ANTIBODY STRUCTURE AND FUNCTION

ANTIBODY STRUCTURE AND FUNCTION

WHAT ARE IMMUNOGLOBULINS?

* Antigen specific proteins produced by B lymphocytes

* Belong to immunoglobulin super family (BCR, TCR, MHC)

* Bound to surface of B lymphocytes* Function as binding (receptor) sites for specific antigens

* Antigen receptor sites on mature B lymphocytes* IgM* IgD

* Membrane-bound form of immunoglobulins

WHAT IS THE IMMUNOGLOBULIN SUPERFAMILY

* Proteins with structural feature first defined in immunoglobulins

* Characteristic structural feature* Sequence of Domains providing stable conformation

* Domain (loop 60 – 70 a.a.)

* Polypeptide (100 to 110 amino acids) chain folded into sandwich (2 slices of bread) held together by disulfide bond

* IG superfamily members* Antibodies, B cell receptors, T cell receptors, MHC molecules and

others

The Making of an AgentHow Antibodies are Generated

* Activation of a B cell and Clonal Expansion

1) Antigen binds theBCR on a B-cell, activating it

2) B-cell begins to divide (Clonal Expansion), forming either plasma cells(antigen factories) or memory B-cells.Abs has 107-1011 diversity

IgMIgD IgG, IgA

STRUCTURE OF ANTIBODIES

* Antibodies are glycoproteins composed of* Polypeptide chains and carbohydrate

* Monomeric structure* Polypeptide chains

* 2 identical heavy chains

* 2 identical light chains

* Polypeptide chains joined by disulfide bonds

* Carbohydrate

Switch region

STRUCTURE OF ANTIBODIES

* Polypeptide chains have variable and constant regions* Variable

* N (amino)-terminal of polypeptide chain

* Antigen binding site

* Constant * C (carboxyl)-terminal of polypeptide chain

* Binding sites for cell surface receptors and complement

* Structure represented by the letter “Y”

* Y shaped molecule cleaved by protease papain* Fragment antigen binding (Fab)

* Fragment crystallizable (Fc)

Cys, Prolin rich

Variable domain of Antibodies

VH

FR1 FR2 FR3 FR4

CDR1 CDR2 CDR3

VL

FR1 FR2 FR3 FR4

CDR1 CDR2 CDR3

CLASSES (ISOTYPES) OF IMMUNOGLOBULINS

* Classes based on constant region of heavy chains* Immunoglobulin A (IgA)

* Immunoglobulin D (IgD)

* Immunoglobulin E (IgE)

* Immunoglobulin G (IgG)

* Immunoglobulin M (IgM)

* Differentiation of heavy chains* Length of C region, location of disulfide bonds, hinge region,

distribution of carbohydrate

* Classes have different effector functions

No hing

No hing

CLASSES (ISOTYPES) OF IMMUNOGLOBULINS

* Additional classification based on light chains* Kappa 60% in human 90% in mice

* Lambda 40 % in human and 10% in mice

* Each IG has either kappa or lambda, not both* IgG kappa

* IgG lambda

* No functional differences between light chains

J chain

The AssignmentAntibody Isotype

* Mammals express 5 different isotypes of antibodies (IgA, IgD, IgE, IgG and IgM) with different functions and locations

* Class of antibody is defined by the heavy chain

The AssignmentAntibody Isotype

* Basic antibody is composed of 1 Ig unit, i.e. is a monomer

* Some are dimeric (IgA) or pentameric (IgM)

* Isotype changes during development of B-cell

ANTIGEN BINDING SITES OF IMMUNOGLOBULINS

* Antigen binding sites vary with size and shape of antigen

* Part of antigen to which antibody binds* Antigenic determinant (Epitope)

* Antigen-Antibody binding based on non-covalent forces* Hydrogen bonds

* Affinity* Strength of binding of one molecule to another by a single binding site

* Avidity* Overall strength of binding between two molecules

CONSTRUCTION OF LIGHT CHAIN AND HEAVY CHAIN VARIABLE REGIONS

* Light chain* Constructed from 2 segments

* 1 (V) segment

* 1 (J) segment

* Heavy chain* Constructed from 3 segments

* 1 (V) segment

* 1 (D) segment

* 1 (J) segment

SURFACE IMMUNOGLOBULINS ASSOCIATED WITH PROTEINS TO COMPLETE ANTIGEN RECEPTOR

* In ER, IgM and IgD associated with transmembrane proteins* Ig-alpha* Ig-beta

* Transmembrane proteins* Transport M and D to B cell surface* Communication of antigen binding to inside of B cell

* Tails interact with intracellular signaling molecules

* Complex of IgM and IgD with Ig-alpha and Ig-beta forms* B-cell receptor

DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN

* Following antigen activation of B-cells, additional diversification occurs in V domain by* Somatic hypermutation

* Somatic hypermutation* Introduction of random single nucleotide substitutions (point

mutations) throughout V regions of H and L chains

* Mechanism poorly understood

* More common in hypervariable regions (CDRs)

OUTCOME OF SOMATIC HYPERMUTATION

* Gives rise to some antibodies with higher* Affinity for antigen

* Affinity* Strength of binding of one molecule to another by a single binding

site

* Higher affinity antibodies are produced as immune response proceeds* Affinity maturation

THE PRIMARY HUMORAL IMMUNE RESPONSE

* Immune response initially produces IgM antibodies then switches to IgG antibodies

* Question* Why switch from IgM to IgG?

* Answer* Limited effector mechanisms for IgM* Range of effector mechanisms for IgG

* Mechanism* Isotype or class switching

ISOTYPE OR CLASS SWITCHING

* Process by which B cell changes class of IG produced while preserving antigenic specificity

* Involves somatic recombination which attaches different heavy chain constant region to variable region

* Occurs only during active immune response

* Mechanisms involves recombination between* Switch sequences (regions)

CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF IMMUNOGLOBULINS

Classes Subclasses

IgG IgG1, IgG2, IgG3, IgG4

IgA IgA1, IgA2

IgM

IgD

IgE

IgGA2m1, has no disulfid bond between L,H but has L,L. IgA2 has more sucrose than IgA1 , but the type of sucrose in IgA1 and IgD is N.Acetylgalactosamine

S-IgA is responsible for immune exclusion.

S IgA production in lamina properia milieu

The role of SC component in SIgA2m1 is protection of Ab because Ab dose not contain disulfide bond between H,L.

“Protease A” from N. gonorrhoeae, Strep. Sanguis, H.influenza, Strep. Pneumoniae digest hing region of IgA1. Hing is not in IgA2.

FUNCTIONS AND PROPERTIES OF ANTIBODY

* Neutralization* Direct inactivation of pathogen or toxin thereby preventing its interaction with human cells

* Opsonization* Coating of pathogens for more efficient phagocytosis

* Activation of complement* More efficient phagocytosis* Direct killing

Gate Keeper* Increase the concentration of Ab and complement proteins out of the arteries .

Mucosal immunity* SIgADestructive roles* Immune complex disease, Auto immunity and Allergy

The MissionAntibody Function

* Antibodies are the main component of the Humoral Immune System

* They bind antigen and flag them for elimination via 1 of 3 ways:

Neutralization: Viruses and intracellular bacteria require ahost cell in order to replicate Antibodies prevent their entry into the cell by binding the antigen, making it harderfor it to pass through the cell membrane. Antibodies cannot attack pathogens hidden within cells

Opsonization: Upon binding to an antigen, antibodies flag the foreign agent for destruction or elimination by other immune cells, such as natural killer cells or macrophages

Activation of Complement: Similar to opsonisation, antibody will flag the antigen for elimination. However, elimination is initiated by a cascade of proteins which collect on the cell membrane and form a hole, leading to cell lysis

IgM ANTIBODY OF THE IMMUNE RESPONSE

* First isotype produced in primary response* May or may not be produced in secondary response

* Produced before B cells undergo somatic hypermutation

* Occurs as pentamer with J chain* Found primarily in blood and lymph

* Multiple binding sites confers high avidity and compensates for low affinity of monomers

* Highly effective in complement activation

* Functions as rheumatoid factor

IgG ANTIBODY OF THE IMMUNE RESPONSE

* Second isotype produced in primary response

* Primary isotype of * Secondary immune response

* Memory immune response

* Represents approximately 75% of total serum IG

* Four subclassses (1-4)* Different effector functions

* Transported across placenta

* Functions as rheumatoid factor

IgA ANTIBODY OF THE IMMUNE RESPONSE

* Two subclasses (IgA1 and IgA2) and two forms (monomeric and dimeric)

* Monomeric* Located in blood and extracellular spaces* Predominately IgA1

* Ratio of IgA1 to IgA2 is 10:1

* Functions as rheumatoid factor

* Dimeric* Located in mucous membranes and secretions* Predominately IgA2* Ratio of IgA2 to IgA1 is 3:2 * J chain like IgM

IgE and IgD ANTIBODIES OF THE IMMUNE RESPONSE

* IgE (Gate keeper)* Binds with high affinity to receptors on mast cells, basophils and

activated eosinophils

* Longer half-life when cell bound

* Initiates a strong inflammatory reaction to parasites

* Involved in allergic reactions

* IgD* Antigen receptor on mature B-cells

* No other known function

Antigenic marker on Immunoglobulin

Idiotypic:,VH,VL

Allotypic:CH, CL

Isotypic: CH, CL

Network theory* Idiotype determinants are immunogen for hosts.

* Niels K.Jerne express that after increase in Ab level, the Ab stimulate anti- idiotype Ab that suppress Ab response against Antigen.

* Anti Idiotype is produced in the high level of specific Ab.

Antibody Production

* Antibodies are generated by immunizing (injecting) animals with purified antigen. The animal responds by producing antibodies that specifically recognize and bind to the antigen.

* Antibody reagents may be polyclonal or monoclonal.

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Polyclonal Antibodies

* Polyclonal antibody reagents are produced as different classes of immunoglobulins by many B-cells clones and react with various epitopes on an antigen. (Figure 2)

* They are more tolerant of small changes in the nature of the antigen since they often recognize multiple epitopes.

* They may be generated in a variety of animals like rabbit, goat, sheep, horse, etc. The rabbit is the most commonly used animal for generating polyclonal antibodies.

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Polyclonal Antibodies

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Polyclonal antibodies reacting with various epitopesEach antibody is made by a different B-cell

Figure 2

Polyclonal Antibody Production

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antigen

A rabbit is injected (intradermally or subcutaneously) with a purified dose of antigen.

Monoclonal Antibodies

* Monoclonal antibodies are derived from a single B-cell and are produced as a single class of immunoglobulin.

* They are raised by fusion of the specific B-cells with immortal myeloma (B-cell) cancer cells to form a hybridoma.

* A hybridoma can multiply indefinitely and continuously produce a specific monoclonal antibody.

* They react with a specific epitope on a given antigen (Figure 3), giving less background staining.

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Monoclonal Antibodies

Monoclonal antibodies reacting with similar epitopes

Figure 3

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The antibody-producing B-cells are harvested from the spleen or lymph nodes.

Monoclonal Antibody Production

B-lymphocytes

spleen

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The B-cells are fused with mouse myeloma cells forming immortal hybrid cells or hybridomas.

Monoclonal Antibody Production

myeloma cells

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B-lymphocytes

Humanization of Antibodies

Mouse Chimaeric

Humanized Human

Bispecific antibodiesAffibody

Plastic antibody

Thanks