Download 4.1mb

October 5, 2004
IMMUNITY
ADAPTIVE
CELL MEDIATED
INNATE
HUMORAL
IMMUNITY
ADAPTIVE
CELL MEDIATED
INNATE
HUMORAL
ANTIBODIES
Structure
Sequence diversity
Nature of reaction with Ag
Different functional properties
If bacteria are injected into blood specific
substances are found that exhibit different
biologic properties
Agglutinin - specifically clump only the bacteria used for
immunization
Opsonin - facilitate engulfment
Antitoxin - neutralize toxin associated with the immunizing
bacteria
Cytolysis - lyse bacteria
Precipitins - form flocculate precipitates when added to
supernatant from the bacteria
If bacteria are injected into blood specific
substances are found that exhibit different
biologic properties
Agglutinin - specifically clump only the bacteria used for
immunization
Opsonin - facilitate engulfment
Antitoxin - neutralize toxin associated with the immunizing
bacteria
Cytolysis - lyse bacteria
Precipitins - form flocculate precipitates when added to
supernatant from the bacteria
Antibody (Ab)
A molecule produced by animal in response to
foreign substance (Ag) which has the property of
binding the eliciting agent
Immunoglobulin (Ig)
Antibody (Ab)
A molecule produced by animal in response to
foreign substance (Ag) which has the property of
binding the eliciting agent
Immunoglobulin (Ig)
Antibodies are found both as cell-associated
receptors (BCR) and as circulating effector
molecules
Antigen (Ag)
Any molecule capable of being bound by the
combining site of an Ab or the T-cell receptor
Immunogen
A substance capable of eliciting an immune
response; all immunogens are antigens but not all
antigens are immunogens.
We will now consider the experiments that were
done to determine antibody atructure
Immune serum from rabbits before and after the
antibodies were removed by precipitation with Ag
(ovalbumin)
The antibodies migrated as a g-globulin
Showed they w
gamma globulin
When purified antibodies (produced by dissolving
immunoprecipiates) were analyzed in an
ultracentrifuge they ran with a 7S sedimentation
co-efficient indicating a m.w. of approx. 150,000
gamma globulin
7S = 150,000 molecular weight
gamma globulin
7S = 150,000 molecular weight
Molecular analysis of
precipitates showed
the valence = 2
Two molecules of Ag were precipitated by each Ab
gamma globulin
7S = 150,000 molecular weight
valence = 2
gamma globulin
7S = 150,000 molecular weight
valence = 2
papain --> 2Fab + 1Fc
Fab binds Ag but no precipitate
Fc forms crystals
MW of Fab and Fc = 50,000
Ratio: 2 Fab and 1 Fc
Digestion of antibodies with the enzyme papain
yielded two fragments, the Fab (fragment antigen
binding), and the Fc (fragment crystallizable)
gamma globulin
7S = 150,000 molecular weight
valence = 2
papain --> 2Fab + 1Fc
pepsin--> 1 (Fab')2 of 100,000
binds 2 molecules of Ag
can precipitate
(Fab')2 break SS-->2Fab
gamma globulin
7S = 150,000 molecular weight
valence = 2
papain --> 2Fab + 1Fc
pepsin--> 1 (Fab')2 of 100,000
Ab--> cleave disulfides-->
2H(50000) + 2L(25000)
Antiserum
Anti-L reacts only with Fab
Anti-H reacts with Fab and Fc
Anti-Fab reacts with H and L
If break disulfide bonds and
separate on the basis of size
you get two products
gamma globulin
gamma globulin7S = 150,000 molecular weight
valence = 2 weight
7S = 150,000 molecular
papain --> 2Fab + 1Fc
valence = 2
pepsin--> 1 (Fab')2 of 100,000
papain --> 2FabAb-->
+ 1Fc
cleave disulfides-->
2H(50000) + 2L(25000)
pepsin--> 1 (Fab')
2 of 100,000
Antiserum
Anti-L reacts only with Fab
Ab--> cleave disulfides-->
Anti-H reacts with Fab and Fc
2H(50000) + 2L(25000)
Anti-Fab reacts with H and L
Antiserum
Anti-L reacts only with Fab
Anti-H reacts with Fab and Fc
Anti-Fab reacts with H and L
gamma globulin
7S = 150,000 molecular weight
valence = 2
papain --> 2Fab + 1Fc
pepsin--> 1 (Fab')2 of 100,000
Ab--> cleave disulfides-->
2H(50000) + 2L(25000)
Antiserum
Anti-L reacts only with Fab
Anti-H reacts with Fab and Fc
Anti-Fab reacts with H and L
gamma globulin
7S = 150,000 molecular weight
valence = 2
papain --> 2Fab + 1Fc
pepsin--> 1 (Fab')2 of 100,000
Ab--> cleave disulfides-->
2H(50000) + 2L(25000)
Antiserum
Anti-L reacts only with Fab
Anti-H reacts with Fab and Fc
Anti-Fab reacts with H and L
This is the structure of the fundamental building block of an Ab
molecule. Some antibodies (such as IgG) are made of one of
these building blocks. Others (IgA and IgM) are made of
multiple copies of this basic building block.
One problem with further analysis was the
heterogeneity of antibodies: note the narrow peak
of the homogenous protein albumin compared to
the broad peak of the Ig
It was a major breakthrough when it was realized
that multiple myeloma is a tumor of plasma cells
generally synthesizing a single species of Ig and
that the myeloma proteins are homogeneous Abs
produced by the plasma cells
Bence-Jones proteins are monoclonal (homogeneous)
L chains in patients with multiple myeloma
How are antibodies capable of
binding many different
antigens?
Light chains are of two types:


anti-
++++
-
anti-
-
++++
Light chains are of two types:


anti-
++++
-
anti-
-
++++
Take two kappa (or lambda) lights chains from two
different patients with multiple myeloma and produce
proteolytic fragments
Light chains are of two types:


anti-
++++
-
anti-
-
++++
Take two kappa (or lambda) lights chains from two
different patients with multiple myeloma, produce
proteolytic fragments.
Analyze the products by 2-D peptide map (chromatography
in one dimension, electrophoresis in the other).
You find that about half of the cleavage products are
identical between the two chains while the other half differ.
The shaded spots indicate peptides shared by the two  or  chains.
Note the difference between  and .
The conclusion is that the lights chains have a portion that is variable
and a portion that is constant.
Amino acid sequence analysis shows that the variable portions is at one
end of the light (and heavy) chains
VL
CL
( or)
VH
CH
(many different options)
VL
CL
( or)
VH
CH
(many different options)
More extensive analysis of the sequences of
variable regions shows that they in fact contain
relatively conserved regions and other regions
which are hypervariable
There are 20 different amino acids.
If all are found with equal frequency
variability is 20/.05 = 400.
If only one amino acid is found the
variability is 1/1 =1/
More extensive analysis of the sequences of
variable regions shows that they in fact contain
relatively conserved regions and other regions
which are hypervariable.
There are 20 different amino acids.
If all are found with equal frequency
variability is 20/.05 = 400.
If only one amino acid is found the
variability is 1/1 =1/
Hypervariable regions are also called complementarity
determining regions (CDRs). The prediction is that the
CDRs contact antigen.
X-crystallography shows that the Ab molecule folds into compact
domains. The CDRs are loops extending from the variable regions so
that they are easily accessible for interaction with Ag. The other amino
acids in the variable region are the “framework” amino acids and provide
a scaffold to maintain the CDRs in the proper orientation.
Hypervariable regions are also called complementarity
determining regions (CDRs). The prediction is that the
CDRs contact antigen.
Light chains have two domains: one variable and one constant
Heavy chains have four or five domains, one of which is variable with
the remains constant
All heavy chains have at least one carbohydrate moiety attached
The forces are weak and
operate at short distances
Therefore the closer the
approach (the better the fit)
the stronger the interaction
A complex of an Ab and a
protein Ag shows:
the reaction occurs between two
interacting surfaces
the better the fit, the stronger the interaction
most but not all of the interaction with Ag
occurs through the CDRs
on a protein Ag the amino acids comprising
the epitopes are adjacent in 3D space but not
necessarily in linear sequence
Another characteristic of the antibody molecule is a flexible hinge
When a divalent hapten reacts with Abs, trimers, tetramers and other
large complexes form. Because of the flexibility of the hinge region
Digestion with papain removes the Fc
There are several different possible structure of the constant
regions of the heavy chains
Classes Heavy Chain Subclasses (human)

IgM
g
IgG
IgG1, IgG2, IgG3, IgG4
IgA

IgA1, IgA2

IgE
IgD

Classes + subclasses = isotypes
Light chains have two isotypes: and
Each isotype is encoded by a separate gene
IgM
H chain has four constant region domains and no hinge
In addition to H and L also contains J chain
(H2L2)5J or (H2L2)6
Usually the first Ig made in response to antigen
Increased valence leads to increased avidity
IgG
IgG
IgG
Most abundant serum immunoglobulin
IgA
J
SC
Monomer
Dimeric IgA
Secretory IgA
Present in secretions where it provides protection at epithelial surfaces
Frequently found as a polymer with J chain
In secretions it also has a fourth chain, secretory component, a product
of the epithelial cells
IgE
Present at low concentrations
Responsible for allergy
Binds to receptors on mast cells
IgD
Principle role seems to be as a membrane Ig
All isotypes can exist as both secreted and
membrane bound antibodies
All isotypes can exist as both secreted
and membrane bound antibodies.
The two forms differ in their carboxy
terminal sequence.
Secreted antibodies have a hydrophilic
terminus.
Membrane antibodies have a hydrophobic
sequence which inserts into the plasma
membrane and a short cytoplasmic tail.