ANTIBODIES

INTRODUCTION
An ANTIBODY is defined as “an immunoglobulin
capable of specific combination with the antigen that
caused its production in a susceptible animal.”The higher
vertebrates, including man, have evolved a defense
system to protect themselves against the invasion of
foreign substances-a virus, a bacterium or a protein.
lmmunoglobulins, a specialised group of proteins are
mostly associated with y-globulin fraction (on
electrophoresis) of plasma proteins. Some
immunoglobulins however, separate along with P and a-
globulins. Therefore, it should be noted that y-globulin
and immunoglobulin are not synonymous.
lmmunoglobulin is a functional term while y-globulin is a
physical term. Antibodies are produced by plasma cells
which is present of B-cell membrane.
When a host is challenged by foreign material (bacteria,
virus, toxins, etc.) the first response of certain host
immune cells called macrophages is to engulf these
invaders (antigens) and process them biochemically. This
biochemical processing essentially creates a blueprint
that is used for the development of an immune response
that results in the production of antibodies . The unique
feature of antibodies produced in response to an antigen
is that they are synthesized in such a way that they are
highly specific for that antigen. Thus, they can chemically
interact and bind only with that particular antigen,
neutralize it, and/or aid in its destruction and removal
from the body.
(A) Foreign material is represented here by a virus that
has gained access to the body.
(B) The virus is phagocytosed by a macrophage.
(C) The virus is broken down into subunits by enzymes
contained in the phagocytic vacuole.
(D) An antigen-presenting molecule escorts antigenic
subunits from the virus to the macrophage surface.
(E) The antigenic molecule derived from the virus is
presented to a T cell.
(F) The B cell encounters the virus and expands its
antibody production specific to the viral epitope.
(G) The macrophage and the T cell release chemicals that
stimulate B cells.
(H) Antibodies are released into circulation.
(I) Antibodies neutralize the virus in circulation by
binding to the viral epitopes and inhibiting viral
attachment to target cells.

The largest and diverse population of antibodies is

generated by random combinations of set of a gene
segments that encode different binding sites , followed
by random mutation in this area of the antibody gene
,which creat further diversity .
Antibodies can occur in two physical forms, a soluble
form that is secreted from the cell to be free in the blood
plasma, and a membrane-bound form that is attached to
the surface of a B cell and is referred to as the B-cell
receptor (BCR).
Antibodies are glycoprotein molecules that recognise a
particular epitope on an antigen,bind specically to it and
finally facilitate the clearance of that antigen.
Antibody genes also re-organize in a process called class
switching that changes the one type of heavy chain
crystallised fregment (Fc) to another, creating a different
isotype of the antibody that retains the antigen-specific
variable region. This allows a single antibody to be used
by different types of Fc receptors, expressed on different
parts of the immune system.
They are typically made of basic structural units—each
with two large heavy chains and two small light chains.
There are several different types of antibody heavy
chains that define the five different types of crystallisable
fragments (Fc) that may be attached to the antigen-
binding fragments. The five different types of Fc regions
allow antibodies to be grouped into five isotypes. Each Fc
region of a particular antibody isotype is able to bind to
its specific Fc Receptor (FcR), except for IgD, which is
essentially the BCR, thus allowing the antigen-antibody
complex to mediate different roles depending on which
FcR it binds. The ability of an antibody to bind to its
corresponding FcR is further modulated by the structure
of the glycan(s) present at conserved sites within its Fc
region.The ability of antibodies to bind to FcRs helps to
direct the appropriate immune response for each
different type of foreign object they encounter.

HISTORY
1. The first use of the term "antibody" occurred in a
text by Paul Ehrlich. The term Antikörper (the
German word for antibody) appears in the
conclusion of his article "Experimental Studies on
Immunity", published in October 1891, which states
that, "if two substances give rise to two different
 Antikörper, then they themselves must be
different".However, the term was not accepted.
2. The earliest reference to antibodies came from Emil
von Behring and Shibasabura Kitasato in 1890.In a
landmark publication they showed
3.
4. that the transfer of therapeutic serum from animals
immunized against diphtheria to animals suffering
from it could cure the infected animals. The potential
for treatment in humans was immediately apparent
and Behring was later awarded the Nobel Prize for
this work in 1901.
5. In 1900 Paul Ehrlich, proposed the side-chain theory,
where he hypothesized that side chain receptors on
cells bind to a given pathogen. He was the first to
propose a model for an antibody molecule in which
the antibody was branched and consisted of multiple
sites for binding to foreign material, known as
antigen, and for the activation of the complement
pathway. This model agreed with the ‘lock and key’
hypothesis for enzymes proposed by Emil Fischer
6. Astrid Fagraeus in 1948 described that plasma B cells
are specifically involved in antibody generation and
by 1957 Frank Burnet and David Talmage had
developed the clonal selection theory. This stated
that a lymphocyte makes a single specific antibody
molecule that is determined before it encounters an
antigen, which was in contrast to the instructive
theory developed by Linus Pauling in 1940 where the
antigen acted as a template for the antibody
7. By 1959 Gerald Edelman and Rodney Porter
independently published the molecular structure of
antibodies, for which they were later jointly awarded
the Nobel Prize in 1972.
8. The first atomic resolution structure of an antibody
fragment was published in 1973 and this was quickly
followed by the invention of monoclonal antibodies
in 1975 by Georges Köhler and César Milstein
signaling the start of the modern era of antibody
research and discovery.
STRUCTURE
In simplistic terms antibodies perform two main
functions in different regions of their structure. While
one part of the antibody, the antigen binding fragment
(Fab), recognizes the antigen, the other part of the
antibody, known as the crystallizable fragment (Fc),
interacts with other elements of the immune system,
such as phagocytes or components of the complement
pathway, to promote removal of the antigen.
1. Classes of L Chains
The L chains are similar in all classes of immunoglobulins.
The L chain has a molecular weight of approximately
25,000 and the H chain of 50,000. There are two classes
of L chains, designated kappa (k) and lambda (λ) . Both
are normally expressed in every individual. However,
each B cell expresses (and each antibody contains) only
one type of L chain—either κ or λ but not both. Kappa (κ)
and lambda (λ) are named after Korngold and Lapari who
originally described them. Kappa (κ) and lambda (λ)
chains occur in a ratio of about 2:1 in human sera.
2. Classes of H Chains
The H chains are structurally and antigenically distinct for
each class and are designated by the Greek letter
corresponding to the immunoglobulin class as shown
below:
Immunoglobulin class H chain
IgG gamma (γ) gamma (γ)
IgA alpha (α) alpha(α),
IgM mu (μ) mu (μ),
IgD delta (δ) delta (δ),
IgE epsilon (ε) epsilon (ε)

In humans there are five classes of heavy chains

designated by lower case Greek letters: . The properties
of these heavy chains determine respectively the five
immunoglobulin classes—IgG, IgA, IgM, IgD. Each
immunoglobulin class differs in its general properties,
halflife, distribution in the body, and interaction with
other components of the host’s defensive systems.

3. Constant and Variable Regions

The antigen-combining site of the molecule is at its
amino terminus. All immunoglobulin chains possess a
constant (“C”) region that determines the biologic action
of an antibody, and a variable (“V”) region that binds a
unique epitope. Both light and heavy chains contain two
different regions. Of the 220 amino acids,those constitute
carboxy-terminal half of L chain occurs in a constant
sequence. This part of the chain is called Constant
regions (CL). Only two sequence patterns are seen in the
constant region—those determining kappa (κ) and
lambda (λ) specificities. On the other hand, the amino
acid sequence in the amino terminal half of the chain is
highly variable, the variability determining the
immunological specificity of the antibody molecules. It is,
therefore, called variable region (VL). Within the light
chain variable domain are hypervariable regions or
complementarity-determining regions (CDRs) that differ
in amino acid sequence more frequently than the rest of
the variable domain.The H chain also has Constant
regions (CH) and variable regions (VH). While in the L
chain the two regions are of equal length, in the H chains
the variable region constitutes approximately only a fifth
of the chain and is located at its amino terminus. The
variable regions (VL and VH) from different antibodies do
have different sequences. It is the variable regions (VL,
VH) that when folded together form the antigen binding
sites. The other domains of the heavy chains are termed
constant domains and are numbered CH1, CH2, CH3, and
sometimes CH4, starting with the domain next to the
variable domain. It is the portion of H chains present in
Fab fragment. H chains carry a carbohydrate moiety,
which is distinct for each class of immunoglobulins.
4.Fab and Fc Portions
i. Papain (proteolytic enzyme from papaya) cleaves the
lg , so that two Fab (fraction antibody) portions and one
Fc (fraction crystallizable) portion are produced. The
antigen binding part of the antibody is in the Fab
fragment.
ii. The cleavage takes place in the hinge region, where lg
molecule can have mobility in 3 dimensional space, so as
to adjust for tight grip on the antigen. Carbohydrate
groups of the lg molecule are also situated in the hinge
region. The area capable of complement binding lies in
the Fc portion.
iii. Another proteolytic enzyme, pepsin cleaves lg at
another site so as to yield F(ab)2, where two Fab
portions are combined together Fab part can combine
with antigen very weakly, but combination with F(ab)2 is
stronger.
Fc Fragment:
The Fc fragment is composed of the carboxy terminal
portion of the H chains. It can be crystallized, and is
therefore called Fc (fragment-crystallizable). This third
fragment does not bind antigen, but contributes to the
antibody’s biologic activity, such as binding complement,
transplacental passage, skin fixation and catabolic rate.
Functions of Fc Binds complement leading to
complement fixation.
• Binds to cell receptors (FcRs).
• Determines passage of IgG across the placental
barrier.
• Determines skin fixation and catabolic rate.
Antigenic determinants that distinguish one class of
antibody from another are also located on Fc fragment.
5. Immunoglobulin Domain
Each immunoglobulin peptide chain has internal disulfide
links in addition to interchain disulfide bonds which
bridge the H and L chains. These interchain disulfide
bonds form loops in the peptide chain, and each of the
loops is compactly folded to form a globular domain,
each domain having a separate function. The variable
region domains, VL and VH, are responsible for the
formation of a specific antigen binding site. The CH2
region in IgG binds C1q in the classical complement
sequence, and the CH3 domain mediates adherence to
the monocyte surface. The areas of the H chain in the C
region between the first and second C region domains
(CH1 and CH2) is the hinge region. It is more flexible and
is more exposed to enzymes and chemicals. Papain acts
here to produce one Fc and two Fab fragments.
IMMUNOGLOBULIN CLASSES
Human serum contain five classes of immunoglobulins—
IgG, IgA, IgM, IgD and IgE in the descending order of the
concentration.
Immunoglobulin G (IgG)
9. This is the major immunoglobulin in human
serum, accounting for about 80 percent of the total
immunoglobulin pool.
10. It has a sedimentation coefficint of 7S and a
molecular weight of 150,000.
11. It contains less carbohydrate than other
immunoglobulins.
12. IgG is the predominant immunoglobulin in
blood,lymph, peritoneal fluid, and cerebrospinal
fluid, and it is distributed nearly equally between
extraand intravascular spaces.
13. Four subclasses of IgG (Ig1, Ig2, Ig3, Ig4) have been
recognized.
- Each subclass possesses a distinct type of γ chain
which can be identified with specific
 antiserum.
-They constitute about 65 percent, 23 percent, 8
percent and 4 percent respectively of the total
human IgG.

· the subclass produced is dependent on the type of

cytokine present. IgG1 and IgG3 exhibit high affinity
for Fc receptors on phagocytes, while IgG2 exhibits
very low affinity and IgG4 has moderate affinity for
Fc receptors IgGs are capable of exiting the
circulatory system and enter tissues.
· IgG1, IgG3, and IgG4 can cross placental barrier to
provide protection for newborns. IgGs are efficient at
activating the complement system, and are very
effective for opsonization using Fc receptors on
phagocytes.
· Through its Fc region IgG can also bind to natural
killer cells and participate in antibody-dependent
cytotoxicity. IgG has a half-life ranging from 7 to 23
 days, depending on its subclass.

Functions of IgG
IgG is a very versatile molecule. It may be considered a
general purpose antibody, protective against those
infectious agents which are active in the blood and
tissues.
· Transfer from mother to fetus
· Opsonization
· Fixing to guinea pig skin
· Suppresses the homologous antibody synthesis
· Immunological reactions
2. Immunoglobulin A (IgA):
14. It is the second most abundant class, constituting
about 10 to 13 percent of serum immunoglobulins.
15. The normal serum level is 0.6 to 4.2 mg per ml.

16. It has a half-life of 6-8 days.

17. In the blood IgA are present in low levels in
monomeric form. They are most active at mucosal
surfaces where they are present in dimeric form and
 provide the primary defense at mucosal surfaces.
18. More IgA is produced in mucosal linings than all
other types of antibody combined. Its major function
is to act as a neutralizing antibody.
19. IgA is the primary immunoglobulin found in external
secretions, such as mucus, tears, saliva, gastric fluid,
colostrum and sweat. It exists in different forms in
these various solutions.
20. IgA occurs in two forms. Serum IgA and secretory
IgA

IgA1 may account up to 85% of the total IgA in serum.

Selective IgA deficiency is one of the most common
immunodeficiency diseases that increases susceptibility
to infections.
IgA deficiencies are commonly seen in patients with
autoimmune diseases and allergic disorders. IgA has a
half-life of about 5 days.
Functions of IgA
Prevention of organisms entry into body tissues
Phagocytosis and intracellular killing
Alternative pathways activation
Newborn protection
3. Immunoglobulin M (IgM)
21. About 10 percent of normal serum Igs consists of this
class.
22. This class of immunoglobulin is first to be produced
in response to infection and is found either on
membranes of B cells or as a 5-subunit
macromolecule secreted by plasma cells.
23. It is also the first immunoglobulin class to be
synthesized by the neonates.
24. The surface IgM differs from the secreted form in its
Fc region. Surface IgM binds directly as an integral
membrane protein and not to the IgM Fc receptor.
 25. Secreted IgM is a pentameric molecule where
multiple immunoglobulins are covalently linked with
disulfide bonds. This structure provides multiple
binding sites.
26. Each monomer consists of two light chains (either κ
or λ) and two heavy chains. Because of its
pentameric nature IgM is particularly suited for
activating complement and causing agglutination.
27. IgM has a half-life of about 5 days.

Functions of IgM
· Agglutination ,precipitation,complement activations
· produce early in immune response and it is the first
effective defense against bacteremia.
 · complement fixing
· fetal immunity
· acute infection
Immunoglobulin D (IgD)
28. IgD has a monomer structure similar to IgG.
29. Its molecular weight is 180,000 daltons.
30. IgD is an immunoglobulin found in trace amounts in
the blood serum (0.03 mg/ml).
31. Half-life is about 3 days.
32. IgD antibodies are abundant in combination with
IgM on the surface of B cells and bind antigens, thus
signaling the B cell to start antibody production.
33. Two subclasses of IgD (IgD1 and IgD2) are known.
Function of IgD
· involved with development and maturation of the
antibody response.
· its presence on lymphocyte surface of
immunocomplement cells forms and important
function for B cell activation and immunoregulation.
5. Immunoglobulin E (IgE)
34. This group of antibodies is effective at mucosal
surfaces, blood, and tissues.
35. It is present as monomer consisting of two heavy
chains (ε chain) and two light chains.
36. The ε chain contains 4 Ig-like constant domains.
37. In serum, it is present in low concentrations
contributing to only about 0.002% of total serum
antibodies.
38. Most IgE is tightly bound to its receptors on mast
cells and basophils via the Fc region.
39. It plays a crucial role in hypersensitivity reactions
and its production is strictly controlled by cytokines.
40. IgE has a half-life of about 2 days.
Function of IgE
· Involved in many allergic reactions .
· Its role in ADCC help to expel paracites

Role of Different Immunoglobulin Classes

IgG: Protects the body fluids
IgA: Protects the body surfaces
IgM: Protects the blood stream
IgE: Mediates type I hypersensitivity
IgD: Role not known.

ANTIGEN-ANTIBODY INTERACTIONS
The reactions between antigen and antibody occurs in
three stantigenes:
1. Primary stage: In primary or initial interaction there is
no visible effect and the reaction is rapid, occurs even at
low temperatures and obeys the laws of physical
chemistry and thermodynamics. The reaction is
reversible, the combination between antigen and
antibody being effected by the weaker intermolecular
forces such as van der Waals‘ forces, ionic bonds and
hydrogen bonding, rater than by the firmer covalent
bonding. Free and bound antigen or antibody can be
estimated separately in the reaction mixture by using a
number of physical and chemical methods.
2. Secondary stage: The primary interaction in most
instances, but not all, is followed by the secondary stage,
leading to demonstrable events such as precipitation,
agglutination, lysis of cells, killing of live antigens,
neutralization of toxins and other biologically active
antigens, fixation of complement, immobilization of
motile organisms and enhancement of phagocytosis.
When such reactions were discovered, it was believed
that a different type of antibody was responsible for each
type of reaction and the antibodies were designated by
the reactions they were thought to produce. The
antibody causing agglutination was called agglutinin, that
causing precipitation precipitin and the corresponding
antigen, agglutinogen, precipitinogen, and so on.
Zinsser’s unitarian hypothesis (by the 1920s) replaced
this view which held that an antigen gave rise to only one
antibody, which was capable of producing all the
different reactions depending on the nature of the
antigen and the conditions of the reaction. Both these
views are extreme and fallacious. The truth is that a
single antibody can cause precipitation, agglutination and
most of the other serological reactions but it is also true
that an antigen can stimulate the production of different
classes of immunoglobulins which are different in their
reaction capacities as well as in other properties .
3. Tertiary reactions: Some antigen-antibody reactions
occurring in vivo initiate chain reactions that lead to
neutralization or destruction of injurious antigens, or to
tissue damage. These are tertiary reactions and include
humoral immunity against infectious diseaseas well as
clinical allergy and other immunological diseases.

GENERATION OF ANTIBODIES

MONOCLONAL AND POLYCLONAL ANTIBODY

When designing procedures, it is important to
d i fferentiate between monoclonal and
polyclonal antibodies, as these differences are
the foundation of both advantages and
limitations for their use.
MONOCLONAL ANTIBODY:
41. Monoclonal antibodies (mAb or moAb) are identical
immunoglobulins, generated from a single B-cell
clone. These antibodies recognize unique epitopes,
 or binding sites, on a single antigen.
42. The use of monoclonal antibodies to treat diseases is
called immunotherapy therapy because each type of
monoclonal antibody will target a specific targeted
antigen in the body.
Advantages:
· Highly specific recognition of only one epitope of an
antigen
· Immortal hybridoma cell lines have the ability to
produce unlimited quantities of antibodies
· High consistency among experiments
· Minimal background noise and cross-reactivity
· Excellent for affinity purification
Disadvantages:
· Developing a monoclonal takes time and requires
high technical skills.
· They can produce large amounts of specific
antibodies but may be too specific to detect in across
a range of species.
· Vulnerable to the change of epitope. Even a slight
change in conformation may lead to dramatically
reduced binding capacity.
POLYCLONAL ANTIBODY:
43. Polyclonal antibodies ( pAbs) are created in the body
by B Cells. Their primary purpose is to act against
certain antigens in the body. They do this by
identifying different epitopes on a given antigen.
44. polyclonal antibodies are composed of a mixture of
antibodies that represents the natural immune
response to an antigen, they are prone to a higher
risk of batch-to-batch variability than monoclonal
antibodies.
Advantages:
· Short production time and low cost.
· Highly stable and tolerant of pH or buffer changes.
· High affinity. Since the antibodies bind to more than
one epitope, they can help amplify the signal from
target protein even with low expression level. This
makes these antibodies ideal for
immunoprecipitation and chromatin
immunoprecipitation.
· Tolerant of minor changes of antigen. Polyclonal
antibodies are less sensitive to antigen changes
(slight denaturation, polymorphism, heterogeneity of
glycosylation) than monoclonal antibodies.
Disadvantages:
· Prone to batch to batch variability.
· Multiple epitopes make it important to check
immunogen sequence for any cross-reactivity.

BIOLOGICAL EFFECTS OF ANTIBODIES

The membrane attack complex (MAC) cytolysis:MAC is
formed on the surface of pathogenic bacterial cell as a
result of the activation of the complement system (both
alternative and the classical pathways). The MAC forms
transmembrane channels in bacterial walls, disrupting
their phospholipid bilayer and leading to cell lysis and
death.
Immobilization:An antibody can be directed against cilia
or flagella of motile bacteria or protozoa that results in
cessation of their motility and blocks their ability to move
around and spread infection.
Cytolysis:Certain antibodies can cause disruption of the
microbial membrane that result in death of bacterial
cells. This requires the participation of the complement
system.
Neutralization of exotoxins: Antitoxin antibodies can be
generated against microbial toxins. The F(ab) region of
the antibody made against epitope of the binding site of
an exotoxin can block the exotoxin from binding to the
exotoxin receptor on the host cell membrane.
Agglutination of microorganisms:The F(ab) sites of IgM
and IgA antibodies can link microorganisms together and
cause them to agglutinate. The agglutinated
microorganisms can be phagocytosed more effectively.