This document provides information about antibodies (immunoglobulins). It discusses the structure of antibodies, including the variable and constant regions of the heavy and light chains. The five classes of antibodies (IgG, IgA, IgM, IgD, IgE) are described along with their functions. IgG is the most abundant antibody and can activate complement and mediate phagocytosis. IgA is found in secretions and provides mucosal immunity. IgM is the first antibody produced during infection and is a potent complement activator. Abnormal immunoglobulins produced in diseases are also mentioned.

1. Dr. Meenakshi Sharma
Assistant Professor
Department of Microbiology
Mayo Institute Of Medical Sciences
Antibodies - Immunoglobulins

2. Index
 Introduction
 Structure of Antibody
 Functions of Immunoglobulins
 Immunoglobulin classes
 Antigenic determinants of
Immunoglobulins
 Abnormal Immunoglobulins

3. Introduction
 Antibodies are
 Glycoprotein molecules that recognise a particular
epitope on an antigen
 Bind specifically to it and facilitates its clearance.
 Present on B cell membrane and secreted by
plasma cells.
 Secreted antibodies circulate in blood, where they
eliminate/neutralise the antigen
 Sera having high antibody levels following
infection or immunisation –IMMUNE SERA

4.  Fractionation of immune sera separates the
serum proteins by half saturation with ammonium
sulphate separates the serum proteins into:-
 Soluble albumin
 Insoluble globulins
 Globulins can be separated into water soluble
Pseudoglobulins and water insoluble Euglobulins.
 Most antibodies have been found to be
euglobulins

5. Tiselius (1937), swedish biochemist

6.  Most antibodies were found in gamma globulin
frations, hence named immunoglobulins (Ig).

7.  In 1964, WHO endorsed the term
‘immunoglobulin’ which was internationally
accepted .
 The definition includes besides antibody
globulins, the abnormal protiens found in
myeloma, macroglobulinemia, cryoglobulinemia
and the naturally occuring subunits of
immunoglobulins.
 All antibodies are immunoglobulins but all
immunoglobulins may not be antibodies.

8.  Immunoglobulin (Ig) constitute 20-25% of total
serum proteins.
 Based on physiochemical and antigenic
differences 5 classes: IgG, IgA, IgM, IgD and IgE

9. Structure of Antibody
 Y shaped heterodimer,
composed of 4
polypeptide chains
2 identical light (L)chains, of
molecular weight 25000 Da each
and
2 identical heavy (H) chains
each having molecular weight
50000 Da or more

10.  All 4 H and L chains are bound to each other by
disulfide bonds and noncovalent interactions such
as salt linkages, hydrogen bonds and
hydrophobic bonds All chains have 2 ends
 an amino terminal end (NH3) and
 a carboxy terminal end (COOH)
 There are 5 classes of H chains
and 2 classes of L chains

11. H chains
 5 classes
 Structurally and antigenically distinct
 Each designated by Greek letter corresponding
to immunoglobulin class
 5 classes of Ig (IgG, IgA, IgM, IgD and IgE)
classified based on AA sequence of heavy chains

12. L chains
 2 types
 Kappa (κ) and lambda (λ) named after Korngold
and Lapari
 In humans, L chains, 60% kappa and 40%
lambda
 Both light chains of Ab molecule should be same
type, either κ or λ, never both

13. Type of heavy chain in each Ig class
Immunoglobulin class Heavy chain type
IgG γ (gamma)
IgA α (alpha)
IgM μ (mu)
IgD δ (delta)
IgE ε (epsilon)

14. Variable and constant regions
 Each H and L chain – comprises of 2 regions
Variable region
Constant region
 Depending upon whether AA sequences of the
regions show variable or uniform pattern among
different antibodies

15. Variable region
 The 1st 110 AA residues near amino terminal end
(NH3) end of both L and H chains constitute
variable region – V L and VH respectively.
 Hypervariable region: maximum sequence
variation is concentrated in a few discrete regions
called hypervariable regions
 Less variable stretches are termed as framework
regions

16.  HV regions form the antigen binding site which
are complementary to the structure of the epitope
and are called complementarity determinig
regions (CDR’s)
 Each Fab fragment has six CDR’s (three in H and
3 in L)
 The framework region acts as a scaffold support
to six CDR loops.
 Paratope: site on hypervariable regions that make
actual contact with epitope

17. Constant region
 The sequence of aminoacids beyond the variable
region is realtively constant throughout the
antibody molecule and is caleed the C constant
region .
 Length = 104 AA for L chain, 330 AA for γ, α, and
δ heavy chains and 440 AA for μ and ε heavy
chains.
 Carbohydrate moieties are linked to constant
region of H chains
 Carboxy terminal constant region of heavy chains
mediates the effector functions.
 The C region of light chains does not attach to the
cell membrane and does not participate in its
effector functions.

18. Immunoglobulin domains
 H and L chain are further
folded into
domains
Within a domain, a loop like
structure of 60 AA is present
which is formed due to an
intrachain disulfide bond
Light chain – contains one
variable (VL) and one constant
domain (CL)
Heavy chain – one variable
(VH ) and 3-4 constant domains
(CH)

19. Hinge region
In heavy chain (γ,α,δ), the
junction formed between CH1
and CH2 domain = Hinge region
Rich in proline and cysteine
Very flexible, allows Ig
molecule to assume different
positions, helps Ab reach
towards Ag
Sensitive to various enzymatic
digestions

20. Enzymatic digestion
 Enzymatic digestion – generates various
fragments

21.  In the presence of cysteine, Cleave Ig above
disulfide bridge of hinge region into two fractions :
 Insoluble fraction which crystallises in cold called Fc for
crystallisable
 Soluble fraction which while unable to precipitae can still
bind the antigen called Fab (antigen binding)
 Results in 3 fragments each
 Two Fab fragments
 One Fc fragment
Papain digestion

22. Pepsin digestion
Cleaves Ig molecule at point below
disulfide bridge of hinge region
One F(ab’)2 fragment; 2 Fab subunits
bound together
Many smaller fragments

23. Functions of Immunoglobulins
 Antigen binding (by Fab region)
Primary function of an antibody; protects host
Fab fragment – bears variable region; involved in interaction
with Ag
Valency of Ab = No. of Fab region it possesses.
 Effector functions (by Fc region)
 Variety of secondary “effector functions” are produced
like
Fixation of complement
Binding to various cell types

24. Immunoglobulin classes
 Based on 5 types of heavy chains = 5 classes of
Ig
 IgG, IgA, IgM, IgD and IgE

25. IgG
 70-80% of total Ig in body
 Maximum daily production, longest half life of 23
days
 Highest serum concentration
 Has 4 subclasses – IgG1, IgG2, IgG3 and IgG4
 Subclasses vary in biological function, length of
hinge region and No. of disulfide bridges

26. Functions
 Can cross placenta: provide immunity to fetus and
newborn
 Complement fixing: Fc region can bind to
complement factors, activate classical pathway of
complement system
 Phagocytosis: IgG1 and IgG3 bind to Fc receptors
on phagocytes with high affinity and enhances the
phagocytosis of antigen bound to it
 Mediate precipitation and neutralisation reaction
 Plays major role in neutralisation of toxins as it can
easily diffuse into extravascular space
 Is raised after long time following infection and
represents chronic or past infection

27. IgA
 Second most abundant
 Constitutes 10-15% of total serum Ig
 Half life of 6-8 days
 2 subclasses: IgA1 and IgA2
 Occurs in two forms :Serum IgA and Secretory
IgA
 Serum IgA : monomeric 7 S molecule, Interacts
with Fc receptors expressed on immune effector
cells, to initiate various functions like ADCC,
degranulation of immune cells, etc

28.  Secretory IgA
 Dimeric in nature, larger molecule than serum IgA
 2 IgA monomeric units joined by J chain
 Also secretory component present,belived to
protect IgA from denaturation by bacterial
proteases
 Major Ig in colostrum, saliva & tears

29. Functions
 Secretory IgA provides an important defence
mechanism against organisms like salmonella,
vibrio cholera and viruses like polio, influenza etc.
 Breast milk rich in IgA – protects the newborn
against infection during first month of life.
 IgA does not fix complement but can activate the
alternative complement pathway.
 Promotes phagocytosis and intracellular killing of
microorganisms.

30. IgM

31. IgM
 Highest MW (Millionaire molecule)
 Present only in intravascular compartment
 Not in body fluids or secretions
 Exists in monomeric and pentameric forms (10
Fab regions and 10 valencies)

32. Functions
 Acute infection: 1stAb to be produced following
infection.
 Represents acute or recent infection. Also called
primary immune response Ab
 Complement fixing: most potent activator of
classical complement pathway. Multiple
complement binding sites (5 Fc regions)
 Present on B cell surface. Serves as B cell
receptor for Ag binding
 Acts as Opsonin: binds as antigen which is then
easily recognised and removed

33. Functions
 Protection against intravascular organisms
 Mediate agglutination: 20 times more effective in
bacterial agglutination than IgG
 Fetal immunity: 1stAb to be synthesised in fetal
life. Indicator of IU infection

34. IgE
 Lowest serum concentration, shortest half life and
minimum daily production
 Affinity for surface of tissue cells (mast cells)
 Functions
Potent mediator of Type 1 hypersensitivity
Elevated in helminthic infection

35. IgD
 Found as membrane Ig on surface of B cells
 Acts as B cell receptor along with IgM

37. Antigenic determinants of
Immunoglobulins
 Since Ab are glycoproteins, they can themselves
function as potent immunogens
 Can induce Ab response in hosts other than
parent host
 Not entire Ig molecule is immunogenic, contains
antigenic determinants at specific sites
 Based on location of Ag determinants: Ig
molecules divided into isotype, idiotype and
allotype

40. Abnormal Immunoglobulins
 Bence Jones proteins
 Produced in neoplastic condition of plasma cells
called Multiple Myeloma
 Also called light chain disease
 Cancerous plasma cells produce excess light chain
(BJP) accumulated in pt serum and urine
 Waldenstrom’s Macroglobulinemia :B cell
lymphoma, produce excess IgM
 Heavy chain disease
 Cryoglobulinemia