Antibody

ANTIBODY
Presented By- Sunita
PH.D (Ist year)
MBGE Deptt.

CONTENTS
 Introduction
 Properties of antibody
 Basic structure of antibody
 Types of antibody
 Class of antibody
 Enzymatic experiments with antibody
 Effectors function of antibody
 Conclusion

INTRODUCTION
 Antibodies are the antigens binding proteins present on B cell
membrane and secreted by plasma cells.
 Antibodies are active serum proteins formed in response to an
antigen and react specifically with that antigen.
 All antibodies are immunoglobulin's but all immunoglobulin's
may not be antibodies.

PROPERTY OF ANTIBODY
 Antibody is a glycoprotein.
 Antibody (Ab) also know as Immunoglobulin (Ig).
 Antibody is a “Y” shaped molecules.
 Antibody is made up of four polypeptide chain.
 Antibody is heterodimeric structure.
 Antibodies contain disulphide bonds.
 Each light chain is made of 214 Amino Acids.
 Each heavy chain is made up of 450 – 700 Amino acid.

STURCTURE OF ANTIBODY
 Antibodies are heavy (~150 kDa) globular plasma proteins.
 There are four polypeptide chains:-
 Two identical heavy chain and two light chains connected by
disulfide bonds.
 Light Chain (L) consists polypeptides of about 22,000 Da.
 Heavy Chain (H) consists larger polypeptides of around 50,000
Da or more.
 There are five types of Ig heavy chain (in mammal) denoted by
the Greek letters: α, δ, ε, γ, and μ.
 There are two types of Ig light chain (in mammal), which are
called lambda (λ) and kappa (κ).

 Hinge Region
 Rich in proline residues
(flexible)
 Hinge found in IgG, IgA and
IgD
 Proline residues are target
for proteolytic digestion
(papain and pepsin)
 Rich in cysteine residues
(disulfide bonds)
 IgM and IgE lack hinge
region

 An antibody is made up of a variable region and constant.
 CONSTANT REGION:- The region that has a constant structure
is called the constant region.
 Constant region in the c-terminal end.
 VERIABLE REGION:- The region show the variability is called
the variable region, variable region is located at the
extremity in the N terminal end.
 COMPLEMENTARITY DETERMINING REGIONS: antigen
binding site is complimentarily to the structure of epitope these
area are called CDR.
 Framework :-the VL and VH domain exhibit,less variation
these stretches are called framework.

HEAVY AND LIGHT CHAIN
 All 4 Heavy and Light chains are bound to each other by
disulfide bonds and non covalent interactions such as salt
linkages.
 All chains have 2 ends– an amino terminal end (NH3) and a
carboxy terminal end (COOH).
 There are 5 classes of Heavy chains and 2 classes of L chains.

DIFFRENCE BETWEEN HEAVY AND
LIGHT CHAIN
 5 classes
 Each designated by Greek
letter
 5 classes of Ig (IgG,
IgA, IgM, IgD and IgE)
classified based on AA
sequence of heavy
chains
 2 types
 Kappa (κ) and lambda (λ)
 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
HEAVY CHAIN LIGHT CHAIN

TYPE OF ANTIBODIES
 Isotype :- It is distinct form of heavy and light chain which
Present in all member of species, encoded at distinct genetic
loci. (Kappa & lambda and µ,δ).
 Idiotype :- A single antigenic determinant in variable domains
of antibody or T-Cell receptor also called as idiotypic
determinant . (VH and VL )
 Allotype :- An antigenic determinant that varies among
member of a species. The constant region of antibodies possess
allotypic determinant.

ENZYMATIC DIGESTION-GENERATES
VARIOUS FRAGMENT
 Cleave Ig above
disulfide bridge of hinge
region.
 Results in 3 fragments
each.
 Two Fab fragments
soluble fragments which
bind to Antigen.
 One Fc fragment
insoluble, crystallized in
cold.
 Cleaves Ig molecule at
point below disulfide
bridge of hinge region.
 One F(ab’)2 fragment;
2 Fab subunits bound
together.
 Many smaller fragments.
Mercaptoethanol:- Reduction
(Eliminates Disulfide Bonds)
And Alkylation Showed.
Papain digestion Pepsin digestion

IMMUNOGLOBULIN CLASSES
 Based on 5 types of heavy
chains = 5 classes of Ig
 IgG, IgA, IgM, IgD and IgE

IMMUNOGLOBULIN IgG
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, all different
Subclasses vary in biological function, length of hinge region
and No. of disulfide bridges.

FUNCTION
 Can cross placenta: provide immunity to fetus and newborn.
 Plays major role in neutralization of toxins as it can easily
diffuse into extra vascular space.

IgM
 5% to10% of total serum immunoglobulin
 IgM is pentameric structure
 IgM is first Ig class produce in a primary response
 Highest molecular weight(180,000 Da)
 Not in body fluids or secretions

FUNCTION
 Acute infection: 1st Ab to be produced following infection. Represents
acute or recent infection. Also called primary immune response Ab
 Present on B cell surface. Serves as B cell receptor for Ag binding
 Acts as Opsonin: binds as antigen which is then easily recognized
and removed
 Fetal immunity: 1st Ab to be synthesized in fetal life.

IgA
 Second most abundant Ig molecules
 Constitutes 10-15% of total serum Ig
 IgA are the dimeric structure
 2 subclasses: IgA1 and IgA2
 Serum IgA and Secretary IgA
 All J-chain containing IgA are called secretory IgA.

IgA FUNCTION
 Dimeric in nature
 Also secretory component present
 Location: found in milk, saliva, tears, etc
 Function: mediates local or mucosal immunity
 Effective against bacteria like Salmonella, Vibrio, etc
 Breast milk rich in Secretory IgA; protect to infant

IgE
 Lowest serum concentration,0.3%
 shortest half life and minimum daily production
 life span of IgE is 1 to 5 days

IgD
 First discovered Ig molecules
 Total serum Ig 0.2%
 Life span of IgD immunoglobulin is 2 to 8 days
 Membrane bound immunoglobulin express by
mature B cell

EFFECTOR FUNCTION OF ANTIBODY
 Antibody do not kill or remove pathogen solely by
binding to them then effectors function and antibody are
effect the pathogen that result the removal the pathogen
and death.
 COMPLEMENT SYSTEM
 OPSONIZATION
 NEUTRILIZATION
 ADCC (ANTIBODY-DEPENDENT-CELL-MEDIATED-CYTOIXICITY)

COMPLEMENT SYSTEM
 A collection of antiserum glycoprotein called the complement.
 The complement activation pathway is a protein fragment
called c3b
 Complement system represents a group of about 30 proteins.
 The activation of the complement cascade may be initiated by
several protein that circulate in our body.
 The complement works as a cascade system.

 Their activation via major three pathways.
 Classical pathway:-antibody dependent pathway and
triggered by formation of soluble antigen-antibody complex
or by binding of the antibody to the antigen present on the
target cell surface.
 Alternative pathway:- antibody independent pathway
stimulated by antigen directly e.g.. Bacterial cell surface
components.
 Mannose binding Lectin pathway:-Also antibody
independent but resembles classical pathway.

Classical pathway
 The formation of antigen and antibody complex(immune
complex).
 The initiation stage of activation involves components
C1,C2,C3,C4 which are present in plasma inactive form.
 Three subcomponent of C1 –C1q,C1r,C1s.
 C1q molecules are 18 polypeptide chain that associate to
form six collagen-triple helical arm.
 C1q binding site in CH2 domain in fc portion of antibody
molecule.
 Each C1r and C1s are monomer contain catalytic domain.

The building of a C3 activation
complex
 Once C1 is activated, it activates 2 other complement proteins,
C2 and C4 by cutting them in half.
 C2 is cleaved into C2a and C2b.
 C4 is cleaved into C4a and C4b.
 Both C2a and C4b bind together on the surface of the bacteria
C2b and C4a diffuse away.

C3 Activation complex
 C2a and C4b bind together on the surface to form a
C3 activation complex.
 The function of the C3 activation complex is to
activate C3 proteins.
 This is done by cleaving C3 into C3a and C3b.

 C3b is an opsonin
 Opsonins are molecules that bind both to bacteria
and phagocytes.
 Opsonization increases phagocytosis by 1,000
fold.
C3b

c3a
 C3a increases the inflammatory response by binding
to mast cells and causing them to release histamine.

Building the C5 activation complex
 Eventually enough C3b is cleaved that the surface
of the bacteria begins to become saturated with it.
 C2a and C4b which make up the C3 activation
complex .
 When C3b binds to C2a and C4b it forms a new
complex referred to as the C5 activation complex.

The C5 activation complex
 The C5 activation complex (C2a, C4b, C3b)
activates C5 proteins by cleaving them into C5a
and C5b
 Many C5b proteins are produced by the
C5activation complex. These C5b begin to coat the
surface of the bacteria.

The function of C5a
 C5a disperses away from the bacteria.
 Binds to mast cells and increases inflammation.
 Most powerful chemotactic factor known for
leukocytes

Building the Membrane Attack
complex
 C5b on the surface of bacteria binds to C6
 The binding of C6 to C5b activates C6 so that it
can bind to C7
 C7 binds to C8 which in turn binds to many C9’s
 Together these proteins form a circular complex
called the Membrane attack complex (MAC)

Membrane Attack complex
 The MAC causes Cytolysis.
 The circular membrane attack complex acts as a
channel in which cytoplasm can rush out of and
water rushes in.
 The cells inner integrity is compromised and it dies

Alternative pathway
 The alternative pathway is part of the non-specific
defense because it does not need antibodies to
initiate the pathway.
 The alternative pathway is slower than the Classical
pathway

Initiation of The Alternative pathway
 C3 contains in
unstable thioester
bond.
 This unstable bond
makes C3 subject to
slow spontaneous
hydrolysis to C3b and
C3a
 The C3b is able to
bind to foreign
surface antigens.

FACTOR B
 C3b on the surface
of a foreign cells
binds to another
plasma protein
called factor B

Factor D
 The binding of C3b
to factor B allows a
protein enzyme
called Factor D to
cleave Factor B to
Ba and Bb.
 Factor Bb remains
bound to C3b while
Ba and Factor D
disperse away.

The C3 activation complex
 Properdin, also called factor P, binds to the
C3bBb complex to stabilize it.
 C3bBbP make up the C3 activation complex
for the alternative pathway

C3 ACTIVATION COMPLEX
 The C3 activation
complex causes the
production of more
C3b.
 This allows the initial
steps of this pathway
to be repeated and
amplified
 2X106 molecules can
be generated in 5
minutes

C5 ACTIVATION
 When an additional
C3b binds to the C3
activation complex it
converts it into a C5
activation complex.
 The C5 activation
complex cleaves C5 into
C5a and C5b.
 C5b begins the
production of the MAC.

LECTIN MANNOSE PATHWAY
 First step is binding of Mannose
Binding Lectin (MBL) to mannose
residue on the surface of microbes.
 To the MBL bound to microbe, MBL
–Associated Serine Proteases
(MASP-1and MASP-2) will bind.
 MASP 1 and 2 is similar in structure
and function to C1s and C1r
 This complex will cleave C4 and
C2

Lectin Binding Pathway
 Independent of antibodies.
 Lectins are proteins that recognize and bind to
specific carbohydrate targets.
 Activated by Mannose binding lectin (MBL) – lectin
that binds to mannose residues on the microbes.
 MBL is similar to C1q in structure and function.

 The lectin pathway is activated by the binding of
mannose binding lectin (MBL) to mannose residue on
glycoprotein or carbohydrate on the surface of
microorganisms.
 MBL is a acute phase protein and its concentration
increase during inflammatory response.
 Its function in the complement pathway similar to
C1q.
 After MBL binds to cb residue on the surface of
pathogen.

 MBL associate serine protease,MASP-1and MASP-2
Bind to MBL.
 The active complex formed by this association cause
cleavage and activation of C4 and C2.
 MASP-1 and MASP-2 similar to C1r and C1s.
 This means C2 and C4 form a C5 convertase .

Opsonisation
Opsonization → enhance the activity of phagocytosis.
 Promotion of phagocytosis of antigen by
macrophage and neutrophils.
 Protein molecules called fc receptor which can bind
the constant region of Ig molecules that are present
on the surface of macrophage and neutrophils.
 The principal opsonins are IgG opsonin and C3b
opsonin.

phagocytosis
 Phagein = "to eat", cytos = "cell“,
 osis = “process”
 Phagocytosis is the process of engulfment and
destruction of foreign particle particles such as
bacteria.

Neutralization
 The antibody bind to specific microbe or microbial
toxin.
 Block pathogen entry into the cell and neutralize their
infectivity.
 Microbes and microbial toxin bind to surface molecule
of host.
 Antibody bind to the pathogen.
 Neutralization encourage or prevent a pathogen form
initiating an infection.

single-chain variable fragment (scFv)
 An scfv fragment consist of the smallest functional antigen –
binding domain of an antibody ( 30 kDa).
 The variable heavy (VH)and variable light(VL) chains are
joined together by a flexible peptide linker (fusion protein).
 A short linker peptide of ten to about 25 amino acids.
 The linker is usually rich in glycine for flexibility,
 They are bind to the epitope of antigen.
 The two-hybrid system, phage display is used for the high-
throughput screening of protein interactions. ( determination of
interaction partners of a protein).

Antibody dependent cell mediated
cytotoxicity
 The linking of antibody bound to target cell with fc receptor.
 a number of cell type NK cell can direct the cytotoxic
activities of the effectors cell against the target cell this
process is called antibody dependent cell mediated
cytotoxicity.
 When macrophage, neutrophils bind to target cell way of fc
receptor and that result the level of lytic enzyme.
 Their cytoplamic lysosomes and release of these lytic enzyme.
At the site of the fc mediated contact may in damage to target
cell ,start the process of degranulation.
 Macrophages NK cell have been secrete tumor nacrosis factor.
(TNF)

Antibody

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