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 Agent How Antibodies are Generated Activation of a B cell and Clonal Expansion 1) Antigen binds the BCR 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 Activated B-cells undergo clonal expansion, where they either become antibody factories called Plasma cells, or they differentiate into memory B-cells. Memory B-cells survive for years and are essential for long term immunity. This allows the immune system to remember an antigen and respond faster the next time it is faced with this antigen. 4

IgD IgG, IgA IgM

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

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 Assignment Antibody 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 Assignment: Isotypes Mammals express five different isotypes of antibodies, known as: IgA, IgD, IgE,IgG and IgM. The "Ig" prefix stands for immunoglobulin and the letter denotes the class of antibody. Each isotype has different functions and locations. The heavy chain defines the class of antibody, i.e. if the antibody is an IgA, IgD, IgE, IgG, or an IgM. 22

The Assignment Antibody 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 The Assignment: Isotypes The basic antibody is composed of 1 Ig unit, that is, it is a monomer. However, secreted antibodies can also be dimeric with two Ig units as in the case of IgA, or pentameric with five Ig units, like IgM. Antibody isotype changes during development and with activation of the B-cells. Immature B-cells that have never been exposed to antigen are known as naïve B-cells and express only cell surface bound IgM. As the B-cell begins to reach maturity, it expresses both IgM and IgD. After reaching maturity, the B-cell is ready to interact with circulating antigen. Once an antigen is bound, the B-cell becomes activated and begins to divide and differentiate into the antibody factory, a plasma cell. In this activated form, the B-cell can now secrete antibody, allowing it to circulate in your blood and serum. Some daughter cells of the activated B-cells undergo isotype switching, whereby we get production of IgE, IgA or, most commonly, IgG. 23

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 Constructed from 2 segments 1 (V) segment 1 (J) segment Heavy chain Constructed from 3 segments 1 (D) 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 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 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 SIgA Destructive roles Immune complex disease, Auto immunity and Allergy

The Mission Antibody Function Antibodies are the main component of the Humoral Immune System They bind antigen and flag them for elimination via 1 of 3 ways: Function Antibodies constitute the main component of the humoral immune system. They circulate freely in the bloodstream and search for harmful foreign agents. Antibodies protect the immune system from invading antigens by binding to them and flagging them for elimination via one of three ways: 37

Viruses and intracellular bacteria require a Neutralization: Viruses and intracellular bacteria require a host cell in order to replicate Antibodies prevent their entry into the cell by binding the antigen, making it harder for it to pass through the cell membrane. Antibodies cannot attack pathogens hidden within cells Neutralization: Viruses and intracellular bacteria require a host cell in order to replicate and pass on disease. These antigens generally enter the cell either by fusing with receptors on the cell. Antibodies prevent this action by binding to the antigen, making it harder for it to pass through the cell membrane. Antibodies cannot attack pathogens within cells.

antigen, antibodies flag the foreign agent for destruction 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 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 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. 19

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. 21

Polyclonal Antibodies Figure 2 Polyclonal antibodies reacting with various epitopes Each antibody is made by a different B-cell 22

Polyclonal Antibody Production antigen A rabbit is injected (intradermally or subcutaneously) with a purified dose of antigen. 24

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. 30

Monoclonal Antibodies Figure 3 Monoclonal antibodies reacting with similar epitopes 32

Monoclonal Antibody Production spleen B-lymphocytes The antibody-producing B-cells are harvested from the spleen or lymph nodes. 36

Monoclonal Antibody Production myeloma cells B-lymphocytes The B-cells are fused with mouse myeloma cells forming immortal hybrid cells or hybridomas. 37

Humanization of Antibodies Mouse Chimaeric Humanized Human

Bispecific antibodies Affibody Plastic antibody

Thanks