This topic covers the brief introduction of Ag and Ab in detail. Types and functions of Ig is explained in detail. Paraproteinemias is explained with simple pictures. by Dr. N.Sivaranjani, MD

1. Immunochemistry
Dr. N.Sivaranjani,M.D.
Assistant professor
Dept of Biochemistry

2. Immunology
• Study of immunity and immune systems of
vertebrates
• Immunis means exempt / free from burben
• Immunity involves the resistance shown and
protection offered by host organism against the
infectious disease.
• 3 salient features of immunological reaction :
Recognition of self
Specificity
Memory of response

3. Antigen (Ag)
Any substance which invokes an immunological response
is an antigen or immunogen.
Certain components of cell membranes act as specific
antigens.
Most are proteins or large polysaccharides from a
foreign organism.

4. Epitope:
Small part of an antigen that interacts with an antibody.
Antigenic determinant site
Any given antigen may have several epitopes.
Each epitope is recognized by a specific antibody.

5. Epitopes: Antigen Regions that Interact with
Antibodies

6. Antibodies (Ab)
Proteins that recognize and bind to a particular antigen with
very high specificity.
Produce in response to exposure to the antigen.
One virus or microbe may have several antigenic determinant
sites, to which different antibodies may bind.
Each antibody has at least two identical sites that bind
antigen: Antigen binding sites.
Belong to a group of serum proteins called immunoglobulins
(Igs).

8. Immunoglobulins

9. Immunoglobulins
• Specialized group of proteins mostly associated
with γ globulin fraction.
• Immunoglobulins is a functional term while γ
globulin is a physical term.
• Abbreviated as Ig
• Ig are produced by plasma cells in response to an
antigen and which function as antibodies.

10. Structure of immunoglobulins
All immunoglobulin molecules basically consist of :
two identical heavy chains and
two identical light chains
held together by disulfide linkages (Inter-chain and
Intra-chain ) and non-covalent interactions.
It is Y shaped tetramer (H2L2)
Each heavy chain contains 450 amino acids
Light chain has 212 amino acids
Heavy chain of Ig are linked to carbohydrates hence Ig are
glycoproteins.

11. Variable & Constant Regions
• Each chain of Ig has two regions – constant and variable
region.
• Light chain
Amino terminal half – is variable region (VL )
carboxy terminal half – is constant region (CL)
• Heavy chain
One quarter of amino terminal region – variable region
(VH)
Remaining three quarters – constant region
(CH1 , CH2 , CH3 )

12. • Amino acid sequence of variable regions of light and heavy
chains is responsible for specific binding of immunoglobulin
(antibody) with antigen.
• There are certain hypervariable regions interspersed
between the relatively invariable regions.
• Light chain has 3 hypervariable regions
• Heavy chain has 4.
• The hypervariable regions more specifically determine the
antigen-antibody binding.

13. Heavy chains
Light chain
Light chain
Antigen binding siteAntigen binding site
COOHCOOH
Hinge Region
Hinge Region
Carbohydrate
ss
ss
ss ssCL
CL
CH2
CH3 CH3
CH2
Structure of Ig
Fab
Fc

14.  Proteolytic cleavage of Ig :
 Papain enzyme split the Ig at the site
Between CH1 and CH2 region
(hinge region)into two fragments.
 Fab (fraction antibody)
– Ag binding
– Specificity determined by
VH and VL
 Fc (fraction crystallisable)
-- complement binding
Papain
Fc
Fab

15. Pepsin
Fc
F(ab)2
Another proteolytic
enzyme, pepsin cleaves
Ig at another site to
yield F(ab)2

16. Immunoglobulin Classes
• IgG - Gamma heavy chains
• IgM - Mu heavy chains
• IgA - Alpha heavy chains
• IgD - Delta heavy chains
• IgE - Epsilon heavy chains
• Two types of light chains : kappa () and lambda
(λ)
• An Ig contains two  or two λ light chain and
never a mixture.
• Kappa chain (60%) is more common in human.

17. Ig G
Major serum Ig – 75 -80 %
Single Y shaped unit (monomer)
It is the antibody seen in secondary immuno response.
It can transverse blood vessels readily
IgG is only Ig that can cross the placenta and transfer the
mothers immunity to developing fetus.

18. Rh iso immunisation :
• This occurs when mother is Rh negative and fetus is Rh
positive.
• During parturition, fetal RBCs may enter into maternal
circulation , leading to formation of anti-Rh antibodies.
During next pregnancy, these Ab being IgG in class, can
enter into fetal circulation causing fetal hemolysis,
neonatal jaundice and in severe cases, neonatal death or
miscarrige.
• Anti-Rh Ab injected within 24 hrs of delivery of first
child , will avert the isoimmunization and protect future
pregnancy.

19. Opsonization effect - IgG – Fab can bind with
microbes, and also the Fc portion can fix
with macrophage, so that phagocytosis is made
easier
Antibody dependent cell mediated cytolysis
(ADCC) - Antibodies serve as bridge b/w the
infected target cell and effector cell.
ADCC effector cell (Natural killer cells) secrete
lytic enzymes – lysis of infected target cell.

21. Ig M
J Chain
Largest Ig composed of 5 Y shaped units held together by
a J polypeptide chain.
Pentamer –bind with 5 antigenic sites
Due to its large size, IgM cannot transverse blood vesssels,
hence it is restricted to the blood stream.
IgM is first antibody to be produced
in response to an antigen and is most
effective against invading
micro-organism.

22. • Ig M are predominant class of Ab produced in primary
responseto an Ag.
• Natural Ab are IgM in nature.
• A person having blood group A Antigen will have anti B
antibodies in his circulation (isohemagglutinins). These are
produced without any known antigenic stimulation and hence
called natural antibodies.
• IgM Ab cannot cross placenta
• So if the fetus even though it carries an incompatible Ag , is
protected from natural Ab of the mother.

23. IgA
Single (monomer) or double unit (dimer) held together
by J chain
Mostly found in body secretions such as saliva, tears,
sweat, milk and the walls of intestine.
Most predominant Ab in colostrum.
Ig A molecules bind with bacterial Ag present on body
surface and remove them. So IgA prevents the foreign
substances from entering the body cells.

24. J ChainSecretory Piece
The dimer are stabilized against proteolytic enzymes by
secretory piece.
The secretory piece is produced in liver, reaches to the
intestinal mucosal cells, where it combines with Ig A dimer
to form the Secretory IgA which is then released.

26. IgE
Single Y shaped (Monomer)
Normally present in minute conc in blood – 0.3g/ml
IgE levels are elevated in individuals with allergies as
it is associated with the body’s allergic response –
Hay fever, Asthma, Anaphylactic shock.
IgE tightly binds with Fc receptors on basophils and mast
cells which release histamine and cause allergy.
Immediate type Hypersensitivity reaction – peak at 30
min

27. Allergen bound IgE on mast cells induces
degranulation

28. IgD
Single Y shaped unit (Monomer)
Present in low concentration in circulation.
Present on surface of B cells
Their function is not known

29. Immunoglobulin Major Functions
IgG
 Main antibody in the secondary response.
 Opsonizes bacteria, making them easier to
phagocytose.
 Fixes complement, which enhances
bacterial killing. Neutralizes bacterial
toxins and viruses.
 Crosses the placenta.
IgA  Secretory IgA prevents attachment of
bacteria and viruses to mucous membranes.
 Does not fix complement.

30. IgM  Produced in the primary response to an
antigen.
 Fixes complement.
 Does not cross the placenta.
IgD  Found on the surfaces of B cells where it
acts as a receptor for antigen.
IgE  Mediates immediate hypersensitivity
(allergy) by causing release of mediators
from mast cells and basophils upon exposure
to antigen (allergen).
 Does not fix complement.
 Main host defense against helminthic
infections.

31. Production of Ig by multiple genes :
• Ig are composed of light and heavy chain.
• Each light chain is produced by 3 separate genes, namely a
variable region gene , a constant region gene and a joining
region gene.
• For heavy chain – 4 separate gene
variable region gene , a constant region gene and a joining
region gene and diversity region gene.
• Thus multiple genes are responsible for synthesis of any one
of the Ig.

32. Antibody diversity :
• A person is capable of generating Ab to almost an
unlimited range of Ag .
• Humans do not contain millions of genes to separately
code for individual Ig molecules.
• The Ab diversity is achieved by two special process,
namely combination of various structural gene and
somatic mutations.

33. Paraproteinemias (Monoclonal Ig)
• Multiple myeloma
• Bence-jones proteinuria
• Heavy chain disease
• Waldenstrom’s Macroglobulinemia
• Amyloidosis
• Hypergamma globulinemia
• Hypogamma globulinemia

34. Multiple myeloma (plasmacytoma)
• Plasma cell cancer constitutes about 1% of all cancers
• Females are more susceptible than males
• Usually occurs in the age group 45-60 yrs
• Malignancy of a single clone of plasma cells in bone
marrow.
• This results in the overproduction of abnormal Ig mostly
(75%) Ig G and in some cases (25%) Ig A or Ig M.
• Ig D type MM - younger adults - less common (>2%) but
more severe.

35. C - Calcium is increased (Hypercalcemia)
R – Renal failure
A - Anemia
B – Lytic Bone lesion(bone pain & tenderness) -70%
• Spontaneous pathological fracture of weight
bearing bones, rib and vertebrae may occur.
• Paraproteinemia (monoclonal Ig) – leads to
Proteinuria
• Recurrent infections are common - normal Ig is
diminished
• Raised beta-2-microglobulin
• Bone marrow examination – malignant plasma cells
• X ray shows “punched-out osteolytic lesions”.

39. • Electrophoretic pattern
• Serum of MM patients shows a characteristic
pattern in electrophoresis.
• There is sharp and distinct band called M band with
narrow spike (M band or monoclonal band) between
β and  globulins.
• This M band almost replaces the  globulin band due
to the diminished synthesis of normal  globulins.

40. Normal pattern for serum
protein electrophoresis
Multiple myeloma

41. Normal
Multiple myeloma

42. Bence-jones proteinuria
• Henry bence jones first described them.
• 20% of patients with MM,BJP are excreted in
urine which often damages the renal tubules.
• Excess synthesis of light chain (κ or λ)
• BJP have a molecular weight of 20,000 or
40,000 (dimer)

43. Specific test to detect BJP:
• Electrophoresis – best to detect BJP in urine
• Classical heat test
BJP have the special property of precipitation when
urine is heated b/w 40-50˚C and redissolve on
further heating urine at higher than 80 ˚C.
precipitation reappear again on cooling the urine.
• Bradshaw’s test
if BJP are present, white ring of precipitate will
occur if urine is layered on few ml of conc HCL.

44. Heavy chain disease
• Some portion of H chains are deleted during synthesis
• They cannot join with L chains to normal Ig
• Defective heavy chain is excreted in urine
• Gamma chain disease – hepatosplenomegaly and
lympadenopathy
• Alpha chain disease – abdominal lymphoma and
malabsorption.

45. Waldenstrom’s macroglobinemia
• Malignant proliferation of IgM clones
• Males are mostly affected
• Since IgM is macromolecule, they form aggregate or
cryoprecipitate, serum viscosity is increased.
• Sia test :
• A drop of serum is allowed to fall into a
tube containing water.
• Normal serum will spread
• Hyperviscosity serum – form globular precipitate

46. Amyloidosis
• About 20 % patients with myeloma develop amyloidosis
• deposits of light chain fragments in tissue (liver, kidney,
intestine) of MM pateints.