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1
ANTIBODIES
Chapter 4
http://bcs.whfreeman.com/immunology5e/default.asp?s=&n=&i=&v=&o=&ns=0&uid=0&rau=0
Ig M
What do you know about Abs so far?
• B-lymphocytes• Plasma cells• 2 major functions?
– Binding foreign Ags– Effect neutralization or elimination
• Plasma vs. serum?• Ab response to specific Ag?
– Heterogeneous– polyclonal
2
Serum electrophoresis
Tiselius and Kabat: 1939…describe experiment with rabbits
Ab amino acid sequence
• Heterogeneous population• Multiple myeloma• Myeloma cell• Myeloma protein• Bence-Jones proteins• Plasmocytomas: PC• MOPCs: mineral oil PCs
3
Antibody (immunoglobulin) structure: 4 protein chains
Carboxyends
Aminoends
lSSl
lSSl
lSSl
lSSl
light
light
heavy
heavy
Heterodimer
Immunoglobulin structure
IS
S l
IS
Sl
IS
Sl
ISSl
I S S l
IS
Sl
IS
Sl
IS
Sl
ISSl
ISSl
ISSl
IS
Sl
IS
Sl
IS
Sl
I S S lI SS
l
Antibody details
IS
S l
IS
Sl
IS
Sl
ISSl
I S S l
IS
Sl
IS
Sl
IS
Sl
ISSl
ISSl
ISSl
IS
Sl
IS
Sl
IS
Sl
I S S lI SS
l
214 residuesLIGHT CHAIN
446 residuesHEAVY CHAIN
variable
constant
NH3
COO
Antigen binding regions
4
Papain activity
IS
S l
IS
Sl
IS
Sl
ISSl
I S S l
IS
Sl
IS
Sl
IS
Sl
ISSl
ISSl
ISSl
IS
Sl
IS
Sl
IS
Sl
I S S lI SS
lvariable
constant
NH3
COO
papain FC: biological effector functions
FAB
Pepsin
IS
S l
IS
Sl
IS
Sl
ISSl
I S S l
IS
Sl
IS
Sl
IS
Sl
ISSl
ISSl
ISSl
IS
Sl
IS
Sl
IS
Sl
I S S lI SS
l
214 residues
446 residues
variable
constant
NH3
COO
Pepsin
F(ab’)2
FC fragments
5
Domains
IS
S l
IS
Sl
IS
Sl
ISSl
I S S l
IS
Sl
IS
Sl
IS
Sl
ISSl
ISSl
ISSl
IS
Sl
IS
Sl
IS
Sl
I S S lI SS
l
214 residues
446 residues
Variable:Ag binding
Constant:
NH3
COO
VL
VH
VL
VH
CL CL
CH1CH1
CH2
CH3 CH3
CH2
HINGE
Intra-chain disulfide bonds
Inter-chain disulfide bonds
Ab structure
Carbohydrate role: 4-9
6
Light Chain SequencesNH3
COO
1
110
214
Variable
2 types of constant regionsKappa (κ) orLambda (λ)
Heavy chain sequences
V
5 different constant regionsM(μ), A(α), D(δ), G(γ), E(ε)(ISOTYPES OR CLASSES)
NH3
COO
Constant regionsIgA, IgD, IgG IgE, IgM
CH1 CH1 CH1 CH1
Hinge region Hinge region
CH2
CH3
CH2
CH3
CH2 CH2
CH3
CH4
CH3
CH4
9
Light chains: Ig fold structure
CDR’S IN BLUEComplimentary Determining
Regions
Variability equalsnumber of different amino acids at a given
positiondivided by
frequency of most common amino acid at given position
10
Hypervariable regions: CDR’s
Interaction between Ab and flu Ag
Conformation: HIV protease and anti-protease Ab
allosterism
11
Ab-mediated effector functions
• Opsonization– FcR: Fc receptors on macrophage surfaces
• Complement activation• Ab-dependent cell-mediated cytotoxicity
(ADCC)– Fc receptors on natural killer cells
Immunoglobulin activities
Classes and subclasses: p. 96
14
Isotypes/allotypes/idiotypes
Isotypes
Constant-region determinants that identify each heavy chain class and subclass and light chain type and sub-type
Allotypes
15
Allotypes• Alleles of the isotypes within species
γ: Gm {G1m(1), G2m(23), G3m(11), G4m(4a)} :25 different onesα: A2m (1), A2m(2) :2 different onesκ: κm(1), κm(2), κm(3) :3 different ones
Idiotypes
Unique amino acid sequences of VL and VH chains.Determine specificity of the immunoglobulin.
B cell receptor
16
Human Fc receptorsPart of secretory component
Ig superfamily: common primordial domain gene structures
More superfamily
17
Monoclonal Antibodies
• Ab derived from a single B-cell clone and has a single binding site
• Pure antibody of one specificity• All have same molecular structure
Nucleotide biosynthesis
• De novo pathway• Phosphoribosyl PPi + Uridylate
• Nucleotides• Salvage pathway HGPRT
• Hypoxanthine + thymidine
aminopterin
HAT Media
• Aminopterin (to block de novo pathway)• Hypoxanthine and thymidine ( to allow
salvage pathway)
18
Monoclonal Antibody ProductionAg
HGPRT+, Ig+
Myeloma cells
HGPRT-, Ig-spleen
Can’t use salvage pathway
Myeloma cells+ spleen cellsin polyethylene glycol
fusion
HAT media
Unfused myeloma cells dieUnfused spleen cells dieHybridomas live HGPRT+, Ig+, immortal growth
+ +
Assay for desired Ab,select positive clones
HypoxanthineAminopterinThymidine
Monoclonal Ab
Abzymes
• Antibody acting like an enzyme and catalyzing a reaction
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