structure of immunoglobulins

1. ANTIBODIES/
IMMUNOGLOBULINS
Dr V S Vatkar
Asso Prof
Microbiology department
D Y Patil Medical College , Kolhapur

2. INTRODUCTION
 Introduction of antigen (Ag) into an animal-
certain substance appears in the serum &
tissue fluids are called ANTIBODIES
 Glycoprotein molecule, that recognises a
particular epitope on an Ag , binds
specifically to it in some observable manner
(agglutination, precipitation, CFT etc)

3.  Sera having high Antibody (Ab) levels
following infection or immunization is
called IMMUNE SERA
 Secreted Ab circulate in blood & they
eliminate or neutralizes the Ag or their
effects like phagocytosis, Antibody
dependant cell mediated cytotoxicity
(ADCC), opsonisation etc

4. FRACTION OF IMMUNE SERA: DONE BY HALF LIFE
SATURATION WITH AMMONIUM SULPHATE
Separate serum protein
Soluble ALBUMIN Insoluble GLOBULINS
Water soluble
PEUDOGLOBULINS
Insoluble EUGLOBULINS
Most Ab were found to be EUGLOBULINS

5.  Tiselius (1937): separate serum protein into
α, β & gamma globulins based on their
electrophoretic mobility
 In 1938 he showed the Ab activity was
associated with gamma globulin factor,
known as IMMUNOGLOBULINS (Ig)
 In 1964 WHO endorsed the generic term
IMMUNOGLOBULINS

6. Ig : synthesised by PLASMA Cells
& to some extent by
LYMPHOCYTES.
They contribute 20-25% of total
serum
5 classes of Ig have been
recognised : IgG, IgM, IgA, IgE &
IgD

7. STRUCTURE OF IMMUNOGLOBULIN (IG)
PORTER, EDELMAN, NISONOFF & HIS COLLEAGUES PUT A DETAIL PICTURE OF
RABBIT’S IgG ANTIBODY TO EGG ALBUMIN
IgG Ab
Split into 2 fractions by
Papaine in presence of cysteine
Insoluble fraction which
crystallisable in cold
Soluble fraction unable to
Ppt with egg albumin still binds
With it , called Fab
Fc- crystallisable
fragment
Fab – Ag binding fragment

8. STRUCTURE OF IMMUNOGLOBULIN (IG)
 Each Ig molecule: 1 Fc & 2 Fab
 Fab portion: bivalent & ppt with Ag,
fragment called : F(ab)2
 Fc portion: designed into small
fragments
 Glycoprotein- each molecule consist of
two pairs of polypeptide chains of diff
size
 Smaller chains: light (L) chains

9.  Molecular weight: L chain- 25,000
H chain- 50,000
 L chain is attach to H chain by disulphide
bond
 L chain: similar in all classes of Ig
 Two types: (K)kappa 60% & (λ) lambda
40%
 Molecule either have K or λ chain (never
both)
 K & λ : named after Kongold & Lapari

10. H CHAIN : STRUCTURALLY & AGENICALLY DIFF FOR
EACH CLASS
IgG: γ (gamma) H chain
IgM : μ H chain
IgA : α H chain
IgD : δ H chain
IgE : ε H chain

12.  Ag combining site : at AMINO-TERMINUS (N
terminal)
 Composed of both L & H chains
 214 aa present in L chain & 107 aa in H
chain
 First 110 aa from N terminal: quite variable
(VARIABLE REGION)
 VL: variable region on L chain
 VH: variable region on H chain
 At this region Ag binds to Ab

13. Carboxy terminus (C terminal) :
relatively constant region
 CL : constant light chain
 CH : constant heavy chain
 This region : mediated the effector functions
 C region of light chain does not attach to cell
membrane & does not participate in its
effector functions

14. IMMUNOGLOBULIN DOMAIN
 L &H chains contain several homologous
units of about 110 aa residues.
 Within each unit intrachain disulphide bond
(-s-s-) forms a loop of 60 aa is called a
DOMAIN
 L chain contains: 1 VL & 1CL domain
 H chain contains : 1 VH & 3 -4 CH
domains depending on the class of Ig
(CH1, CH2, CH3 etc)

15. HYPERVARIABLE & FRAMEWORK REGION:
 variable region of L & H chain domain
shows maximum sequence variation of aa
at terminal end of amino terminus:
HYPERVARIABLE REGION (binding site of
Ab to Ag)
 Also called as COMPLEMENTARY
DERMINING REGION (CDR)
 Each Fab fragment has 6 CDR (3 on L
chain & 3 on H chain)

18. IMMUNOGLOBULIN CLASSES
Ig CLASS H CHAIN ½ LIFE SERUM
CONC
IgG
Intra & Extra
Vascular
Gamma (γ)
chain
23 days 80%
IgA
Extravascular
α chain 6-8 days 10-13%
IgM
Intravascular
μ chain 5 days 5-8%
IgD
Mostly
Intravascular
δ chain 3 days 3 mg/100ml
IgE
Mostly
Extravascular
ε chain 2 days Few nano
gm/ml

19. IgG
 Major serum Ig
 80 % of total serum
 Mol wt – 1,50,000
 Equal distribution in intravascular &
extravascular compartments
 Contains less –CHO than any other Igs
 Half life : approx. 23 days
 Levels increase in chronic infections like
malaria, kala azar or myeloma

20.  Normal serum conc: 8-16 mg/ml
 Only maternal Ab transported across the
placenta & provides natural passive
immunity to new born, not synthesised by
fetus insignificant amount
 Participate in most of the immunological
reactions like CFT, Precipitation, neutralization
of toxins & viruses
 Passive administration of IgG Ab suppresses
the homologous Ab synthesis (mainly used
when mother : Rh –ve & baby Rh +ve, anti D
IgG : given during delivery

21.  Four classes of
IgG: IgG1, IgG2,
IgG3 & IgG4 due to
presence of γ1, γ2,
γ3 & γ4
 Distribution in
human serum
 IgG1: 65%
 IgG2: 23%
 IgG3: 8%
 IgG4: 4%

22. IgA
 Second most abundant class
 10-13% of serum Ig
 Normal serum level: 0.6-4.2 mg/ml
 Half life: 6-8 days
 Found on mucosal surfaces & in secretions
 Major Ig in COLOSTRUM, SALIVA & TEARS
 Two forms: serum IgA & Secretory IgA

23.  Serum IgA:
monomer
 Mol wt: 1,60,000
 Secretory IgA: two
monomer joined with J
chain,
 Mol wt : 4,00,000
 Synthesised by
PLASMA Cells
situated near mucosal
or glandular
epithelium
 Glycine rich
polypeptide
 Secretory IgA: binds
to receptors on the
surface epithelium &
forms the Ab paste
on mucosal surfaces
 Relatively resistant
to digestive enzymes
& reducing agents

24. FUNCTION OF IgA:
 Inhibit the adherence of micro-organisms on
the mucosal surface & preventing their entry to
body tissue
 Provides imp defence mechanism against :
salmonella, vibrio & viruses like polio, influenza
etc
 Promotes phagocytosis & intracellular
killing of micro-organisms
 Breast milk: rich in IgA, helps to protect new
borne inf during first month of life

25.  Two classes:
 IgA1 & IgA2

26. IgM
 Constitute : 5-6% of serum
 Normal level: 0.5-2 mg/ml
 Half life: 5 days
 Short lived: disappears earlier than IgG
 80% intravascular, protects blood invasion by
micro-organisms
 Mol wt: 9,00,000 (heavy molecule), also called
as millionaire mol.
 Polymer of 5 subunits, joined with each other by
J chain

27.  Earliest Ig synthesised by FETUS, about
to 20 wks of age
 NOT TRANSPORTED ACROSS
PLACENTA
 Presence of IgM in fetus or in new born
indicates resent inf or intrauerine inf like
toxoplasma, syphilis, CMV, rubella, HIV etc
 Deficiency : always associated with
septicemia
 Many natural Ab to micro-organisms are
usually IgM, typhoid O Ag & regain Ab in

28.  A single mol of IgM
can bring about
immune hemolysia
where as 1000 IgG
mol are required for
the same effect
 500-1000 times
more effective than
IgG in opsonisation
& 20 times in
bacterial

29. IgD
 Structurally resembles with IgG
 Serum conc: about 3 mg/100ml
 Mostly intravascular
 Half life: 3 dys
 Serves as recognising receptor for Ag
 Combination of cell memb bound IgD or IgM
with corresponding Ag leads to stimulation
of B cells activation or cloning to produce Ab

30. IgE
 Half life: about 2 days
 Mol wt : 1,90,000
 Resembles with IgG structurally
 Heat labile (inactivated at 56 0C 1 hr)
 Serum levels: few nano grams/ml
 Affinity towards surface of tissue cells mainly
MAST Cells
 Mostly extra vascular

31.  Elevated levels: allergy: atopy (type I
Hypersensitivity) conditions such as asthma,
hay fever , eczema, intestinal parasitic inf etc
 Produced by linings of URT & Intestinal tract
 IgE deficiency: always associated
with IgA deficiency
 Protects by degranulation of mast
cells & releases inflammatory
response

32. IgG – protects BODY FLUIDS
IgA – protects BODY SURFACE
IgM – protects BLOOD STREAM
IgE – mediates reaginic
hypersensitivity
IgD – recognition mol on the
surface of B lymphocytes

33. ABNORMAL IMMUNOGLOBULIN
 Discoved by Bence Jones (1847):
 Protein found in MULTIPLE MYELOMA:
identified in urine: property of coagulation
when heated at 50 0 C but redissolves at 70
0 C
 Plasma cell disorder: unchecked
proliferation of one clone of plasma cell,
resulting extensive production of particular Ig
synthesised by clone cell (called as
MONOCLONAL Ab)

34.  Affect plasma cells synthesising IgG, IgA,
IgE & IgD
 IgM producing cells: Waldenstrom’s
macroglobulinemia
 H chain disorder: lymphoid leukemia,
overproduction of Fc part
 Cryoglobulinemia: gel / ppt formed on
cooling the serum & redissolves on warming

35.  Not always associated with ds, but often
found in myeloma, macroglobulinemias &
autoimmne conditions such as SLE
 Most cryoglobulins contain IgG, IgM & their
mix ppt

36. IMMUNOGLOBULIN SPECIFICITIES
 Isotype: genetic variations or diff in the
constant region of H chain Ig classes &
subclasses in the species
 Allotypes: multiple alleles that exist for
some of the genes
 Idiotypes: specific Agenic determinants on
the H chain & L chain variable regions
(paratopes)