Slide 1

IMMUNOGLOBULINS AND THEIR FUNCTIONS PRESENTED BY : roshni maurya

WHAT ARE ANTIBODIES? (Ab)Proteins that recognize and bind to a particular antigen with very high specificity.Made in response to exposure to the antigen.One virus or microbe may have several antigenic determinant sites, to which different antibodies may bind.Each antibody has at least two identical sites that bind antigen: Antigen binding sites.Valence of an antibody: Number of antigen binding sites. Most are bivalent.Belong to a group of serum proteins called immunoglobulins (Igs).

2

WHAT ARE IMMUNOGLOBULINS?(Ig)In 1964,endorsed by WHO, the generic term immunoglobulin was internationally accepted for proteins of animal origin endowed with known antibody activity and for certain proteins related to them by chemical structure.

Ig are synthesized by plasma cells, to some extent by lymphocyte.

Ig provide a structural and chemical concept, while the term antibody is a biological and functional concept.

All Ab are igs, but all igs may not be antibodies.Constitute 20-25% of total serum proteins.

3

4

WHAT IS THE IMMUNOGLOBULIN SUPERFAMILYProteins with structural feature first defined in immunoglobulins

Characteristic structural featureSequence of Domains providing stable conformation

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

IG superfamily membersAntibodies, B cell receptors, T cell receptors, MHC molecules and others

5

STRUCTURE OF ANTIBODIESAntibodies are glycoproteins composed ofPolypeptide chains and carbohydrate

Monomeric structurePolypeptide chains2 identical heavy chains2 identical light chainsPolypeptide chains joined by disulfide bondsCarbohydrate

6

STRUCTURE OF ANTIBODIESPolypeptide chains have variable and constant regionsVariableN (amino)-terminal of polypeptide chainAntigen binding site Constant C (carboxyl)-terminal of polypeptide chainBinding sites for cell surface receptors and complementStructure represented by the letter YY shaped molecule cleaved by protease papainFragment antigen binding (Fab)Fragment crystallizable (Fc)

7

8

9

H chains are distinct for each of the five Ig classes or isotypes and are designated for the respective classes of Ig, namely IgG, IgA ,IgM , IgD IgE.L chains are one of two types designated and and only one type is found in Ig.Human ImmunoglobulinLight Chain TypesKappa Lambda

L and H chains are subdivided into variable and constant regions. The regions are composed of three-dimensionally folded, repeating segments called domains. An 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.(IgG and IgA have three CH domains,whereas IgM and IgE have four.)

The variable regions are responsible for antigenbinding,whereas the constant regions are responsible for various biologic functions eg, complement activation and binding to cell surface receptors.The variable regions of L have three extremely variable (hypervariable / hot spots) amino acid sequence at the amino-terminal end that form the antigen-binding site .H chain have 4 such region.

Immunoglobulin Fragments: Structure/Function Relationships

Ag Binding

Complement Binding SitePlacental TransferBinding to Fc Receptors

Structure of the Variable RegionHypervariable (HVR) or complementarity determining regions (CDR)

FR1

FR2

FR3

FR4

HVR1

HVR2

HVR3

Variability Index257550100Amino acid residue

150100500Framework regions

A. Immunoglobulin classes - The immunoglobulins can be divided into 5 different classes based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class will have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences).1. IgG - Gamma heavy chains2. IgM - Mu heavy chains3. IgA - Alpha heavy chains4. IgD - Delta heavy chains5. IgE - Epsilon heavy chains

Immunoglobulin Classes, Subclasses, Types and Subtypes

. Immunoglobulin Subclasses - The classes of immunoglobulins can de divided into subclasses based on small differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a subclass will have very similar heavy chain constant region amino acid sequences. Again these differences are most commonly detected by serological means.1. IgG Subclassesa) IgG1 - Gamma 1 heavy chainsb) IgG2 - Gamma 2 heavy chainsc) IgG3 - Gamma 3 heavy chainsd) IgG4 - Gamma 4 heavy chains2. IgA Subclassesa) IgA1 - Alpha 1 heavy chainsb) IgA2 - Alpha 2 heavy chains

CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF IMMUNOGLOBULINSSubclasses are numbered according to plasma concentration

17

C. Immunoglobulin Types - Immunoglobulins can also be classified by the type of light chain that they have. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain. These differences are detected by serological means.1. Kappa light chains2. Lambda light chainsD. Immunoglobulin Subtypes - The light chains can also be divided into subtypes based on differences in the amino acid sequences in the constant region of the light chain.1. Lambda subtypesa) Lambda 1b) Lambda 2c) Lambda 3d) Lambda 4E. Nomenclature - Immunoglobulins are named based on the class, or subclass of the heavy chain and type or subtype of light chain. Unless it is stated precisely you are to assume that all subclass, types and subtypes are present. IgG means that all subclasses and types are present.F. Heterogeneity - Immunoglobulins considered as a population of molecules are normally very heterogeneous because they are composed of different classes and subclasses each of which has different types and subtypes of light chains. In addition, different immunoglobulin molecules can have different antigen binding properties because of different VH and VL regions.

FUNCTIONS AND PROPERTIES OF ANTIBODYNeutralizationDirect inactivation of pathogen or toxin thereby preventing its interaction with human cells

OpsonizationCoating of pathogens for more efficient phagocytosis

Activation of complementMore efficient phagocytosisDirect killing

19

20

IgGStructureMonomer (7S)

IgG1, IgG2 and IgG4

IgG3

IgGPropertiesMajor serum IgMajor Ig in extravascular spacesThe only antibody to cross the placenta Fixes complementBinds to Fc receptorsPhagocytes - opsonizationNK cells ADCC

drmsaiem BIOLOGICAL & CHEMICAL PROPERTIES OF IgsIgG. Is the major immunoglobulin in human serum, accounting for approximately 80%. Concentration of approximately 12 mg/ml

IgG is a monomer consisting of identical pairs of H and L chains linked by disulfide bridges.

Four subclasses of IgG have been identified, based on H chain differences: IgG1, IgG2, IgG3, and IgG4.

IgG is the only immunoglobulin that can cross the placenta in humans and protect the infant during the first months of life. IgG molecules are capable of binding complement by the classical pathway (except for the IgG4, which activate by the alternative pathway).

drmsaiemBIOLOGICAL AND CHEMICAL PROPERTIES OF IMMUNOGLOBULINSIgG is the major antibody produced in the secondary immune response

IgG has a half-life of approximately 23 days

Effective antitoxic immunity is exclusively IgG.

IgG is the major opsonizing immunoglobulin in phagocytosis; neutrophils have receptors for the Fc fragments of IgGI and IgG3.

IgG appears late but persists for longer period. It appears after initial response which is IgM in nature.

24

IgMStructurePentamer (19S) composed 5 H2L2 units plus one molecule of J chainExtra domain (CH4)J chain

C4

J Chain

IgMProperties3rd highest serum IgFirst Ig made by fetus and B cellsProduced early in the primary responseThe most efficient IgFixes complement

Tail Piece

Agglutinating IgBinds to Fc receptorsB cell surface Ig

IgM ANTIBODY OF THE IMMUNE RESPONSEFirst isotype produced in primary responseMay or may not be produced in secondary responseProduced before B cells undergo somatic hypermutationOccurs as pentamer with J chainFound primarily in blood and lymphMultiple binding sites confers high avidity and compensates for low affinity of monomersHighly effective in complement activationFunctions as rheumatoid factor. Represents about 8% to 10% of the total serum Igs . Concentration of I.2 mg/ml., half life is 5 days

27

Fixation of C1 by IgG and IgM Abs

C1rC1s

C1q

C1rC1s

C1qNo activationActivation

IgAStructureSerum monomerSecretions (sIgA)Dimer (11S), sIgA molecule consists of two H2L2 units plus one molecule each of J chain and secretory component(SC or SP)

J ChainSecretory Piece

IgA ANTIBODY OF THE IMMUNE RESPONSETwo subclasses (IgA1 and IgA2) and two forms (monomeric and dimeric)

MonomericLocated in blood and extracellular spacesPredominately IgA1Ratio of IgA1 to IgA2 is 10:1Functions as rheumatoid factor

DimericLocated in mucous membranes and secretionsPredominately IgA2Ratio of IgA2 to IgA1 is 3:2 J chain like IgM

30

Origin of sIgA: The SP is a polypeptide synthesized by epithelial cells that provides for IgA passage to the mucosal surface. It also protests IgA from being degraded in the intestinal tract.

YY

YY

YY

31

IgAProperties2nd highest serum IgMajor secretory Ig ( saliva, tears, respiratory, intestinal, and genital tract secretions.)Does not fix complement unless aggregatedBinds to Fc receptors on some cells

IgDStructureMonomerTail piece

Tail Piece

IgDProperties4th highest serum IgB cell surface IgDoes not bind complement

IgEStructureMonomerExtra domain (CH4)

C4

IgEStructurePropertiesLeast common serum IgAllergic reactionsParasitic infectionsDoes not fix complement

IgGIgAIgMIgDIgESedimentation coefficient (S)771978Molecular weight150,000160,000900,000180,000190,000Serum concentration(mg/l)1221.20.030.00004Half life (days)23652-81-5Daily production (mg/kg)34243.30.40.0023Intravascular distribution(%)4542807550Carbohydrate (%)38121312Complement fixation Classical ++-+++-- Alternative-+---Placental transport+----Present in milk++---Selective secretion by seromucus gland-+---Heat stability (56 C)++++-

ABNORMAL IMMUNOGLOBULINSThe earliest description of an abnormal immunoglobulins was the discovery Bence Jones (1847) of the protein that bears his name . Bence Jones protein is found typically in multiple myeloma.

It can be identified in urine by its characteristics property of coagulation when heated to 50C but redissolving at 70C. Bence Jones are light chain immunoglobulins and so may occur as kappa or lambda forms.

Multiple myeloma may affect plasma synthesising IgG , IgA, IgD or IgE. Similarly involvement of IgM producing cells is known as Waldenstroms macrolobulinemia. In this condition , there occurs excessive production of the respective myeloma protein (M protein) and of their chains (Bence Jones protein).

Cryoglobulinemia is a condition in which there is the formation of a gel or a precipitate on cooling the serum, which redissolve on warming. Most cryoglobulins consists of either IgG , IgM or their mixed precipitates.

How Do B Cells Produce Antibodies?B cells develop from stem cells in the bone marrow of adults (liver of fetuses).After maturation B cells migrate to lymphoid organs (lymph node or spleen). Clonal Selection: When a B cell encounters an antigen it recognizes, it is stimulated and divides into many clones called plasma cells, which actively secrete antibodies.Each B cell produces antibodies that will recognize only one antigenic determinant.

Clonal Selection of B Cells is Caused by Antigenic Stimulation

Consequences of Antigen-Antibody BindingAntigen-Antibody Complex: Formed when an antibody binds to an antigen it recognizes.Affinity: A measure of binding strength.1. Agglutination: Antibodies cause antigens (microbes) to clump together.IgM (decavalent) is more effective that IgG (bivalent).Hemagglutination: Agglutination of red blood cells. Used to determine ABO blood types and to detect influenza and measles viruses. 2. Opsonization: Antigen (microbe) is covered with antibodies that enhances its ingestion and lysis by phagocytic cells.

Consequences of Antibody Binding

Humoral Immunity (Continued)3. Neutralization: IgG inactivates viruses by binding to their surface and neutralize toxins by blocking their active sites.4. Antibody-dependent cell-mediated cytotoxicity: Used to destroy large organisms (e.g.: worms). Target organism is coated with antibodies and bombarded with chemicals from nonspecific immune cells.5. Complement Activation: Both IgG and IgM trigger the complement system which results in cell lysis and inflammation.

Consequences of Antibody Binding

Immunological MemoryAntibody Titer: The amount of antibody in the serum.Pattern of Antibody Levels During InfectionPrimary Response: After initial exposure to antigen, no antibodies are found in serum for several days. A gradual increase in titer, first of IgM and then of IgG is observed. Most B cells become plasma cells, but some B cells become long living memory cells.Gradual decline of antibodies follows.

Immunological Memory (Continued)Secondary Response:Subsequent exposure to the same antigen displays a faster and more intense antibody response.Increased antibody response is due to the existence of memory cells, which rapidly produce plasma cells upon antigen stimulation.

Antibody Response After Exposure to Antigen

drmsaiemAllo-antibodies & Auto-antibodiesAntigens that initiate the immune cascades results in the formation of either allo-abs or auto-abs.

Allo-antibodies are produced after exposure to genetically different or non-self, antigens of the same species.

Auto-antibodies are produced in response to self antigens.

drmsaiemLaboratory Examination of Reaction between Ags and Abs In vitro testing for the detection of Ags or Abs may be accomplished by a variety of immunologic techniques. Such techniques as agglutination, precipitation, agglutination inhibition, and hemolysis are the most commonly used methods to detect the presence of blood group Ags or Abs.

In blood bank testing, agglutination reactions are the major manifestation of the blood group Ag-Ab response. Typing for ABO, Rh, and other blood group Ags is accomplished by agglutination reaction.

drmsaiemLaboratory Examination of Reaction between Ags and AbsThere are two stages for agglutination to develop;

Stage 1: known as sensitization, Ab binding occurs. Antigenic determinants on the red cell membrane combine with the antigen combining site (Fab region) on the variable regions of the immunoglobulin heavy and light chains.

Stage 2: a lattice structure composed of multiple Ag-Ab bridges between Abs and red cell Ags is formed visible agglutination is present during this stage.

CONCLUSIONIn general ,IgG protects the body fluids, IgA protects body surfaces and IgM the blood stream, while IgE mediates reaginic hypersensitivity. IgD is a recognition molecule on the surface of B lymphocytes.

thank u!

Sheet1ClassesSubclassesIgGIgG1, IgG2, IgG3, IgG4IgAIgA1, IgA2IgMIgDIgE

Sheet2

Sheet3