 IMMUNOGLOBULIN---------------------

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IMMUNOGLOBULINS
 In 1964, the WHO endorsed the generic term
“Immunoglobulin which was internationally
accepted as-- proteins of animal origin endowed
with known antibody activity and certain other
proteins related to them by close structure. All
antibodies are immunoglobulin but all
immunoglobulin may not be antibodies .Antibodies
are substance which are formed in the serum and
tissue fluid in response to an antigen and react with
that antigen specifically in an observable manner.
 All antibodies are immunoglobulins (Ig), but all Ig
may not be antibodies.
 Ig constitute 20 -25% of total serum proteins.
 Five classes of Ig have been recognized –
 IgG
 IgA
 IgM
 IgD
 IgE
STRUCTURE OF IMMUNOGLOBULIN

 Each molecule is split by papain into 3 parts –

 One Fc
 Two Fab pieces
STRUCTURE
 Ig are glycoproteins.
 Each molecule consist of 2 pairs of polypeptide
chains of different sizes.
 Smaller chains are called light (L) chains.
 Larger chains are called heavy (H) chains.
 Molecular weight of L chain – 25,000kDa and H
chain 50,000kDa.
 L chain is attached to H chain by disulphide bond.
 The two H chains are joined by 1-5 S-S bonds.
H - CHAINS
 Structurally and antigenically distinct for each
class.
 Designated by Greek letter corresponding to the Ig
class. Immunoglobulin Class H chain

IgG γ (gamma)

IgA α (alpha)

IgM μ (mu)

IgD δ (delta)

IgE ε (epsilon)
L - CHAINS
 Similar in all classes of Ig.
 Occur in two varieties –
 kappa (κ)
 lambda (λ)
 A molecule of Ig may have either kappa or lambda
chains, but never both together.
 Kappa and lambda chains occur in a ratio of 2:1 in
human sera.
ANTIGEN BINDING SITE
 Antigen combining site of the molecule is at its
amino terminus.
 Composed of both L and H chains.
 Of 214 aminoacid residues that make up the L
chain, about 107 that constitute the carboxy
terminal half occur in a constant sequence.
 This part of chain is called ‘constant’ region.
 Aminoacid sequence in the aminoterminal half of
the chain is highly variable.
 The variability determines the immunological
specificity of the antibody melocule.
 It is called ‘variable’ region.
 H chain also has ‘constant’ and ‘variable’ regions.
 In L chains the two regions are of equal length.
IMMUNOGLOBULINS
 Immunoglobulins are synthesised by plasma cells
and to some extent by lymphocytes.

 Ig constitute 20 to 25% of serum proteins.

 Igs provides a structural and chemical concept
whereas antibody is biological and functional
concept.
 Immunoglobulin Structure

Disulfide bond
 Heavy & Light
Chains
Carbohydrate
 Disulfide bonds
 Inter-chain CL
 Intra-chain VL

CH2 CH3
CH1
Hinge Region
VH
 IgM
J Chain
 Structure
 Pentamer (19S)
 Extra domain (CH4)
 J chain
Cµ4
IgA
 Structure
 Serum - monomer
 Secretions (sIgA)
 Dimer (11S)
 J chain
 Secretory component

Secretory Piece J Chain

IMMUNOGLOBULIN CLASSES
 Human sera contain
 IgG
 IgA
 IgM
 IgD
 IgE
in decreasing order of concentration.
IgG
 Major serum immunoglobulin (80%).
 Normal serum concentration – 8 to 16 mg/ml
 Molecular weight – 150,000 (7S).

 Distributed approx equally between intravascular

and extravascular compartments.
 Contains less carbohydrate than other Ig.
 Half life – approx 23days
IgG (contd….)
 Only maternal Ig to be transported across placenta.
 Provides natural passive immunity in the newborn.
 Not synthesized by the fetus in any significant
amount.

 Binds to microorganisms and enhances their

phagocytosis.
IgG (contd….)
 Participates in most immunological reactions such
as complement fixation, precipitation,
neutralization of toxins and viruses.
 Considered a general purpose antibody, protective
against infectious agents which are active in blood
and tissues.
 If passively administered suppresses homologous
antibody synthesis by a feedback process.
 This is utilised in isoimmunisation of women by
administration of anti-Rh(D) IgG during delivery.
IgG (contd….)
 It is a late antibody and appears after the initial
immune response by IgM.
 Four subclasses each possessing a distict type of
gamma chain are recognised –
 IgG1 (65%)
 IgG2 (23%)
 IgG3 (8%)
 IgG4 (4%)
IgA
 Second most abundant class – 10 to 13% of serum
Ig.
 Normal serum level – 0.6 to 4.2 mg/ml
 Half life – 6 to 8 days
 Major Ig in colostrum, saliva and tears.
 Occurs in two forms –
 Serum IgA
 Secretory IgA
IgA
 Structure
 Serum - monomer
 Secretions (sIgA)
 Dimer (11S)
 J chain
 Secretory component

Secretory Piece J Chain

Serum IgA :-
 Monomeric 7 S molecule

 Molecular weight – about 160,000

Secretory IgA (SIgA) :-

 Dimer (11 S) formed by two monomer units joined

together at their carboxyterminals by a glycopeptide

termed J chain.
 Found on mucosal surfaces and in secretions.

 Molecular weight – about 400,000

IMMUNOGLOBULINS.
 IgA –serum IgA is monomer.
 SIgA is dimer joined together at
carboxyterminal by glycopeptide called J chain.
SIgA is synthesised by plasma cells near mucosal or
glandular epithlium.Secretoty component is a glycine
rich polypeptide produced by glandular epithelium.
It protects IgA from denaturation by bacterial
protease in site like intestinal epithelium.SIgA forms
an antibody paste and prevent entry of organism into
body tissue.
 IgA does not fix complement but can activate
alternative complement pathway.
 Promotes phagocytosis and intracellular killing of
microorganisms.
 Two IgA subclasses –
 IgA₁ : lacks interchain disulphide bonds between
heavy and light chains
 IgA₂ : minor component of serum IgA but
dominant form in secretions
IgM
 Constitutes 5-8% of serum Ig.
 Normal level – 0.5 to 2 mg/ml
 Half life – about 5 days
 Heavy molecule (19 S); molecular weight –
900,000 to 1,000,000
 Also called ‘the millionaire molecule’.
 Polymers of five four-peptide subunits, each
bearing an extra CH domain.
 IgM
J Chain
 Structure
 Pentamer (19S)
 Extra domain (CH4)
 J chain
Cµ4
IgM (contd….)
 Polymerisation of the subunits depends on presence
of J chain.
 Valency of 10 observed only with small haptens.
 With larger antigens, effective valency falls to 5,
probably due to steric hindrance.
 80% of IgM is intravascular.
 Phylogentically oldest Ig class.
 Earliest Ig to be synthesized by the fetus (beginning
about 20 weeks of age).
IgM (contd….)
 Not transported across placenta presence of
IgM in fetus or newborn intrauterine infection.
 Its detection is useful in diagnosis of congenital
infections (syphilis, rubella, HIV infection,
toxoplasmosis).
 Relatively short lived, disappear earlier than IgG.
 Hence, demonstration in serum indicates recent
infection.
 Treatment of serum with 0.12 M 2-
mercaptoethanol selectively destroys IgM without
affecting IgG.
 Can be used for differential estimation of IgG and
IgM.
 Isohemagglutinins (anti-A, anti-B), other natural
antibodies to microorganisms, antibodies to typhoid
‘O’ antigen, reagin antibodies in syphilis are
usually IgM.
 Unique structural features of IgM appear suited to
the biological role of providing protection against
microoragnisms.
 Single molecule of IgM can bring about immune
hemolysis, whereas 1000 IgG molecules are
required for same effect.
 Also 500-1000 times more effective than IgG in
opsonisation, 100 times in bactericidal action and
20 times in bacterial agglutination.
 In neutralisation of toxins and viruses, it is less
active than IgG.
 Responsible for protection against blood invasion
by microorganisms as largely confined to
intravascular space.
 Deficiency is associated with septicemia.
 Monomeric IgM is the major antibody receptor on
the surface of B lymphocytes for antigen
recognition.
IgD
 Resembles IgG structurally.
 Present in concentration of 3mg/100ml of serum.
 Mostly intravascular.
 Half life – about 3 days.
 Occur on surface of unstimulated B lymphocytes
and serve as recognition receptors for antigens.
 Combination of cell membrane bound IgD/IgM with
corresponding antigen leads to specific stimulation
of B cell – either activation or suppresion.
IgE
 Discovered in1966.
 8 S molecule with molecular weight about 190,000.
 Half life – about 2 days
 Resembles IgG structurally.
 Exhibits unique properties –
 Heat lability (inactivated at 56⁰C in 1 hour)
 Affinity for the surface of tissue cells (mast cells)
of same species (homocytotropism).
IgE (contd….)
 Chiefly produced in the linings of respiratory and
intestinal tracts.
 IgE deficiency has been associated with IgA
deficiency in individuals with impaired immunity.
 Responsible for anaphylactic type of
hypersensitivity.
 Special role in defence against helminthic
infections.
 IgG protects the body fluids.
 IgA – body surfaces
 IgM – blood stream
 IgE – mediates reaginic hypersensitivity
 IgD – recognition molecule on the surface of B
lymphocytes.
ABNORMAL IMMUNOGLOBULINS

 Earliest description of abnormal Ig was discovery of

Bence Jones protein.
 It is typically found in multiple myeloma.
 Identified in urine by its characteristic property of
coagulation when heated to 50⁰C but redissolving at
70⁰C.
 They are light chains of Ig and may occur as kappa
or lambda forms.
 But in one patient, the chain is either kappa or
lambda only and never both.
IMMUNOGLOBULINS
 IgM ---first to appear,- -recent infection.
 IgG----- past infection.
 IgG protects the body fluids.
 IgM protects the blood stream.
 IgE mediates reaginic hypersensitivity.
 IgA—body surfaces.
 IgD recognition molecule on the surface of B
lymphocyte.
 IgG, IgD and IgE resembles structurally.
 THE-------------------------------
 END====.S
ANTIGEN COMBINES WITH ITS SPECIFIC ANTIBODY IN AN OBSERVABLE MANNER
AND THE REACTION IS SPECIFIC.
IN VITRO IT FORMS THE BASIS OF SEROLOGICAL TEST AND IN VIVO IT FORMS THE
BASIS OF IMMUNITY/ HYPERSENSITIVITY/AUTOIMMUNITY.

.
characteristics
 Antigen –antibody reaction is firm but reversible. It
depends upon affinity and avidity. Affinity is the
intensity of attraction between antigen and
antibody. Avidity is the strength of bond after
formation of ag-ab complex.
 Antigen and antibody can combine in varying
proportion. Antibody is usually bivalent.
 No denaturation of ag. Or ab.
Reaction occurs in two stages.
 Primary stage—initial interaction between antigen
and antibody is rapid but with no visible effect.
Week bond, no covalent bond . Reversible.
 Secondary stage—primary stage is followed by
secondary stage in most of the cases with visible
effect.
Ag-Ab.
 Incomplete or monovalent antibodies do not cause
agglutination. They may act as blocking antibodies
inhibiting agglutination by the complete antibodies
added subsequently.
 Agglutination is more sensitive than precipitation
for detection of antibodies.
 Precipitation is very sensitive in detection of
antigen.
 Titre is the highest dilution that gives positive test.
Types of antigen and antibody reactions.

 Precipitation.
 Agglutination.
 Complement fixation.
 Immunofluorescenes.
 ELISA.
Precipitation reaction
 When a soluble antigen combines with its antibody
in presence of electrolyte at a suitable temperature
and pH it forms an insoluble precipitate it is called
precipitation reaction.
 When it flocculates instead of sedimenting it is
called flocculation.
Mechanism of precipitation
 Marrack hypothesis of lattice formation is widely
accepted.
 According to this multivalent antigen combine
with bivalent antibody in different proportion,
precipitation occurs when a large lattice is formed
consisting of alternating antigen and antibody
molecules . This occurs in the zone of equivalence.
Application of precipitation
 Sensitive test for detection of antigen
 Identification of bacteria e.g.lancefield grouping of
Streptococcus
 V.D.R.L.in syphilis as slide flocculation,Kahn test
as tube flocculation
 Forensic application in identification of blood
seminal stain
 Testing for food adulterants
 To standerdize toxin and antitoxin
Types of precipitation reactions
 Ring test e.g. Ascoli’s thermo precipitation test.
 Flocculation tests.
 Immunodiffusion tests-this is type of precipitation
test done in agar gel .Advantage of test in agar gel
is--reacting band is visible can be stained ,the
number of different antigen can be observed and
identity, cross reaction etc can be observed.
Immunodiffusion tests
 Single diffusion in one dimension Oudin
procedure.
 Double diffusion in one dimension. Oakley-
Fulthorpe procedure
 Single diffusion in two dimensions. For estimation
of Ig. Class.
 Double diffusion in two dimensions. Ouchterlony
procedure e.g. Elek’s gel precipitation test.
Agglutination reactions
 When a particulate antigen combines with an
antibody in presence of electrolyte , at an optimal
temperature and pH in an observable manner it is
called agglutination reaction . It is more sensitive in
detection of antibody.
.
 Mechanism is same as lattice hypothesis.
Applications
 Slide agglutination.

 Tube agglutination.

 Passive agglutination test.

Slide agglutinations
 When a drop of antiserum is added to a smooth ,
uniform suspension of a particulate antigen in a drop
of saline and mixed with a loop or by gently rocking
the slide agglutination is indicated by clumping of
particle. A control with antigen and N/S should be put
in the same slide.
 Slide agglutination is routinely done for identification
of bacterial isolates from clinical samples.
 It is used for blood grouping and cross matching.
Tube agglutination
 This is a standard quantitative method for
measurement of antibody.
 Tube agglutination is routinely used in the
diagnosis of typhoid ,brucellosis,etc.
 Widal ,WeilFelix ,PaulBunnel.are some of its
examples
Passive agglutination
 By attaching soluble antigen to the surface of
carrier particle a precipitation test can be converted
to an agglutination.
 Carrier particle used are red cells ,latex particle,
bentonite.
 Example--- Rose-Waaler test done in rh arthritis