Antibody & Function

8/2/2019 Antibody & Function

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BCR

SurfaceIg/Ab

Antibody

C omparison of BCR and T CR

960s1984

BCR & T CR:Receptors bind to molecular surfaces (usually proteins)

Receptors exist in trillionsof varieties

Receptors share structural &sequence homology but arenot identical.


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E1 E2 F

albumin

globulins

Mo bility on electr opho resis

A m o u n

t o f p r o

t e i n

K

1939, T iselius & Kabat:

Demonstration of the presence of Ab in a particular serum protein fraction

+ -

Ex p erime n t

Immu n ized rabbit wit h Ovaalbumi n (Ag)

Serum c ollected

Tw o po rti on s P1 & P2

P1 on gel electr opho resis

P2 treated wit h OVA pp trem oved a n d t h en sam p le

on electr opho resis gel

Mo bility vs am oun t of p r otei n p lotted on gra ph


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Immune serum

Ag adsorbed serum

Mo bility

E1 E2 F K

albumin

globulins

A m o u n

t o

f p r o

t e i n

+ -


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Ab150 kd 2

x 50 kDa + 2 x 25 kDa

Gerald Edelman , in 1959

Reducing agent

(Beta-mercaptoethanol/BME )

S-S

BME

SH HS+

BME+ + +

B iochemical characterization of Ab


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Pa pai n

B y R odney Porter 1959


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R odn ey P orter built a m odel

Ab molecule Y shapedStem and two branches

Branch consist of Lc + Hc (half)

Stem consists of two Hc(remaining halves)

The structure of free tips of the

branches bind specifically to Ag

Separately Lc and Hc areinactive


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Gerald M. Edelman

Born 1929, New York

Rodney R. Porter

191 7 198 5 , EnglandRockefeller University University of Oxford

Oxford, United Kingdom


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C h alle n ge?Ab purified from serum

N-terminal sequencing of Lc/Hc

Frustrating!!!!!

Data made no sense?

Multiple amino acids at each positionSample heterogeneous

Homogeneous source requiredMyeloma proteins produced by B cell tumors

Hilschmann and Craig (196 5 ): Lc SequencedThe first half varies variable region

The second half identical constant region

T h e a n tige n bi ndi n g site must be at t h e ti p of t h e bra n ch.


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R obert o P oljack


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Antibody molecule composed of structurally similar subunits calledImmu no globuli n (Ig)- like d omai n s.

These are a feature of many, many proteins in the immune (and neural!) systems(the Ig superfamily of proteins).

The V-region is one such domain and each C-region another.

Each of these domains contains an internal disulfide bond

Structured as a barrel of F-p leated s h eets (2 sets of anti-parallel strands).

It is important to remember that the structure of the BCR is the same as the

structure of the an tib ody - the only difference is that it is membrane bound.

The membrane bound and secreted forms of immunoglobulin are sp lice varia n ts .

Structural Features of a n tib ody : crystal structure


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Each domain is a barrel- shaped structure

strands running in opposite directions ( antiparallel ) pack together to form twosheets (shown in yellow and green in the diagram of the C domain) Sheets held together by a disulfide bond The strands are lettered sequentially with respect to the order of their occurrence in the amino acid sequence of the domains; the order in each sheet

is characteristic of immunoglobulin domains The strands C' and C'' that are found in the V domains but not in the Cdomains are indicated by a blue shaded background The characteristic four-strand plus three-strand (C-region type domain) or four-strand plus five- strand (V-region type domain) arrangements are typical

immunoglobulin superfamily domain building blocks, found in a whole range of other proteins as well as antibodies and T-cell receptors

Foldi n g p atter n of C L an d V L domai n s

A barrel : hollow cylindrical container


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An tib odiesClass of glycoproteins called immunoglobulins

Y shaped

Arms of Y recognize and bind antigen

Tail of Y responsible for biological activity

Present in body fluids (saliva, tears, urine and milk)

Blood serum has highest concentration

Found floating free in blood serum

Also act as B-cell receptor (antigen binding receptor)


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B ASI C ST RUC T UR E OF I MM UN OG L O BUL IN S

Disulfide b on ds

C arb oh ydrates

Hi n ge R egi on

HeavyC

h ai n Heavy C h ai n

L igh t C h ai n

V H

C H1

C H2

C H3

V L

C L


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B ASI C ST RUC T UR E OF I MM UN OG L O BUL IN S Four chain (polypeptides) structure

Identical light chains (23kD) = 2 Identical heavy chains ( 5 0-7 0kD) = 2 Inter-chain disulfide bonds Intra-chain disulfide bonds Both chains are divided into variable(V) and constant(C)

regions based on variability in the amino acid sequences Light Chain - V L and C L / Heavy Chain - V H and C H Hinge Region is flexible/mobile/swing Carbohydrates (glycoproteins) attached to the C H2 domain


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ST RUC T UR E OF TH E VA R IA BL E R E GIO N

Hy p ervariable (HV R ) or c om p leme n tarity determi n in g regi on s (C DR )

Framew ork regi on s

Region that bind to epitopes Antigen binding site (ABS) Determines the binding of Ig to a specific epitope/antigen Also called com p leme n tarity determi n in g regi on s (C DR )

CDR: short, 6 10 aa

Less variability in aa sequence as compared to HVR


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Five different classes

1. IgG - Gamma heavy chains

2. Ig M - Mu heavy chains3. IgA - Alpha heavy chains4. IgD - Delta heavy chains5 . Ig E - Epsilon heavy chains

Sp ecific structural a n d fu n cti on al p r op erties

Differe n ces i n th e aa seque n ces i n th e c on sta n t regi on of t h e h eavy c h ai n s


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C hain composition of the fiveimmunoglobulin classes in humans


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1. Most abundant class in serum2. 80% of total serum Ig3. C ontains two K-heavy chains4. The subclasses differ in the number of disulfide

bonds and length of the hinge region


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Numbered according to their decreasing average serum concentrationsSubclasses differ i n th e size of t h e h in ge regi on and also in th e n umber a n dpo siti on of t h e in terc h ai n disulfide bonds (thick black lines) linking the heavy chains. Notable feature of human lgG3 is its 11 inter-chain disulfide bonds


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B iological Function

IgG (1, 3, 4) is the only class of Ig that crosses the placenta and protects the develo ping fetus

IgG (3>1>2) E ffective com plement activator

(C lassical pathway )

IgG (1,3>4>2) bind with high affinity Fc rece ptors on phagocytic cells and mediate opsonization.

IgG


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Constitute 5 - 10% of total serumIgs

B-cell membrane bound IgG ismonomeric

Free form of Ab is p en tameric

Pentamers held by S-S bonds between C-terminus of heavychain domains (C4+ C4) &(C3+ C3)

Fc in center and 10 Ag binding

region (Fab) on periphery

Pentamer contains J (joining)-chain (linked by S-S to 2 of 10 chains)


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Fu n cti on s:1. Secret ory IgA p r otects muc ous membra n es fr om bacterial c olon izati on

an d viral i n fecti on (en try site f or p at ho gen s)2. Im po rta n t li n e of defe n se agai n st bacteria suc h as Salmonella, Vibrio

cholerae etc.3 . C om p lex of sIgA a nd p at ho gen rem oved by ph ag ocyt osis/ p eristalsis of gut4. Mo th ers milk = sIgA = p r otects n ew b or n5. IgA is t h e maj or class of Ig i n secreti on s - tears, saliva, mucus


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S tructure and formation of secretory lgA,Only 10 -15 % of the total Ig in serum, but predominant in external secretions

Most serum IgA exists as a monomer, although dimers, trimers, & tetramersare sometimes present.

In contrast, secretory IgA is formed during transport through mucousmembrane epithelial cells ( dimer or tetramer )


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0.2% of t otal serum Ig Its r ole in serum is u n certai n IgD al on g wit h Ig M is maj or membra n e b oun d Igex p ressed on B -cell/ BCR

N o bi ological effect or fu n cti on h as bee n ide n tifiedf or IgD

O n e Disulfide b on d


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IgE is the least common serum Ig It binds very tightly to Fc receptors on basophils

and mast cells even before interacting with Ag Involved in allergic reactions


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35F IGU RE 4 -16

Allergen cross - linkage of receptor -bound lg E on mast cells

Des p ite its e x tremely l ow serum c on c.( 0 .3 ug/ml) Ig E mediates H .S ( h ay fever,

ast h ma, h ives, a n aph ylactic s ho ck )


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Antigenic determinants/ E pitopes onimmunoglobulins

Ab (glycoproteins) can themselves function as potentimmunogens to induce Ab responses.

+ : powerful tools for the study of B -cell development: Ab responses/ Immunotechniques

- : obstacles to passive Ab therapy

Three major catagories of epitopes on Ig molecules:Isotype, Allotype, & IdiotypeE ach located in characteristic portion of the molecule


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ISOTYP E

Is oty p ic determi n ats are c on sta n t regi on determi n an ts C

ollectively defi n es H-c h ai n class a n d subclass &L

-ch ai n ty p ean d subty p e (al ph a, delta, mu, gamma, e p silon , ka pp a,lambda)

E ac h isoty p e in en coded by a se p arate c on sta n t regi on gen e. All members of a s p ecies carry t h e same c on sta n t regi on gen e

(differe n t/multi p le alleles po ssible) Differe n t s p ecies i nh erit differe n t c on sta n t-regi on gen es T h e is oty p ic determi n an ts on an Ab fr om on e s p ecies will be

rec ogn ized as f oreig n in ano th er s p ecies


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Allotype

Although all members of a species inherit the same set of isotypic genes, multiple alleles exist for some of the genes.[ subtle (1~4 a.a.) differences in some, but not all, members of a species]

Ab to allotypic determinants can arise from a blood transfusionor be produced by a pregnant mother in response to paternalallotypic determinants on the fetal Ig.


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IDIOTYP E

VH an d V L domai n s h ave u n ique a .a seq . = able t o rec ogn izediffere n t Ag

E ac h un ique regi on ca n fu n cti on as a n tige n ic determi n an tscalled Idi otop e

R egi on outside Ag bi n di n g site i n V - regi on ca n als o act as

Idi oty p e T h e sum of t h e in dividual idi otop es (eac h in dividual a n tige n ic

determi n an t) is called idi oty p e of t h e a n tib ody An idi otop e ca n be t h e actual a n tige n -bi n di n g site or no t in jecti on of a ho m ogen eous Ab i n to a reci p ien t w ho is

gen etically u n ide n tical t o th e d ono r ca n p r oduce a n ti-idi oty p e Ab


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40F IGU RE 4 -17

Antigenic determinants of immunoglobulinsAntigenic determinants on Ig

( Am on g differe n t i n bred strai n s )


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H ow m Ig/ BCR mediate activating signal after contactwith an Ag?

G eneral structureof

the B -cellreceptor ( BCR )= mIg + Ig a/Ig b

Transmembrane protein complexes

C yt op lasmic tail very s ho rtmIg M mIgD = 3 aamIgA = 14 aa

mIgG mIg E = 28 aa

Too sho rt t o ass ociate wit h sig n ali n g m olecules (PTKs,GPs)

mIg d oes no t c on stitute t h e e n tire Ag bi n di n g rece p tor


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G eneral structure of the B -cell receptor ( BCR )

BCR is a tra n smembra n ep r otei n com p lexC om po sed of mIg +S-S linked Heterodimer

+ Ig a/Ig b

BB --C ell R ece p torC ell R ece p tor


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M embra n e glyc op r otei n s

Affi n ity f or Fc po rti on of Ab

Im po rta n t f or bi ologicalfu n cti on s


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Four distinct roles of Fc binding proteins

The movement of Ab across cell membranes :

poly Ig R for s IgA & to some extent, pentameric IgM The transfer of lg G from mother to fetus across the placenta :

Fc R N (neonatal Fc receptor) Trigger effector functions : Opsonization or AD CC

C ross - linking of Fc R generates immuno -regulatory signals

that affect cell activation, differentiation, etc

BB C ll RC ll R


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F IGU RE 4 -19

The structure of a number of human Fc -receptors

Accessory signal-transducing polypeptide

Fc-binding polypeptide

BB --C ell R ece p torC ell R ece p tor

ff


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46F IGU RE 4 -20

S ome members of the Ig superfamilyIgIg Su p erfamilySu p erfamily

@ T h ey h ave t h e c h aracteristic Ig-f old structure >>> c omm on evoluti on ary a n cestry( ge n e du p licati on & later diverge n ce ) : facilitati n g in teracti on s betwee n membra n ep r otei n s occurri n g betwee n th e faces of b p leated s h eets as well as Ag bi n di n g


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Derived fr om differe n t B -cell li nes N atural mi x ture of a n tib odies secreted agai n st a s pecific

an tige n E

ac h Ab rec ogn izes a differe n t e p it op e Pr oduced by immu n izi ng a n an imal wit h th e a pp r op riate

an tige n T he immu n ized a n imals serum is c ollected

An tib odies ca n then be p urified fr om t he serum L ess e x pen sive

P olycl on al A n tib ody

Mono clon l A n tib odiesMono clon l A n tib odies


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48F IGU RE 4 -21

The conventional polyclonal antiserum containsa mixture of monoclonal antibodies

Mono clon al A n tib odiesMono clon al A n tib odies

mAb is specific for one epitopeon a complex Ag


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Derived from a single B-cell lineMonoclonal antibodies recognize one epitope only.

Process begins by immunizing an animal with an antigen.

The animals spleen is removed.

B-cells are fused with myeloma cells resulting in hybridomas.Hybridomas are screened to find those producing antibodies to the

antigen with which they were immunized.

Each hybridoma cell is derived from one B-cell so the antibodies

that a clonal population of hybridoma cells produce are monoclonalantibodies.

Expensive to produce

Mono clon al A n tib ody (mAb or m oAb) :


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Monoclonal antibody and Hybridoma



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51F IGU RE 4 -22 (a)

( Toxins used to prepare immuno -toxins include ricin, S higella toxin,& diphtheria toxin )


m Ab have important clinical uses in diagnostic , imaging , & thera peutic agents


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F IGU RE 4 -22 (b)

Diphtheria toxin binds to a cell -membrane receptor(L.) &a diphtheria - immunotoxin to a tumor -associated antigen( R . )


mAb or Fab specific f or


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P reP re--clinical and Clinical development programsclinical and Clinical development programs(200 major companies in 2004)(200 major companies in 2004)

C hemicals B iological

978 399

C hemicals vs B iologicals

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