Fatchiyah, Ph.D,

fatchiya@yahoo.co.id

Antigen - Antibodi

B

T

Antigen- specifictriggering

Proliferation

Differentiation

T H2T-cell"help"

T-cell killing: transplants, tumor cells

Delayed type hypersensitivity,(DTH); e.g. tuberculin reaction

Mixed Lymphocyte Reaction (MLR)

Suppression/Regulation (Tolerance)

ANTIBODY

T-CELL FUNCTIONS

ANTIGENAg/Ab complexes

Inactivation of viruses

Allergy

Complement

AUTOIMMUNITY

THREE "LIMBS" OF THE IMMUNE RESPONSE

AFFERENT CENTRAL EFFERENT

APC

Antigenprocessing

(M et al.)

CT

T S/R

AFC

Antigen"presentation"

Inflammation

Killing of bacteria

T H1

HI

CMI

ANTIBODY – Binds antigen

ANTIGEN – Is bound by antibody

ANTIGENIC DETERMINANT (=EPITOPE)Minimum structure to which Ab binds

ADJUVANT – Increases immune response

Figure 4-2

Figure 4-3

LH

L

L

LFd'

(Fc degraded)

FcFab

PAPAIN

PEPSIN

Fd

Intact IgG

L

Fab

F(ab')2

HH

H

H

H

L

IgG: 2 H-chains, 2 L-chainsFab: Ag-binding - specificityFc: non-specific functions

Figure 4-6

Figure 4-4

Figure 4-7

Figure 4-8

Figure 4-9

Figure 4-14

Figure 4-13

Figure 4-15

Figure 4-17

Figure 4-20

Figure 4-21

Preci- agglu- fixes

size binds? pitates? tinates? lyses? C’?

IgG 150K yes yes yes yes yes

F(ab)2 100K yes yes yes no no

Fab 50K yes no no no no

Fc 50K no no no no no

Activities of IgG and its Subunits

C-fixation requires Fc

Precipitation & agglutination require bivalency

Heavy Chain determines class & subclass

All Igs share the same pool of Light Chains, kappa and lambda

All Igs share same basic “IgG-like” structure

Empirical formulae: IgG1=(g1)2,l2

IgA= [(a2)2,k2]2

CHARACTERISTICS OF HUMAN IMMUNOGLOBULINS

serumSub- conc. sedim. # binding H-chain Biological

Class classes mg/ml coeff. size sites class properties

IgG 4 12 7S 150 KDa 2 g C-fixing,(g1, g2, placental X-ferg g3, 4)

IgM - 1 19S 900 KDa 10 m C-fixing,B-cell surface I

IgA 2 2 7,9,11S (160 KDa) n 2,4,6 a secretory Ig( 1

,a a2)

IgD - 0.03 7S 180 KDa 2 d B-cell surface Ig

IgE - 0.0003 8S 200 KDa 2 e "reaginic" Ig

+ X +

Serum Electrophoresis - myeloma, scan

L-chain

H-chain

COOH-terminus

Constant RegionVariable Region

CDR's

NH -terminus2

Compare H-chains and L-chains of different myeloma proteins (within the same class and type),

define Constant and Variable regions

CL

VL

VH

C

CHIII

HII

CHI

● Globular domains & domain interactions

● Ig structure is stabilized by covalent& non-covalent forces

Structure of Mouse IgG2Harris et al., Nature 360:369-72, 1992

CL

VL

VH

C

CH

III

HII

CH

I

Three schematics of IgG

Chains, V- & C-regions, Domains

Ag1Ag2 Ag3

Ab AbAb

GOOD FITNO FITMODERATE FIT(CROSS-REACTIVE, LOWER AFFINITY)

Structural basis for specificity & cross-reaction

Ag/Ab interactions depend on non-covalent forces

Principles of Ag/Ab Reactions:

Abs bind epitopes(heterogeneity of Abs)

Different epitopes --> different Abs(heterogeneity of epitopes)

Binding is reversible (equilibrium)

SerumAb

IgMIgG

1 Ago

1234

weeks

IgM

IgG

1234

weeks2 Ago

PRIMARY AND SECONDARY HUMORAL RESPONSES

SLOW

LOW Ab LEVELS

SHORT-LIVED

MAINLY IgM

LOW AFFINITY

PRIMARY RESPONSESECONDARY RESPONSE

FAST

HIGH Ab LEVELS

LONG-LIVED

MAINLY IgG

HIGH AFFINITY

(kinetics of

clonal selection)

(Ig structure)

Immunological Memory

Manifestations of Antibody

Physical EffectsPrecipitation (soluble Ag)Agglutination (particulate Ag)Binding

Biological EffectsProtectionImmobilizationCytolysisOpsonization

Defining Property of Abs: SPECIFIC BINDING

Amountof ppt.

Amount of added Ab

Antigenexcess Antibody

excess

Equivalence

Ag

Ab

Antibody Precipitation:The Precipitin Curve

Measuring Antibody/Antigen Reactions

Binding is invisible: how to detect & quantitate?

1) Precipitation: Ouchterlony

2) Solid-state binding, labelled antibody: RIA/ELISA

4) Binding of complement: Complement Fixation(hemolysis)

3) Liquid-phase binding: Equilibrium Dialysis

HTf

HTf HSA

BSAAb

completeidentity

non-identity

partialidentity

X

X

Y HSA

BSA

Ouchterlony Analysis:2-Dimensional Precipitation in Agarose

Appendix 2

Ab

HSA

Ab

HSA

Ab

HSA

Ab

HSA

Ab

HSA

Ab

HSA

Ouchterlony: Double Diffusion in Agarose

Ab = rabbit anti-HSA

Reversible binding; linesmay move by dissovlingand re-precipitating…

Ouchterlony: Double Diffusion in Agarose

HSA

Ab

BSA

HSA

Ab

BSA

Ab

HSA

BSA

Ab

HSA

BSA

Ab

HSA

BSA

Ab

HSA

BSA

aX,aY aYX

X

Y HSA

BSA

Ab = rabbit anti-HSA

Reaction of Partial Identity

X

X,Y

HTf

HTf HSA

BSAAb

completeidentity

non-identity

partialidentity

X

X

Y HSA

BSA

Ouchterlony Analysis:2-Dimensional Precipitation in Agarose

BSA

BSA

BSA

BSA

BSA

BSA

HSA

Antiserum "A" 3M Urea

Pass-through;absorbed antiserum "B"

Eluate;Affinity purified antibody "C"

ABSORPTION OF HSA ANTISERUM

HSA BSA

A

HSA BSA

B

HSA BSA

C

(AFFINITY CHROMATOGRAPHY)

aX+aY

aY aX

aYaX+aY aX

X,Y X,Y X,YX X X

See Handout #3

Precipitation in tubes

Ab

Ag

Radial Immunodiffusion(horse serum IgG)

Passive hemagglutinationMHA-TP - MicroHemagglutination for T. Pallidum

Reverse Agglutinationanti-StrepA antibodies coated onto latex beads

ELISA - Enzyme-Linked ImmunoSorbent Assay

Western Blot for HIV

ImmunofluorescenceT-cells in mouse lymph node

Immunofluorescenceanti-nuclear antibodies on HEp-2 cells

globulinsalbumin

g aa

ELECTROPHORESIS OF NORMAL SERUM

Serum Electrophoresis - normal serum, scan

Electrophoresisnormal serum, stain & immunoblot

ElectrophoresisIgGl myeloma, stain & immunoblot