Lecture 4 Antibodies.ppt..........................

ANTIBODIES

1. Effector Mechanisms of Effector Mechanisms of
Humoral ImmunityHumoral Immunity

2. ANTIBODIESANTIBODIES

Antibodies are effectors of humoral immunity.Antibodies are effectors of humoral immunity.

They are mainly produced by plasma cells in They are mainly produced by plasma cells in
response to the presence of specific antigens.response to the presence of specific antigens.
Plasma cells are rich in rough endoplasmic Plasma cells are rich in rough endoplasmic
reticulum and mitochondria which confer their reticulum and mitochondria which confer their
ability to secrete soluble proteins (antibodies)ability to secrete soluble proteins (antibodies)

Antibodies are found in serum(blood), lymph and Antibodies are found in serum(blood), lymph and
other body fluids.other body fluids.

Participates in ag/ab interactions with resultant Participates in ag/ab interactions with resultant
formation of ag/ab complex.formation of ag/ab complex.

ag +ab ag/ab complex.ag +ab ag/ab complex.

3. ANTIBODIESANTIBODIES

An antibody is a specialized protein substance An antibody is a specialized protein substance (glycoprotein –(glycoprotein –
glycosylated protein) glycosylated protein) produced by the host cells in produced by the host cells in response to response to
an antigen an antigen in the host's tissues. in the host's tissues.

Antibodies are capable of Antibodies are capable of reacting specifically with the antigen reacting specifically with the antigen
that that provoked their production. provoked their production.

When the antibody molecule reacts with the antigen, When the antibody molecule reacts with the antigen, the two the two
surfaces fit together like the pieces of a jigsaw puzzle-an surfaces fit together like the pieces of a jigsaw puzzle-an
interaction similar to a lock and key. interaction similar to a lock and key.

This This “recognition” is exquisitely accurate “recognition” is exquisitely accurate and accounts for the and accounts for the
extreme specificity extreme specificity of antibody molecules. of antibody molecules.

It serves as protective agent against organisms. It serves as protective agent against organisms.

4. Immunoglobulins vs antibodiesImmunoglobulins vs antibodies

Antibodies are Antibodies are often referred to as often referred to as immunoglobulins immunoglobulins (Ig)(Ig)..

IgIg make make 20 to 25 % 20 to 25 % of the of the total serum proteinstotal serum proteins. .

The term The term Ig is the Ig is the structural structural and and chemical chemical conceptconcept while while

Antibody is Antibody is biologicalbiological andand functionalfunctional concept. concept.

AllAll abab are are IgIg but but allall Ig Ig (SOME) (SOME) may may not be not be antibodies. antibodies.

Now the term immunoglobulin is used to describe these Now the term immunoglobulin is used to describe these
closely related proteins. closely related proteins.

CLASSES OF IMMUNOGLOBULINS/ANTIBODIESCLASSES OF IMMUNOGLOBULINS/ANTIBODIES

Based on their Based on their sizesize, , carbohydrate content carbohydrate content and and amino acid amino acid
analysisanalysis (chemical composition), (chemical composition), five groups five groups of of
immunoglobulins have been distinguished: immunoglobulins have been distinguished:

IgG, IgA, 1gM, IgD and IgEIgG, IgA, 1gM, IgD and IgE..

5. Characteristics of AntibodiesCharacteristics of Antibodies

ProteinProtein in nature. in nature.

Formed in response to Formed in response to antigenic stimulationantigenic stimulation. .

React with corresponding React with corresponding antigen in a specific and antigen in a specific and
observable manner. observable manner.

6. STRUCTURE OF IMMUNOGLOBULINSTRUCTURE OF IMMUNOGLOBULIN

7. STRUCTURE OF IMMUNOGLOBULINSTRUCTURE OF IMMUNOGLOBULIN

IgsIgs are are glycoproteinsglycoproteins and they are and they are Y- Shaped Y- Shaped molecules.molecules.

A typical molecule is a four polypeptide molecule A typical molecule is a four polypeptide molecule
consisting of :consisting of :

The smaller chains are called The smaller chains are called lightlight (L) chain and larger ones (L) chain and larger ones
heavy heavy (H chains). (H chains).

Two heavy chains Two heavy chains and and two identical light chains two identical light chains held held
together by together by interchain interchain disulphide bonds/linkagesdisulphide bonds/linkages (S—S).(S—S).

Each Heavy Each Heavy (H) (H) chain contains approx chain contains approx ~ 450 amino acids ~ 450 amino acids
and the light chains and the light chains (L) (L) each each 212 a.a 212 a.a approx ~.approx ~.

L chain L chain has molecular weight has molecular weight 25,00025,000 and and H chain 50,000H chain 50,000. .
The two The two H chains H chains are joined together by are joined together by 1-5 S—S bonds 1-5 S—S bonds
(intra chain linkages for stability) (intra chain linkages for stability) depending upon class of depending upon class of
immunoglobulins immunoglobulins

9. STRUCTURE OF IMMUNOGLOBULINSTRUCTURE OF IMMUNOGLOBULIN

Digestion of an immunoglobulin by Digestion of an immunoglobulin by papainpapain and and
pepsinpepsin has aided in the study of their structure.has aided in the study of their structure.

History: History: Porter, Edelman and Nisonoff (1959-64) Porter, Edelman and Nisonoff (1959-64)
developed technique for fragmentation of developed technique for fragmentation of
immunoglobulin molecule byimmunoglobulin molecule by papain papain digestion digestion
which has led to the which has led to the discovery of 5 main classes of discovery of 5 main classes of
immunoglobulin. immunoglobulin.

Porter and colleaguet Porter and colleaguet split rabbit IgG split rabbit IgG molecule by molecule by
papain digestion papain digestion in presence of cystein in presence of cystein into into two two
identical fabidentical fab (Fragment antigen binding; (Fragment antigen binding;
fragments and one Fc (Fragment crystallizable) fragments and one Fc (Fragment crystallizable)
fragment.fragment.

11. 
Digestion With Papain YieldsDigestion With Papain Yields

3 Fragments3 Fragments

2 identical Fab and 1 Fc2 identical Fab and 1 Fc

Fab Because Fragment That is Antigen BindingFab Because Fragment That is Antigen Binding

Fc Because Found To Crystallize In Cold StorageFc Because Found To Crystallize In Cold Storage

Pepsin DigestionPepsin Digestion

F(abF(ab`)2 `)2

No Fc Recovery, Digested EntirelyNo Fc Recovery, Digested Entirely

Mercaptoethanol Reduction (Eliminates Disulfide Mercaptoethanol Reduction (Eliminates Disulfide
Bonds) And Alkylation ShowedBonds) And Alkylation Showed
Enzymatic Digestion Of AntibodiesEnzymatic Digestion Of Antibodies

12. STRUCTURE OF IMMUNOGLOBULINSTRUCTURE OF IMMUNOGLOBULIN

Papain act on the exposed hinge region of the Papain act on the exposed hinge region of the
molecule. molecule.

Fab fragments: Fab fragments: Each fragment contains Each fragment contains a single a single
antigen binding siteantigen binding site. . Fc fragment: is identical in all Fc fragment: is identical in all
IgG molecules.IgG molecules.

Pepsin Pepsin unlikeunlike papain papain strikesstrikes at a at a different point of different point of
the Ig molecule and the Ig molecule and cleaves the Fc cleaves the Fc from the from the
remainders of the molecule leaving remainders of the molecule leaving a large SS a large SS
fragment fragment composed of essentially composed of essentially 2 Fab fragments.2 Fab fragments.

It is It is divalentdivalent and still and still precipitates with antigenprecipitates with antigen..

This fragment is formulated as This fragment is formulated as (Fab)2.(Fab)2.

13. Structural function of ImmunoglobulinStructural function of Immunoglobulin..
Heavy chainsHeavy chains

Each heavy chain has two regions, Each heavy chain has two regions, the the constant constant
regionregion and the and the variable regionvariable region. .

The constant region is identical in all antibodies of The constant region is identical in all antibodies of
the the samesame isotype, isotype, but differs in antibodies of but differs in antibodies of
different isotypes.different isotypes.

The H chains are The H chains are structurally and antigenetically structurally and antigenetically
distinct for each class and designated by Greek distinct for each class and designated by Greek
letter: letter:

IgG (Gamma= IgG (Gamma= γ), IgA (Alpha= ), IgA (Alpha= α), 1gM (Mu= ), 1gM (Mu= µ), ),
IgD (Delta= IgD (Delta= δ) and IgE (Epsilon/Eta= ) and IgE (Epsilon/Eta= ε).).

14. Structural function of Immunoglobulin.Structural function of Immunoglobulin.

Light chainsLight chains

The L chains The L chains are similar in all classes are similar in all classes of immunoglobulins. of immunoglobulins.

They may They may be either kappa or lambda be either kappa or lambda in a molecule of in a molecule of
immunoglobulin. immunoglobulin.

A light chain has A light chain has two successive domainstwo successive domains: : one constant domain one constant domain
and one variable domain. and one variable domain.

The approximate length of a light chain is The approximate length of a light chain is 211 to 217 211 to 217 amino acids. amino acids.

Each antibody contains Each antibody contains two light chains two light chains that are always that are always identical; identical;
only one type of light chain, only one type of light chain, κ=Kappa or λ= Lambda, κ=Kappa or λ= Lambda, is present per is present per
antibody in mammals. antibody in mammals.

Of Of 214 amino acid 214 amino acid residues that make up L chains, residues that make up L chains, 107 constitute 107 constitute
carboxyterminal (constant region).carboxyterminal (constant region).

15. Variable regionsVariable regions
VariabilityVariability is found towards the is found towards the NH
3 end to combat the end to combat the agag..

The The constant dormain constant dormain is found towards the is found towards the COOHCOOH end. end.

The The variable region variable region of the heavy chain and light chains differs of the heavy chain and light chains differs
in in antibodiesantibodies produced by produced by different B cellsdifferent B cells, , but is the same for but is the same for
all all absabs produced by a single B cell or produced by a single B cell or B cell clone.B cell clone.

This This variabilityvariability determines immunological determines immunological specificityspecificity of of
antibody molecule. antibody molecule.

Within the Within the variable domain variable domain the variability is the variability is not uniformlynot uniformly
distributed.distributed.

It has 4 regions within the heavy chain and 3 regions within It has 4 regions within the heavy chain and 3 regions within
the light chains.the light chains.

These regions are reffered to as These regions are reffered to as hypervariable regions hypervariable regions
(CDRs).(CDRs).

16. Variable regionsVariable regions

It is the It is the special folding special folding the 3 dimensionals of the the 3 dimensionals of the
hypervariable regions that determines the nature of antigen hypervariable regions that determines the nature of antigen
binding site binding site (Paratype) (Paratype) of the Ig molecule. of the Ig molecule.

3 D fold of VL and VH domains determine the binding 3 D fold of VL and VH domains determine the binding
specificity specificity

Single monomer has Single monomer has 2 antigen binding sites2 antigen binding sites..

NB; It is the amino acid sequence of variable region of NB; It is the amino acid sequence of variable region of
the light and heavy chain that determines the the light and heavy chain that determines the
immunological specificity of antibodies.immunological specificity of antibodies.

i.e. i.e. The infinite range of antibody specificity of The infinite range of antibody specificity of
antibodies antibodies depends on the variability of amino- acid depends on the variability of amino- acid
sequences at variable region of H chain and L chains which sequences at variable region of H chain and L chains which
form. form.

17. Fab fragments:Fab fragments:

Fab fragmentsFab fragments is an is an amino-terminal,half of heavy amino-terminal,half of heavy
chain and chain and one light chainone light chain..

It does not crystallize.It does not crystallize.

It does not contain carbohydrate and is the It does not contain carbohydrate and is the fragment fragment
of antigen binding. of antigen binding.

It is composed of one constant and one variable It is composed of one constant and one variable
domain from each heavy and light chain of the domain from each heavy and light chain of the
antibody. antibody.

18. Fc regions (Fragment crystallizable)Fc regions (Fragment crystallizable)

It is composed of It is composed of carboxy terminal portion of H chain. carboxy terminal portion of H chain.

The The FcFc is composed of is composed of two heavy chains two heavy chains that contribute that contribute
two or three constant domains two or three constant domains depending on the class of depending on the class of
the antibody. the antibody.

It determines It determines biological properties and functions of biological properties and functions of
immunoglobulins molecules immunoglobulins molecules like like complement fixationcomplement fixation, ,
placental transfer, skin fixation, attachment to placental transfer, skin fixation, attachment to
phagocytic cells, degranulation of mast cells etc.phagocytic cells, degranulation of mast cells etc.

It is It is crystallizablecrystallizable and and contains carbohydrate. contains carbohydrate.

The Fc region also binds to various cell The Fc region also binds to various cell receptors receptors such as such as
Fc receptors Fc receptors and and complement proteins- complement proteins- ByBy doing this, it doing this, it
mediates different mediates different physiologicalphysiological effects including effects including
opsonization , opsonization , cell lysis ,degranulationcell lysis ,degranulation of of mast cells mast cells etc.etc.

20. Hinge regionHinge region

The The namename is derived from the fact that is derived from the fact that studies studies by a by a
variety of variety of techniques, including fluorescence techniques, including fluorescence
polarization polarization have shown that have shown that the Fab fragments can the Fab fragments can
rotate and waggle, coming together or moving apartrotate and waggle, coming together or moving apart. .

As a consequenceAs a consequence, IgG molecules can change their , IgG molecules can change their
shape from a “Y” to a “Tshape from a “Y” to a “T” and vice versa, ” and vice versa, using the using the
region intercalated between Cg1 and Cg2 as a region intercalated between Cg1 and Cg2 as a
“hinge.”“hinge.”

Fd Fragment:Fd Fragment: The portion of H chain present in Fab The portion of H chain present in Fab
fragment is called Fd piece. fragment is called Fd piece. Its function is not known. Its function is not known.

21. Effector functions of antibodiesEffector functions of antibodies

23. NEUTRALIZATION OF MICROBES AND MICROBIAL NEUTRALIZATION OF MICROBES AND MICROBIAL
TOXINSTOXINS

24. CLASS SWITCHING

Prior to class switching, B-cells express:
IgM and IgD as surface proteins but;

Differential splicing allows co-expression of
IgM and IgD with identical V regions on a
single cell.

IgD is lost upon antigen stimulation so
that memory B-cells lack this class of
immunoglobulin

25. Leukocyte FcLeukocyte Fcγγ Receptors Receptors

26. Role of Fcγ Receptors in Phagocytosis andRole of Fcγ Receptors in Phagocytosis and
Activation of PhagocytesActivation of Phagocytes

27. Antibody-Dependent Cell-Mediated CytotoxicityAntibody-Dependent Cell-Mediated Cytotoxicity

28. Antibody-Mediated Clearance of HelminthsAntibody-Mediated Clearance of Helminths

Antibodies; mast cells, Antibodies; mast cells, and and eosinophilseosinophils function with function with
antibodies to mediate the expulsion and killing of some antibodies to mediate the expulsion and killing of some
helminthic parasiteshelminthic parasites

IgE, IgG, and IgA antibodies that coat helminths can IgE, IgG, and IgA antibodies that coat helminths can
bind to Fc receptors on bind to Fc receptors on eosinophilseosinophils and cause the and cause the
degranulation of these cells, degranulation of these cells, releasing the basic protein releasing the basic protein
and other eosinophil granule contents that kill the and other eosinophil granule contents that kill the
parasitesparasites

The high affinity Fcε receptor of eosinophils (The high affinity Fcε receptor of eosinophils (FcεRI) FcεRI)
lacks the signaling β chain and can only signal lacks the signaling β chain and can only signal
relatively weakly through the associated γ chainrelatively weakly through the associated γ chain

29. Inhibitory Signaling by the FcγRIIB ReceptorInhibitory Signaling by the FcγRIIB Receptor

Immune complex–mediated cross-linking of the Immune complex–mediated cross-linking of the
inhibitory FcγRIIB leads to tyrosine phosphorylation of inhibitory FcγRIIB leads to tyrosine phosphorylation of
the the ITIMITIM in the cytoplasmic tail in the cytoplasmic tail

One somewhat empirical but often useful treatment of One somewhat empirical but often useful treatment of
many many autoimmune diseases autoimmune diseases is the intravenous is the intravenous
administration of pooled human IgG administration of pooled human IgG (IVIG). (IVIG). IVIG may IVIG may
engage engage FcγRIIBFcγRIIB to to deliverdeliver inhibitory signals to B inhibitory signals to B
lymphocytes lymphocytes and other cells, thus and other cells, thus reducingreducing antibody antibody
production and production and dampening dampening inflammationinflammation

30. 
Sequencing Of Several Immunoglobulins RevealedSequencing Of Several Immunoglobulins Revealed

100-110 Amino Terminus, Highly Variable (V)100-110 Amino Terminus, Highly Variable (V)

Five Basic Sequence PatternsFive Basic Sequence Patterns

,,, , , , , , 

IgA, IgG, IgD, IgE and IgMIgA, IgG, IgD, IgE and IgM

The Above Classes Are Called IsotypeThe Above Classes Are Called Isotype

Each class can have either Each class can have either  or or  light chains light chains

Minor Differences Led To Sub-classes For IgA and IgGMinor Differences Led To Sub-classes For IgA and IgG

IgA1, IgGA2 and IgG1, IgG2, IgG3, IgG4 IgA1, IgGA2 and IgG1, IgG2, IgG3, IgG4
Sequencing Of Heavy ChainsSequencing Of Heavy Chains

31. Five classes of Antibodies:Five classes of Antibodies:

1. IgG-1. IgG- γ Contains Gamma heavy chain Contains Gamma heavy chain
PropertiesProperties

Four sub classes of IgG have been recognized, on basis of the Four sub classes of IgG have been recognized, on basis of the

number and position of intra chain disulphide linkagesnumber and position of intra chain disulphide linkages

i.e. IgG1 IgG2, IgG3 IgG4. all sub classes are monomers =2 Fabs i.e. IgG1 IgG2, IgG3 IgG4. all sub classes are monomers =2 Fabs

Major Ig in serum/ in extravascular spaces = 75% of the total Ig Major Ig in serum/ in extravascular spaces = 75% of the total Ig
pool.mol wt about 146,000 daltons.pool.mol wt about 146,000 daltons.

The only Placental transfer Ig since there are Fc receptors on the The only Placental transfer Ig since there are Fc receptors on the
Placenta capable of binding the Fc region on the IgG therefore offers Placenta capable of binding the Fc region on the IgG therefore offers
neonates protection .neonates protection .

Roles :Roles :

1. 1. Fixes complement Fixes complement (most important in complement fxn) (most important in complement fxn)

2. Important2. Important as as opsoninopsonin (able to coat surfaces of microbes (able to coat surfaces of microbes
=opsonization) hence enhance phagocytosis=opsonization) hence enhance phagocytosis

3. Precipitin/agglutinin3. Precipitin/agglutinin

32. NEONATAL IMMUNITYNEONATAL IMMUNITY

Neonatal mammals are protected from Neonatal mammals are protected from
infection by maternally produced infection by maternally produced
antibodies transported across the antibodies transported across the
placenta into the fetal circulation and by placenta into the fetal circulation and by
antibodies in ingested milk transported antibodies in ingested milk transported
across the gut epithelium of newborns by across the gut epithelium of newborns by
a specialized process known as a specialized process known as
transcytosistranscytosis

34. NEONATAL IMMUNITYNEONATAL IMMUNITY

Maternal IgG Maternal IgG is transported across the is transported across the
placenta, and placenta, and maternal IgA maternal IgA and and IgGIgG in in
breast milk are ingested by the nursing breast milk are ingested by the nursing
infant. The infant. The transepithelial transport transepithelial transport of of
maternal IgA maternal IgA into breast milk depends on into breast milk depends on
the the poly Ig receptorpoly Ig receptor

35. NEONATAL IMMUNITYNEONATAL IMMUNITY

Transcytosis,Transcytosis,
Transcytosis is a type of
transcellular transport in which
various macromolecules are
transported across the interior of
a cell. Macromolecules are
captured in vesicles on one side
of the cell, drawn across the cell,
and ejected on the other side

36. 
Transport of maternal Transport of maternal IgG IgG across the placenta and across the neonatal intestinal across the placenta and across the neonatal intestinal
epithelium is mediated by an epithelium is mediated by an IgG-specific Fc receptor IgG-specific Fc receptor called the neonatal Fc receptor called the neonatal Fc receptor
(FcRn)(FcRn)

The FcRn is The FcRn is uniqueunique among Fc receptors in that it among Fc receptors in that it resembles a class I MHC molecule resembles a class I MHC molecule
containing a transmembrane heavy chain that is noncovalently associated with β2- containing a transmembrane heavy chain that is noncovalently associated with β2-
microglobulinmicroglobulin
NEONATAL IMMUNITY

38. 2. IgM=Mu=2. IgM=Mu= µ
Properties contains Mu heavy chainProperties contains Mu heavy chain

NB: NB: First First Ig made by fetus Ig made by fetus and and B cellsB cells

Two subclasses of Two subclasses of 1gM1gM are identified, i.e. are identified, i.e. 1gM-I and IgM-21gM-I and IgM-2..

Exists as a Exists as a monomermonomer and as a and as a pentamer (10 ag binding sites)pentamer (10 ag binding sites)

Mostly it is Mostly it is intravascularintravascular and a and a Polymeric form with J chains Polymeric form with J chains

Present in colostrum and mother milk protect newly bornPresent in colostrum and mother milk protect newly born..

It is a very potent activator of complement pathway (Fixes It is a very potent activator of complement pathway (Fixes
complement) becouse it has 10 antigen binding sites’complement) becouse it has 10 antigen binding sites’

Second in concentration in serum after IgGSecond in concentration in serum after IgG

when it attacks a foreign body the five arms can assume a 'crab-when it attacks a foreign body the five arms can assume a 'crab-
like' appearance with all five binding sites attached to the surface like' appearance with all five binding sites attached to the surface
of the invading organism.of the invading organism.

IgM appears on the surface (membrane) of unstimulated B-IgM appears on the surface (membrane) of unstimulated B-
lymphocytes and acts as recognition receptors for antigens. lymphocytes and acts as recognition receptors for antigens.

39. 2. IgM=Mu=2. IgM=Mu= µ
Properties contains Mu heavy chainProperties contains Mu heavy chain

Because of its high number of antigen binding sites (10), it is an Because of its high number of antigen binding sites (10), it is an
effective agglutinator of antigen.effective agglutinator of antigen.

IgM is very effective at IgM is very effective at the lysis the lysis of Gram +ve bacteria of Gram +ve bacteria hence hence
individuals who have IgM deficiency (genetic), have high individuals who have IgM deficiency (genetic), have high
susceptibility to infections susceptibility to infections by pus forming bacteria (pyogenic by pus forming bacteria (pyogenic
bacteria)bacteria)

IgM is the antibody of the ABO blood grouping system therefore IgM is the antibody of the ABO blood grouping system therefore
the antibody which will be involved in heamagglutination the antibody which will be involved in heamagglutination
especially ABO system following a mismatch.especially ABO system following a mismatch.
Blood groop Ag on RBC Abs in Serum
A A b
B B a
AB A $ B None
O None a $ b

40. 3. IgA3. IgA

It has an alpha heavy chainIt has an alpha heavy chain

Found in serum and body secretion: Tears, Found in serum and body secretion: Tears,
saliva, gastric and pulmonary secretionssaliva, gastric and pulmonary secretions

Major secretory Ig on Mucous surfaces give Major secretory Ig on Mucous surfaces give
Local Immunity by coating m.o, bacteria or Local Immunity by coating m.o, bacteria or
viruses preventing their adherence to mucosal viruses preventing their adherence to mucosal
cellscells

Does not fix complement (unless aggregated)Does not fix complement (unless aggregated)

Present in Present in colostrumcolostrum and and mother milk mother milk protect protect
newly born.newly born.

41. 3. IgA3. IgA

It also promotes It also promotes phagocytosis and intracellular killing of phagocytosis and intracellular killing of
organisms. organisms.

IgA exists in IgA exists in serum as monomer serum as monomer and a and a dimmer in the dimmer in the
secretions. Mol wt160,000 daltons, Half life 6 days.secretions. Mol wt160,000 daltons, Half life 6 days.

IgA2 is found in secretions and contains additional structure IgA2 is found in secretions and contains additional structure
unit called unit called transport (T) or secretory (S) piecetransport (T) or secretory (S) piece. .

T piece is synthesized in epithelial cells of gland, intestines T piece is synthesized in epithelial cells of gland, intestines
and respiratory tract. It is attached to IgA molecule during and respiratory tract. It is attached to IgA molecule during
transport across the cells. T piece links together two IgA transport across the cells. T piece links together two IgA
molecules at Fc portion.molecules at Fc portion.

J chain is also found in IgA. J chain is synthesized by J chain is also found in IgA. J chain is synthesized by
lymphoid cell. It stabilises and protects IgA from lymphoid cell. It stabilises and protects IgA from
proteolytic damage particularly in G.I.T.proteolytic damage particularly in G.I.T.

42. 4. IgD:4. IgD: Delta= Delta= δ

IgD IgD is present in the least concentration of 0.03 mg per ml is present in the least concentration of 0.03 mg per ml
and it has half-life of 3 days. and it has half-life of 3 days.

It is mostly intravascular.It is mostly intravascular.

Its function is less defined Its function is less defined than other isotypes. than other isotypes.

It is primarily It is primarily an Antigen receptor on B cells an Antigen receptor on B cells that have not that have not
been exposed to antigens where it Functions as antigen been exposed to antigens where it Functions as antigen
receptor. It may also be important in receptor. It may also be important in maturation of Bcell maturation of Bcell
following antigen contact to plasma cells.following antigen contact to plasma cells.

Appears to have a role in Appears to have a role in activating and suppressing activating and suppressing
lymphocyte activity and found in large quantities in the cell lymphocyte activity and found in large quantities in the cell
walls of many B-cells. walls of many B-cells.

IgD has 2 binding sites.IgD has 2 binding sites.

43. 5) IgE:5) IgE: Epsilon/Eta= Epsilon/Eta= ε

IgE is a IgE is a reaginic antibody reaginic antibody responsible for responsible for immediate immediate
hypersensitive hypersensitive reactions. reactions.

Also known as Also known as reaginicreaginic antibody. antibody.

It It is mostly intravascular is mostly intravascular in distribution. in distribution.

Normally it is found in Normally it is found in traces in serumtraces in serum

IgE has 2 binding site/monomer IgE has 2 binding site/monomer

It has affinity for surface of tissue cells (particularly It has affinity for surface of tissue cells (particularly
mast cells) mast cells) of the same species. Most importantly of the same species. Most importantly
activates histamine secreting cellsactivates histamine secreting cells. .

44. 5) IgE:5) IgE: Epsilon/Eta= Epsilon/Eta= ε

It is capable of binding It is capable of binding basophils and mast cellsbasophils and mast cells since since
its its Fc region Fc region has a high affinity for them, following has a high affinity for them, following
second contact with antigen known as allergens second contact with antigen known as allergens
triggering their degranulationtriggering their degranulation leading to allergy leading to allergy

Elevated levels Elevated levels are seen in are seen in atopic condition atopic condition like like
asthma, hay fever and eczemaasthma, hay fever and eczema

It also appears to play a role in It also appears to play a role in parasitic infectionparasitic infection--
Children having parasitic infection in intestine show Children having parasitic infection in intestine show
elevated levels of 1gE. elevated levels of 1gE.

It does not pass through placenta or fix complement. It does not pass through placenta or fix complement.

45. Properties of ImmunoglobulinsProperties of Immunoglobulins
PropertyProperty IgG IgG

IgAIgA IgMIgM IgEIgE IgDIgD
Heavy chain Heavy chain

symbolsymbol
γγ αα µµ εε δδ
Molecular Molecular
weightweight
150 150
KDaKDa
170-400 170-400

KDaKDa
900900
KDaKDa
190190
KDaKDa
180180
KDaKDa
PercentagePercentage
in serumin serum
75 %75 % 15 %15 % 10 %10 % 0.004 %0.004 % % 0.2 % 0.2
ComplementComplement
fixationfixation
YesYes NoNo YesYes NoNo NoNo
Transplacental Transplacental

passagepassage
YesYes NoNo NoNo NoNo NoNo
OpsonizationOpsonization YesYes NoNo NoNo NoNo NoNo

46. The structures of the five types of antibody The structures of the five types of antibody
molecules molecules

47. SUMMARY OF THE FUNCTIONS OF
ANTIBODIES

The primary function of all immunoglobulins is specific
binding of antigens. Immunoglobulins are able to
recognize specific Ag and differentiate that which is self
from non-self. They are also able to remember contact
with the same Ag in future.

PRECISE FUNCTIONS

1. Agglutination: Agglutination of particulate antigens
i.e. viruses, bacteria etc. IgM is the most potent agglutinin
since it is pentameric in nature (10 binding sites).

48. SUMMARY OF THE FUNCTIONS OF
ANTIBODIES
PRECISE FUNCTIONS

2.Opsonization: Opsonin (coating of a surface of
bacteria to facilitate phagocytosis by polymorphs).
IgG is the most potent opsonin. The polymorphs that
are mainly used in phagocytosis are: Neutrophils and
monocytes which later graduate to become
Macrophages in the tissues. Several bacteria have
developed mechanisms of evading phagocytosis

49. SUMMARY OF THE FUNCTIONS OF
ANTIBODIES
PRECISE FUNCTIONS

3. Activation of complement fixation:
Ag + Ab ≈ Ag + Ab complex
Ag+ Ab complexes are responsible for activation of
complex. Most potent immunoglobulin here is IgG and
IgM. Complement is important in inflammation and lysis
of bacteria and fungi.

4. Hypersensitivity reaction: Are important in the
allergic response, especially IgE.

50. SUMMARY OF THE FUNCTIONS OF
ANTIBODIES
PRECISE FUNCTIONS

5.Neutralization: Neutralization of particular toxins
that are released by bacteria i.e.is important in
neutralization of toxics released by tetanus bacteria

6.Hinder attachment: They may also hinder
attachment of virus to the target cells (receptors on host
cells).

7. Mucosal protection: Are also important on
mucosal protection mainly at the point or at the route
of disease entry especially IgA

51. SUMMARY OF THE FUNCTIONS OF
ANTIBODIES
PRECISE FUNCTIONS

7.Are also important in Antibody dependent
cellular cytotoxicity (ADCC): Immunoglobulins
bind organisms by Fab region and polymorphs, NK
cells can attach via Fc region and cause killing of
pathogenic organisms by releasing of the toxic
substances and kill the organism especially IgE in
defense against helminthic infections.

52. Primary and Secondary antibody responsePrimary and Secondary antibody response
Primary antibody respone Secondary antibody responsePrimary antibody respone Secondary antibody response
* first exposure to antigen * Subsequent exposure* first exposure to antigen * Subsequent exposure
* lag period: days or weeks * Lag period: hours* lag period: days or weeks * Lag period: hours
(slow onset) (rapid onset)(slow onset) (rapid onset)

* Small amount immunogl. * large amount immunogl.* Small amount immunogl. * large amount immunogl.
low Ab level with gradual increase high Ab with rapid increaslow Ab level with gradual increase high Ab with rapid increas

•Ab Persist for short duration * Ab Persist for short duration * Persist for long periods Persist for long periods rapidly rapidly
•Weeks then decline (monthes or years)Weeks then decline (monthes or years)
* Antibody * Antibody is IgM is IgM * Antibody is * Antibody is IgGIgG

53. Primary and Secondary antibody responsePrimary and Secondary antibody response
Amount of
antibodies
in serum
Time (months)
1 2 3 4 5 6
1
st
injection of Ag
2
nd
injection of Ag
Primary response
to Ag
Secondary response
to Ag

54. Immunoglobulin Class (Isotype) SwitchingImmunoglobulin Class (Isotype) Switching

Process occurring after activation of the B cell, Process occurring after activation of the B cell,
which allows the cell to produce which allows the cell to produce different classes of different classes of
antibody (IgA, IgE, or IgG). antibody (IgA, IgE, or IgG).

-Initially, naïve B cells express only -Initially, naïve B cells express only cell-surface cell-surface
IgM and IgDIgM and IgD with identical antigen binding regions. with identical antigen binding regions.

Immunoglobulin class switching Immunoglobulin class switching can be used to can be used to
change the change the class of the heavy chainclass of the heavy chain, , but not of the but not of the
light chain. Class switching therefore occurs in light chain. Class switching therefore occurs in
the heavy chain gene the heavy chain gene locuslocus by a mechanism called by a mechanism called
class switch recombination (CSR).class switch recombination (CSR).((Some B cells do not Some B cells do not
differentiate to form plasma cells which produce IgM (IgM differentiate to form plasma cells which produce IgM (IgM
antibody is produced during Primary response) but undergo antibody is produced during Primary response) but undergo
secondary DNA rearrangement forming IgG, IgA and IgE.) secondary DNA rearrangement forming IgG, IgA and IgE.)

55. Immunoglobulin Class (Isotype) SwitchingImmunoglobulin Class (Isotype) Switching

Only the constant region of the antibody heavy Only the constant region of the antibody heavy
chain changes during class switching; chain changes during class switching; the variable the variable
regions, and therefore antigen specificity, remain regions, and therefore antigen specificity, remain
unchanged. unchanged.

Each Each isotypeisotype is adapted for is adapted for a distinct functiona distinct function, ,
therefore, therefore, after activation, an antibody with a IgG, after activation, an antibody with a IgG,
IgA, or IgE effector function might be required to IgA, or IgE effector function might be required to
effectively eliminate an antigen. effectively eliminate an antigen.

Class switching Class switching is triggered by is triggered by cytokinescytokines; the ; the
isotype isotype generated depends on which generated depends on which cytokines are cytokines are
present in the B cell environment. present in the B cell environment.

56. Antibodies Act As ImmunogensAntibodies Act As Immunogens

Antigenic Determinants on Abs Fall in 3 Antigenic Determinants on Abs Fall in 3
CategoriesCategories

IsotypicIsotypic

AllotypicAllotypic

IdiotypicIdiotypic

IsotypicIsotypic

Constant Region Of AbConstant Region Of Ab

If you inject Ab in a different species Anti-If you inject Ab in a different species Anti-
Isotype is generatedIsotype is generated

If within same species, No Anti-isotypeIf within same species, No Anti-isotype

57. 
AllotypeAllotype

Even though same isotypes within one species Even though same isotypes within one species
small differences (1-4 a/a) arise in different small differences (1-4 a/a) arise in different
individuals (form of polymorphism)individuals (form of polymorphism)

If injected with such Ab you generate anti-If injected with such Ab you generate anti-
allotype Aballotype Ab

Ex. During pregnancyEx. During pregnancy

Blood transfusionBlood transfusion

Antibodies Act As ImmunogensAntibodies Act As Immunogens

58. 
IdiotypeIdiotype
Unique VUnique V
HH AND V AND V
LL binds antigen but can also behave binds antigen but can also behave
as antigenic determinantas antigenic determinant

If you inject a monoclonal antibody into a If you inject a monoclonal antibody into a
genetically identical recipient then anti-idiotypic genetically identical recipient then anti-idiotypic
antibodies are generatedantibodies are generated

No anti-isotypic and no anti-allotypic Abs will be No anti-isotypic and no anti-allotypic Abs will be
generatedgenerated
Antibodies Act As ImmunogensAntibodies Act As Immunogens

60. Monoclonal Antibody Monoclonal Antibody
ApplicationsApplications

Diagnostic TestsDiagnostic Tests

Abs are capable to detect tiny amouns (pg/mL) of moleculesAbs are capable to detect tiny amouns (pg/mL) of molecules

Ex. Pregnancy hormonesEx. Pregnancy hormones

Diagnostic ImagingDiagnostic Imaging

mAbs that recognize tumor antigens are radiolabeled with mAbs that recognize tumor antigens are radiolabeled with
iodine I-131iodine I-131

ImmunotoxinsImmunotoxins

mAbs conjugated with toxinsmAbs conjugated with toxins

mAbs To Clear PathogensmAbs To Clear Pathogens

www.elusys.comwww.elusys.com

61. 
Immunize Animal With AntigenImmunize Animal With Antigen

Multiple Clones Are Generated, Good For In VivoMultiple Clones Are Generated, Good For In Vivo

For Clinical Diagnosis, Research, One Clone That For Clinical Diagnosis, Research, One Clone That
Reacts To Single Epitope Is PreferredReacts To Single Epitope Is Preferred

Solution By Kohler and MilsteinSolution By Kohler and Milstein

Fuse A Myeloma Cell (Cancerous) With A Normal Fuse A Myeloma Cell (Cancerous) With A Normal
Plasma CellsPlasma Cells

Resulting Clones Can Be Cultured IndefinitelyResulting Clones Can Be Cultured Indefinitely

Produces An Antibody Recognizing One EpitopeProduces An Antibody Recognizing One Epitope
Monoclonal AntibodiesMonoclonal Antibodies

63. Factors influencing antibody production.Factors influencing antibody production.

AgeAge::Embryo is immunologically immature,immuno-competence is not Embryo is immunologically immature,immuno-competence is not
complete at birth. Full competence is acquired by the age of 4 yrs. In old complete at birth. Full competence is acquired by the age of 4 yrs. In old
age capacity to produce antibodies is reduced.age capacity to produce antibodies is reduced.

Route of administrationRoute of administration: : Human immune response is better after Human immune response is better after
parenteral administration of antigen than oral but igA Antibody parenteral administration of antigen than oral but igA Antibody
oral/nasal is suitableoral/nasal is suitable..

Nutrition: Nutrition: Protein calorie malnutrition suppresses both humoral as well Protein calorie malnutrition suppresses both humoral as well
as cellular response.as cellular response.

Immuno-suppressive agentImmuno-suppressive agent: : e.g x-ray, suppresses antibody formation.e.g x-ray, suppresses antibody formation.

Adjuvant:Adjuvant: this is a substance that enhances immunogenicity of an this is a substance that enhances immunogenicity of an
antigen. This will therefore enhance antibody productions.antigen. This will therefore enhance antibody productions.

Multiple antigens:Multiple antigens: When 2 or more antigens are administered, their When 2 or more antigens are administered, their
effect can be diminished due to competition or enhanced e.g. DPT.effect can be diminished due to competition or enhanced e.g. DPT.

Size and no. of doses:Size and no. of doses: An antigen is only effective above a critical dose. An antigen is only effective above a critical dose.
Massive antigen stimulus appears to inhibit ag-ab system Secondary Massive antigen stimulus appears to inhibit ag-ab system Secondary
antigenic stimulus produces increased ab response.antigenic stimulus produces increased ab response.