ANTIBODYANTIBODY

Babitha EliasBabitha Elias

DEFINITIONDEFINITION

Antibodies are substances which are Antibodies are substances which are formed in the serum or tissue fluids in formed in the serum or tissue fluids in response to an antigen.response to an antigen.

Antibodies react with antigen Antibodies react with antigen specifically and in an observable specifically and in an observable manner.manner.

Immunoglobulin – proteins of animal Immunoglobulin – proteins of animal origin, endowed with known Ab origin, endowed with known Ab activity & for certain other proteins activity & for certain other proteins related to them by chemical structure.related to them by chemical structure.

Properties of antibodiesProperties of antibodies Chemical nature of antibodies is Chemical nature of antibodies is

globulin – immunoglobulinglobulin – immunoglobulin Constitute about 20 – 25% total serum Constitute about 20 – 25% total serum

proteins.proteins. Based on sedimentation studies – 7S Based on sedimentation studies – 7S

(M.W – 1,50,000- 1,80,000 KD). (M.W – 1,50,000- 1,80,000 KD). Heavier antibodies – 19S globulins Heavier antibodies – 19S globulins (M.W – 900,000)(M.W – 900,000)

Kabat showed, on electrophoretic Kabat showed, on electrophoretic mobility, the antibodies belong to mobility, the antibodies belong to gammaglobulins gammaglobulins

Kabat, 1939Kabat, 1939

Structure of immunoglobulinsStructure of immunoglobulins Porter, Edelman and NisonoffPorter, Edelman and Nisonoff Antibody molecule consists of two Antibody molecule consists of two

identical heavy chains (H) and two identical heavy chains (H) and two identical light chains (L).identical light chains (L).

Heavy chains are longer & light chains Heavy chains are longer & light chains are shorter.are shorter.

Both chains are polypeptide in nature.Both chains are polypeptide in nature. 2 heavy chains are held together by 2 heavy chains are held together by

disulphide bonds.disulphide bonds. Each light chain is attached to a heavy Each light chain is attached to a heavy

chain by disulphide bond.chain by disulphide bond.

The H chains The H chains are structurally and are structurally and antigenically distinct in different antigenically distinct in different classes of immunoglobulins.classes of immunoglobulins.

5 different classes of 5 different classes of immunoglobulins depending on the immunoglobulins depending on the presence of heavy chain.presence of heavy chain. Ig G – Ig G – γγ Ig M - Ig M - μμ Ig A - Ig A - αα Ig E - Ig E - εε Ig D - Ig D - δδ

The L chainsThe L chains are similar in all classes of are similar in all classes of immunoglobulins.immunoglobulins.

Present in two forms – kappa (K) and lambda Present in two forms – kappa (K) and lambda (L).(L).

Effect of enzymes on ImmunoglobulinsEffect of enzymes on Immunoglobulins

Papain DigestionPapain Digestion Papain can digest immunoglobulin molecule Papain can digest immunoglobulin molecule

into 3 fragments – into 3 fragments – one Fcone Fc (Fragment (Fragment crystallisable) and crystallisable) and 2 identical Fab2 identical Fab (fragment (fragment antigen binding)antigen binding)

The 2 Fab fragments possess the antigen The 2 Fab fragments possess the antigen binding sites but the Fc fragment lacks the binding sites but the Fc fragment lacks the ability to bind antigen.ability to bind antigen.

Each Fab fragment consists of a light Each Fab fragment consists of a light (L) chain & a part of H chain(L) chain & a part of H chain

Portion of H chain in Fab fragment – Portion of H chain in Fab fragment – Fd regionFd region

Fc fragment consists of both the H Fc fragment consists of both the H chains.chains.

It determines the biological It determines the biological properties of the Ig – complement properties of the Ig – complement fixation, placental transfer etc.fixation, placental transfer etc.

Pepsin digestionPepsin digestion Pepsin cleaves immunoglobulin into Pepsin cleaves immunoglobulin into

1 Fc1 Fc portion & portion & 2 Fab2 Fab fragments held fragments held together in position.together in position.

The Fab is bivalent & still it can The Fab is bivalent & still it can precipitate with antigen – F(ab’)2precipitate with antigen – F(ab’)2

Pepsin degrades the Fc portion into Pepsin degrades the Fc portion into smaller fragments. smaller fragments.

Pepsin digestion

H chains & L chainsH chains & L chains Consists of two portions – a variable Consists of two portions – a variable

region (V) & a Constant region (C).region (V) & a Constant region (C). In the L chain, the 2 regions are of In the L chain, the 2 regions are of

equal length, while in the H chain, equal length, while in the H chain, the V region constitute only a fifth of the V region constitute only a fifth of the chain.the chain.

Variable regions are in the amino Variable regions are in the amino terminus (NHterminus (NH22) and Constant region ) and Constant region at carboxyterminus (COOH)at carboxyterminus (COOH)

IMMUNOGLOBULIN CLASSESIMMUNOGLOBULIN CLASSES

IMMUNOGLOBULIN G (Ig G)IMMUNOGLOBULIN G (Ig G) Major serum immunoglobulin Major serum immunoglobulin

– 80% of total amount.– 80% of total amount. The normal serum The normal serum

concentration – 8-16 mg/ml.concentration – 8-16 mg/ml. Molecular weight – 150,000 Molecular weight – 150,000

(7S)(7S) Half life – 23 daysHalf life – 23 days It can cross the placenta – It can cross the placenta –

Transplacental Transplacental

It is distributed equally between the It is distributed equally between the intravascular & extravascular intravascular & extravascular compartments.compartments.

Ig G appears late but persists long. It Ig G appears late but persists long. It appears after the initial immune appears after the initial immune response.response.

It participates in precipitation, It participates in precipitation, complement fixation, & complement fixation, & neutralization of toxin & viruses.neutralization of toxin & viruses.

Ig G binds to microorganisms & Ig G binds to microorganisms & enhances phagocytosis.enhances phagocytosis.

Catabolism of Ig G –Catabolism of Ig G – When Ig G level is raised, the synthesis of Ig G When Ig G level is raised, the synthesis of Ig G

against that particular Ag is catabolised rapidly & against that particular Ag is catabolised rapidly & result in deficiency of particular Ab. Eg: myeloma result in deficiency of particular Ab. Eg: myeloma & kala-azar.& kala-azar.

In hypogammaglobulinaemia, Ig G antibody given In hypogammaglobulinaemia, Ig G antibody given for therapeutic purpose will be catabolised slowly.for therapeutic purpose will be catabolised slowly.

Passively administered Ig G suppresses the Passively administered Ig G suppresses the homologous antibody synthesis by feed back homologous antibody synthesis by feed back mechanism. mechanism.

4 sub classes of Ig G – Ig G1, Ig G2, Ig G3, Ig 4 sub classes of Ig G – Ig G1, Ig G2, Ig G3, Ig G4G4

It is protective against microorganisms which It is protective against microorganisms which are active in the blood and tissues.are active in the blood and tissues.

IMMUNOGLOBULIN A (Ig A)IMMUNOGLOBULIN A (Ig A) Second major serum immunoglobulin – 10- Second major serum immunoglobulin – 10-

13%.13%. Normal serum concentration – 0.6-4.2 mg/ml.Normal serum concentration – 0.6-4.2 mg/ml. Half life – 6 – 8 days.Half life – 6 – 8 days. Ig A occurs in two forms – Serum Ig A & Ig A occurs in two forms – Serum Ig A &

Secretory Ig A.Secretory Ig A. Serum Ig A Serum Ig A is a is a monomermonomeric 7S molecule.(MW: ic 7S molecule.(MW:

160,000), While Ig A in the mucosal surfaces 160,000), While Ig A in the mucosal surfaces & secretions (& secretions (Secretory Ig ASecretory Ig A, MW: 400,000) is , MW: 400,000) is a a dimerdimer. It is formed by two monomer units . It is formed by two monomer units joined together by a glycoprotein – joined together by a glycoprotein – J chainJ chain

J chainJ chain – joins the 2 monomers at their – joins the 2 monomers at their carboxy terminalscarboxy terminals. Secretory Ig A & J . Secretory Ig A & J chain are produced by the chain are produced by the plasma cellsplasma cells..

Secretory Ig A contains a secretory piece, Secretory Ig A contains a secretory piece, S piece is synthesised by mucosal & S piece is synthesised by mucosal & glandular epithelial cells. The S piece glandular epithelial cells. The S piece protects Ig A from denaturation by protects Ig A from denaturation by bacterial proteases in sites such as bacterial proteases in sites such as intestinal mucosa.intestinal mucosa.

Ig A is present in secretions such as milk, Ig A is present in secretions such as milk, saliva, tears, sweat, nasal fluids etc. It saliva, tears, sweat, nasal fluids etc. It protects the mucus membranes against protects the mucus membranes against microorganisms..microorganisms..

Ig A cannot fix complement, but can Ig A cannot fix complement, but can activate alternative complement activate alternative complement pathway.pathway.

Ig A is synthesised locally by plasma Ig A is synthesised locally by plasma cells.cells.

2 subclasses of Ig A – Ig A1 & Ig A22 subclasses of Ig A – Ig A1 & Ig A2

IMMUNOGLOBULIN M (Ig M)IMMUNOGLOBULIN M (Ig M) Ig M is a Ig M is a pentamerpentamer consisting of consisting of 5 5

monomersmonomers joined by a J chain. joined by a J chain. It constitutes about 5-8% of total It constitutes about 5-8% of total

serum concentration.serum concentration. Normal level – 0.5-2 mg/mlNormal level – 0.5-2 mg/ml Half life – 5 days.Half life – 5 days. Molecular weight – 19S (900,000-Molecular weight – 19S (900,000-

1,000,000)1,000,000) ‘‘Millionaire molecule’Millionaire molecule’ Distributed intravascularly.Distributed intravascularly.

It is the earliest synthesised Ig by foetus It is the earliest synthesised Ig by foetus in about 20 weeks of age.in about 20 weeks of age.

It appears early in the infection before It appears early in the infection before Ig G. Ig M Abs are short lived – presence Ig G. Ig M Abs are short lived – presence indicates recent infection.indicates recent infection.

It cannot cross placenta – presence in It cannot cross placenta – presence in new born indicates congenital infection.new born indicates congenital infection.

It participates in agglutination, It participates in agglutination, complement fixation, opsonisation & complement fixation, opsonisation & immune hemolysisimmune hemolysis

Ig M provides protection against blood Ig M provides protection against blood invasion by microorganisms.invasion by microorganisms.

Ig M monomers appear on the Ig M monomers appear on the surface of unstimulated B surface of unstimulated B lymphocytes & act as receptors for lymphocytes & act as receptors for antigens.antigens.

Two subclasses – Ig M1 & Ig M2.Two subclasses – Ig M1 & Ig M2.

IMMUNOGLOBULIN E (IgE)IMMUNOGLOBULIN E (IgE) Produced in the lining of respiratory & Produced in the lining of respiratory &

intestinal tracts.intestinal tracts. Also known as Also known as “Reagin”“Reagin” MW- 190,000 (8S)MW- 190,000 (8S) Half life 2-3 days Half life 2-3 days Resembles Ig G structurallyResembles Ig G structurally Heat labile (Inactivated at 56 Heat labile (Inactivated at 56 o o C for 1 C for 1

hr)hr) It has affinity for surface of tissue cells It has affinity for surface of tissue cells

– Mast cells .(Homocytotropism)– Mast cells .(Homocytotropism)

Ig E mediates Type-I hypersensitivity Ig E mediates Type-I hypersensitivity Responsible for asthma, hay fever, Responsible for asthma, hay fever,

eczema, eczema,

Prausnitz-Kustner (P-K) reaction.Prausnitz-Kustner (P-K) reaction. Cannot cross the placental barrier.Cannot cross the placental barrier. Plays role in defense against parasitic Plays role in defense against parasitic

infection.infection.

IMMUNOGLOBULIN D (Ig D)IMMUNOGLOBULIN D (Ig D) It resembles Ig G structurally.It resembles Ig G structurally. Serum concentration – 0.03 mg/mlSerum concentration – 0.03 mg/ml Intravascular in distribution.Intravascular in distribution. MW – 180,000 (7S)MW – 180,000 (7S) Half life – 3days Half life – 3days It also act as receptors for antigen, It also act as receptors for antigen,

like monomeric IgM .like monomeric IgM . 2 subclasses –IgD1, IgD 22 subclasses –IgD1, IgD 2

Fig. 4-13

~80%

~10-15%(High dailyProduction)

~5-10%

~0.002%

~0.2%

Fig. 4-13Fig. 4-13

~80%

~10-15%(High dailyProduction)

~5-10%

~0.002%

~0.2%

Role of different Ig classesRole of different Ig classes

IgG – Protects the body fluidIgG – Protects the body fluid

IgA - Protects the body surfaceIgA - Protects the body surface

IgM – Protects the blood streamIgM – Protects the blood stream

IgE - Mediates reaginic IgE - Mediates reaginic hypersensitivityhypersensitivity

IgD- Recognition molecule on the IgD- Recognition molecule on the surface of B lymphocytessurface of B lymphocytes

ABNORMAL IMMUNOGLOBULINSABNORMAL IMMUNOGLOBULINS

Apart from antibodies, other Apart from antibodies, other structurally similar proteins may be structurally similar proteins may be found in the serum in the following found in the serum in the following conditions.conditions. Multiple myelomaMultiple myeloma Heavy chain diseaseHeavy chain disease CryoglobulinaemiaCryoglobulinaemia

Multiple myelomaMultiple myeloma It is a plasma cell dyscrasia in which It is a plasma cell dyscrasia in which

unchecked proliferation of one clone of unchecked proliferation of one clone of plasma cells resulting in the excessive plasma cells resulting in the excessive production of particular Ig.production of particular Ig.

Multiple myeloma involve plasma cells Multiple myeloma involve plasma cells synthesizing any of the 5 classes of Ig.synthesizing any of the 5 classes of Ig.

Multiple myeloma involving IgM Multiple myeloma involving IgM producing plasma cells – producing plasma cells – “Waldenstrom’s Macroglobulinaemia”“Waldenstrom’s Macroglobulinaemia”

Excessive production of light chains – Excessive production of light chains – Bence Jones proteins.Bence Jones proteins.

It can be identified in urine – proteins It can be identified in urine – proteins get coagulated at 50get coagulated at 50ooC but redissolve C but redissolve at 70at 70ooCC

Heavy chain diseaseHeavy chain disease Abnormal heavy chains are produced Abnormal heavy chains are produced

in excess. This is due to lymphoid in excess. This is due to lymphoid neoplasia.neoplasia.

Cryoglobulinaemia Cryoglobulinaemia It is a condition in which there is It is a condition in which there is

formation of precipitate on cooling formation of precipitate on cooling the serum, which redissolves on the serum, which redissolves on warming.warming.

Found in macroglobulinaemia, Found in macroglobulinaemia, autoimmune conditions like SLE.autoimmune conditions like SLE.

Most cryoglobulins consists of either Most cryoglobulins consists of either Ig G or Ig M.Ig G or Ig M.

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