences are referred to as (kappa) or
(lambda) chains.
• In humans, the light chains are fairly evenly divided
between the two light-chain classes; 60% of human
light chains are whereas only 40% are .
• In mice, the situation is quite different: Only 5% of
mouse light chains are of the light-chain type.
• All light chains have a molecular weight of
approximately 22 kDa.
• even within the variable regions of the light
chain, there were regions of
hypervariability. Since these
hypervariable regions could be shown to
interact with the bound antigen, they were
renamed the complementarity-

9. There are Five Major Classes of Antibody Heavy
Chains
• sequences of the heavy-chain constant regions fall into five basic patterns. Th ese five basic
sequences have been named with Greek letters: μ(mu), δ,(delta), γ(gamma), ε(epsilon), and α
(alpha).
• Each different heavy-chain constant region is referred to as an isotype, and the isotype of the
heavy
• 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.
• antibodies with a heavy chain of the μ(mu) isotype are of the IgM class;
• those with a heavy chain δ(delta), are IgD;
• those with γ(gamma), IgG;
• those with ε(epsilon), IgE; and those with α (alpha) IgA.
• The length of the constant region of the heavy chains is either 330 amino acid residues (for γ, α and
δchains) or 440 amino acids (for μ and ε chains). Correspondingly, the molecular weights of the
heavy chains vary according to their class. IgA, IgD, and IgG heavy chains weigh approximately 55
kDa, whereas IgM and IgE antibodies are approximately 20% heavier.
• The variable region of the heavy chain differs depending on the B cell that produced it, but is the same for all
antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is

10. There are Five Major Classes of Antibody
Heavy Chains
• Minor differences in the amino acid sequences of groups of γ and α heavy
chains led to further subclassification of these heavy chains into sub-
isotypes and their corresponding antibodies therefore into subclasses.
• There are two sub-isotypes of the α heavy chain, α 1 and α 2, thus two IgA
subclasses, IgA1 and IgA2.
• Similarly, there are four sub-isotypes of γ heavy chains, γ 1, γ 2, γ 3, and γ
4, with the corresponding formation of the four subclasses of IgG: IgG1,
IgG2, IgG3, and IgG4.
• exact number, and precise positions of the disulfide bonds between the
heavy chains of antibodies, vary among antibodies of different classes and
subclasses

12. General structures of the five major classes
of antibodies.
• Light chains are shown in lighter shades,
disulfide bonds are indicated by thick
black lines.
• Note that the IgG, IgA, and IgD heavy
chains contain four domains and a hinge
region, whereas the IgM and IgE heavy
chains contain five domains but no hinge
region. The polymeric forms of IgM and
IgA contain a polypeptide, called the J
chain, that is linked by two disulfide
bonds to the Fc region in two different
monomers.
• Serum IgM is always a pentamer; most
serum IgA exists as a monomer, although
dimers, trimers, and even tetramers are
sometimes present.

13. General structure of the four subclasses of human IgG, which differ in the number
and arrangement of the interchain disulfide bonds (thick black lines) linking the heavy
chains. A notable feature of human IgG3 is its 11 interchain disulfide bonds

14. Antibodies and Antibody Fragments Can Serve
as Antigens
• Plasmacytomas are tumors of plasma cells, When a single clone of plasma
cells becomes cancerous, it is called a plasmacytoma for as long as it
remains in a single bone.
• However, once it metastasizes into multiple bone marrow sites, the tumor
is referred to as multiple myeloma.
• Plasmacytoma or myeloma tumors secrete large amounts of monoclonal
antibodies into the serum and tissue fluids of the patients, and these
antibodies can be purified in large quantities.
• Rather than secreting the whole antibody, some of these tumors will
secrete only the light chains, or sometimes both the light chains and the
whole antibodies, into the serum. The homogenous light chains secreted
by these myeloma tumors are referred to as Bence-Jones proteins.

15. • An antigenic determinant is defined as a region of an antigen that makes contact with the antigen-
combining region on an antibody.
• Anti-isotype antibodies are directed against antigenic determinants present on the constant region of
one particular heavy- or light-chain class of antibody, but not on any of the others.
• an anti-isotype antibody may bind only to human μ heavy chains, but not to human δ,(delta),
γ(gamma), ε(epsilon), and α (alpha) constant regions. Alternatively, it may bind to kappa but not to
lambda light chains.
• Some heavy- or light-chain genes occur in multiple allelic forms, and alternative allelic forms of the
same isotype are referred to as allotypes. two antibodies with allotypic differences will vary in just a
few residues of one of the immunoglobulin chains, and these residues constitute the allotypic
determinants.
• Anti-allotype antibodies are generated by immunizing an individual of one species with antibodies
derived from a second animal of the same species bearing an alternative form (allele) of the particular
immunoglobulin gene.
• antibodies directed against the antigen-binding site of a particular antibody are referred to as anti-
idiotypic antibodies.
• Anti-Fab antibodies and anti-Fc antibodies are made by immunizing a different species of animal from
that which provided the antibody fragments with Fab or Fc fragments, respectively

17. Each of the Domains of the Antibody Heavy
and Light Chains Mediate Specific Functions
CH1 and CL Domains
• The CH1 and CL domains serve
to extend the antigen-binding
arms of the antibody molecule,
facilitating interactions with
multivalent antigens and
maximizing the ability of the
antibody to bind to more than
one site on a multivalent
antigen.
The Hinge Regions
• hinge region is rich in proline residues, rendering it particularly
flexible, and as a consequence, the two antigen-binding arms of
IgG, IgD, and IgA antibodies can assume a wide variety of angles
with respect to one another, which facilitates efficient antigen
binding.
• The extended nature of the amino acid chain in the hinge region
contributes to the vulnerability of this part of the molecule to
protease cleavage
• Lacking a hinge region, the heavy chains of IgE make their inter-
heavy chain disulfide bonds between the CH1 and CH3 domains.
In IgM, disulfide bonds bridge the pairs of heavy chains at the
level of CH3 and CH4.
• Although mu and epsilon chains have no hinge regions, they do
have an additional immunoglobulin domain that retains some
hinge like qualities.

18. Carbohydrate Chains
• The two CH2 domains of α,γ and δ
chains and the two CH3 domains of μ
and ε chains are separated from their
partner heavy-chain domains by
oligosaccharide side chains that
prevent the two heavy chains from
nestling close to one another.
• As a result, the paired domains are
significantly more accessible to the
aqueous environment than other
constant region domains.
• This accessibility is thought to
contribute to the ability of IgM and
IgG antibodies to bind to complement
components.
• Immunoglobulins are in general quite
extensively glycosylated, and some
antibodies even have carbohydrates
attached to their light chains.
The Carboxy-Terminal Domains
• Secreted ab have a hydrophilic amino acid
sequence of various lengths at the carboxyl
terminus of the final CH domain.
• In membrane bound immunoglobulin receptors,
this hydrophilic region is replaced by three
regions
1. • An extracellular, hydrophilic
“spacer” sequence of approximately 26
amino acids
2. • A hydrophobic transmembrane segment of
about 25 amino acids
3. • A very short, approximately three
amino acid, cytoplasmic tail
• Immature, pre-B cells express only membrane
IgM.
• Membrane IgD co-expression along with IgM is
one of the markers of differentiation to a
fully mature B cell that has yet to
encounter antigen.
• Following antigen stimulation, IgD is lost
from the cell surface, and the constant
region of the membrane and secreted
immunoglobulin can switch to any one of the
other isotypes.

19. Membrane vs. Secreted forms of immunoglobulin
are created by alternative mRNA splicing