Antigen - Antibodi · Mixed Lymphocyte Reaction (MLR) ... ANTIBODY T-CELL FUNCTIONS ANTIGEN Ag/Ab...
Transcript of Antigen - Antibodi · Mixed Lymphocyte Reaction (MLR) ... ANTIBODY T-CELL FUNCTIONS ANTIGEN Ag/Ab...
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