Antibody

Dr. Wai Mar Linn

M.B.,B.S, DTM&H , MSc

Learning Outcomes

At the end of the lecture , the student must be able to describe

the followings

• Describe the origin, types and classes of antibodies.

• Detail the structure of an immunoglobulin and describe the

roles of various immunoglobulins.

• Explain the Function of antibodies , Antigen-Antibody

Binding and Its Results.

• Describe the genetic recombination leading to the diversity

among immunoglobulins. (Class switching)

Outlines

• Antibody ( Immunoglobulin)

• Antibody structure

• Classes of antibodies

• Function of antibodies

• Antigen-Antibody Binding and Its Results

• Affinity and Avidity

Antibody (Ab) (Immunoglobulins [Ig])

• Antibodies are gamma globulin proteins that react

specifically with the antigen that stimulated their

production.

Antibody

• Antibodies are proteins (immunoglobulins or gamma

globulins) produced by plasma cells in response to

stimulation by a specific antigen, capable of binding to the

antigen that stimulated their production.

• B-cells mature in the bone marrow.

• After the “education” process, immunocompetent but naïve B

lymphocytes leave the bone marrow to populate lymphoid tissues

and circulate through body fluids.

• The activated B-cell clonally proliferates to produce a population of

plasma cells and memory cells, which all recognize the same

antigen.

• The effector cells produced by B-cells are plasma cells, which

secrete antibodies.

• This is the humoral arm of the immune system.

• Memory cells, which are primed to deal with subsequent encounters

with the same antigen.

• Memory cells respond much more quickly and vigorously and form

the basis for the secondary or memory response of the immune

system.

Antibody

• They make up about 20% of the protein in blood plasma.

• Blood contains three types of globulins, alpha, beta, and

gamma, based on their electrophoretic migration rate.

• Antibodies are gamma globulins.

Antibody Structure

• Immunoglobulins made up of light (L) and heavy (H)

polypeptide chains.

• The simplest antibody molecule has a Y shape and consists

of four polypeptide chains: 2 H chains and 2 L chains.

• The four chains are linked by disulphide bonds.

• The terms light and heavy refer to molecular weight; light

chains have a molecular weight of about 25,000, whereas

heavy chains have a molecular weight of 50,000 to 70,000.

• An individual antibody molecule always consists of

2 identical H chains and 2 identical L chains.

Immunoglobulin

Antibody Structure

• L and H chains are subdivided into variable(V) and

constant(C )regions.

• The regions are composed of three-dimensionally folded,

repeating segments called domains.

• A L chain consists of one variable (VL) and one constant

(CL) domain.

• Most H chains consist of one variable (VH) and three

constant (CH) domains.

Immunoglobulin monomer

Antibody Structure

• The variable regions of both the light and heavy chain are

responsible for antigen-binding.

• The constant region of the heavy chain is responsible for

various biologic functions

(e.g.,complement activation and binding to cell surface receptors)

• The complement binding site is in the CH2 domain.

• The constant region of the light chain has no known biologic

function.

• The constant portion of the heavy chains forms the base and

the stem.

Antibody Structure

• Digestion with proteolytic enzyme (papin) cleaves on the

amino-terminal side of the inter-heavy chain disulphide

bonds, yielding two Fab fragments. (Fragment antigen

binding) they retain the ability to recognise the antigen .

• Other fragment can be easily crystallised and was termed

Fc (Fragment crystallisable)

Antibody Structure

• Antibodies have at least two identical antigen binding

(valence) sites.

• A monomer is a single bivalent antibody unit.

• Monomers consist of a combination of two heavy chains

and two light chains.

• Multivalent antibodies are composed of two or more

monomers.

Antibody Structure

• The variable regions of both L and H chains have three extremely

variable (hypervariable) amino acid sequences at the amino-

terminal end that form the antigen-binding site.

• Only 5 to 10 amino acids in each hypervariable region form the

antigen-binding site.

• Antigen–antibody binding involves electrostatic and van der

Waals’ forces and hydrogen and hydrophobic bonds rather than

covalent bonds.

• The remarkable specificity of antibodies is due to these

hypervariable regions.

Antibody Structure

• L chains belong to one of two types, κ (kappa) or λ

(lambda), on the basis of amino acid differences in their

constant regions.

• Both types occur in all classes of immunoglobulins (IgG,

IgM, etc.), but any one immunoglobulin molecule contains

only one type of L chain.

• The amino-terminal portion of each L chain participates in

the antigen-binding site.

• H chains are distinct for each of the five immunoglobulin

classes and are designated γ, α, μ, ε and δ .

• The amino-terminal portion of each H chain participates in

the antigen-binding site; the carboxy terminal forms the Fc

fragment

Antibody Structure

• The variable portion will always be the same on antibodies

produced by the same plasma cell, and will always

recognize the antigen that originally stimulated its

production.

• The structure of the stem, or Fc region determines the class

of an antibody and can be changed by an activated B-cell

(class switching) without the antigen specificity of the

antibody changing.

• Depending on the structure, the Fc region may bind and

activate complement, and may be recognized by Fc

receptors on macrophages, dendritic cells, neutrophils,

eosinophils, mast cells, and NK (natural killer) cells.

Structure of immunoglobulin G (IgG).

The Y-shaped IgG molecule consists of

two light chains and two heavy chains.

Each light chain consists of a variable

region and a constant region. Each

heavy chain consists of a variable region

and a constant region that is divided into

three domains: CH1, CH2, and CH3.

The CH2 domain contains the

complement-binding site, and the CH3

domain is the site of attachment of IgG

to receptors on neutrophils and

macrophages. The antigen-binding site

is formed by the variable regions of both

the light and heavy chains. The

specificity of the antigen-binding site is a

function of the amino acid sequence of

the hypervariable regions

The antigen-binding site is formed by the

hypervariable regions. A: Hypervariable

regions on immunoglobulin G (IgG). B:

Magnified view of antigen-binding site.

Antibody

The most important functions of antibodies are

• to neutralize toxins and viruses

• to opsonize microbes so they are more easily phagocytosed

• to activate complement and

• to prevent the attachment of microbes to mucosal surfaces.

Antigen-Antibody Binding and Its Results

• Antigen-antibody complex formation

• Inactivation of viruses and neutralization of bacterial toxins

• Agglutination of cellular antigens (causing cells to clump,

or agglutinate)

• Opsonization (macrophages have Fc receptors)

• Complement fixation

Antibody

• There are five classes of antibodies: IgG, IgA, IgM, IgE,

and IgD. (GAMED)

• Antibodies are subdivided into these five classes based on

differences in their heavy chains.

IMMUNOGLOBULIN CLASSES

IgG

• Each IgG molecule consists of two L chains and two H

chains linked by disulfide bonds .

• It has two identical antigen-binding sites, it is said to be

divalent.

• IgG has γ Heavy chain

IgG

• IgG is the predominant antibody in the secondary response

and constitutes an important defense against bacteria and

viruses

• IgG is the only antibody to cross the placenta; only its Fc

portion binds to receptors on the surface of placental cells.

It is therefore the most abundant immunoglobulin in

newborns.

• IgG is one of the two immunoglobulins that can activate

complement; IgM is the other

• IgG is the immunoglobulin that opsonizes. It can opsonize

(i.e., enhance phagocytosis) because there are receptors for

the γH chain on the surface of phagocytes.

IgG

• Monomer

• Most prevalent in serum (75% of the total)

• Major antibody of the secondary response

• Has the longest half life

• Pass through the placenta, most abundant Ig in newborn.

• Neutralizes bacterial toxins

• Agglutination

• Participates in complement fixation

• Enhances phagocytosis (opsonization)

• ADCC

IgA

• IgA is the main immunoglobulin in secretions such as

colostrum, saliva, tears, and respiratory, intestinal, and

genital tract secretions.

• serum IgA is a monomer and secretory IgA is a dimer

• IgA has α heavychain

• the dimer is held together by a J chain which is produced

by the antibody producing plasma cells.

• The secretory IgA(sIgA) is always in the dimeric form and

is composed of two basic four chain unit, a J chain and the

secretory component.

• The secretory component is part of the molecule that

transports the dimer produced by a submucosal plasma cell

to the mucosal surface.

IgA

• protects mucosal surfaces.

• It prevents attachment of microorganisms (e.g., bacteria and

viruses) to mucous membranes.

IgM

• present on the surface of both mature and immature B-cells

• As a monomer acting as an antigen receptor

• IgM has μ Heavy chain

• First antibody class secreted in the primary immune

response.

• Secreted as a pentamer, involved in agglutination and

complement fixation.

IgM • In serum, it is a pentamer composed of five H2L2 units

plus one molecule of J (joining) chain

• Pentamer has ten identical Ag binding sites and thus a

valence of 10

IgM

• It has the highest avidity of the immunoglobulins; its

interaction with antigen can involve all 10 of its binding

sites.

• It is the most efficient immunoglobulin in agglutination,

complement fixation (activation), and other antibody

reactions because the pentamer has 10 antigen-binding

sites.

• It and is important in defence against bacteria and viruses.

• It can be produced by the fetus in certain infections.

IgE

• Monomer

• Involved in allergic reactions.

• IgE has Ԑ Heavy chain

• It mediates immediate (anaphylactic) hypersensitivity

• It participates in host defenses against certain parasites

(e.g., helminths [worms])

IgE

• The Fc region of IgE binds to the surface of mast cells and

basophils.

• Bound IgE serves as a receptor for antigen (allergen).

• When the antigen-binding sites of adjacent IgEs are cross-

linked by allergens, several mediators are released by the

cells, and immediate (anaphylactic) hypersensitivity

reactions occur.

• Although IgE is present in trace amounts in normal serum

(approximately 0.004%), persons with allergic reactivity

have greatly increased amounts, and IgE may appear in

external secretions.

• IgE does not fix complement and does not cross the

placenta.

IgE

• worms are too large to be ingested by phagocytes, they are

killed by eosinophils that release worm-destroying

enzymes.

• IgE specific for worm proteins binds to receptors on

eosinophils, triggering the antibody-dependent cellular

cytotoxicity (ADCC) response

IgD

• Monomer

• Present on surface of mature B-cells

especially memory cells.

• IgD has ‘ δ ’ Heavy chain

• Function as an antigen receptor

• present in small amounts in serum.(< 1%)

• Have a very short half - life

• May be important for the secondary immune response

• May be important in regulating the immune response.

Immunoglobulin Isotypes

They are defined by difference in their heavy chain constant

region. Eg: class and sub classes

These determine their biological characteristics.

Eg . Complement activation ,cellular association via Fc

receptor and tissue distribution.

Class Switching

• Gene rearrangement to produce different immunoglobulin

(Ig) classes.

• IgM is formed first because the μ constant region is closest

to the VDJ DNA.

• Later the μ constant region can be switched with a γ, ε, or

α constant region to form the heavy chain of IgG, IgE, or

IgA, respectively.

• Note that the antigenic specificity of the B cell remains the

same because the VDJ DNA remains the same.

V : variable regions; D:diversity segments; J: joining

segments; C :constant regions; S : switch sites.

Affinity

Affinity defines the strength of the interaction between a

single antigen combining site and an epitope.

Avidity

Avidity defines the cumulative interactions of several

combining sites with an antigen.