WHY can immune system recognize so

1
WHY can immune system recognize so many
different epitopes?? Antibody heavy and light
chains are composed of gene segments Variable
regions are unique A limited variety of constant
region sequences are used They must be
rearranged into functional genes before they can
be transcribed
2
p. 106
3
Organization of Ig genes Germline DNA- gene
segments surrounded by noncoding regions These
are rearranged to form functional genes Light
chains- V, J and C segments Heavy chain- V, D,
J, C V regions rearrange first A single V can
rearrange to more than one C
4
Multigene families ? or ? In humans 40 V?, 5
J?, 1 C? Similar number of ? genes in humans
this is rare in mice Heavy-chain gene families
are similar but more complex (D segment) CH
regions formed from exons
5
p. 111
One of many possible combinations
6
Heavy chain DNA D-J and V-DJ rearrangements must
occur separately On a mature B cell, both mIgM
and mIgD are expressed on the cell surface
7
How does rearrangement occur? Each V, D and J is
flanked by RSS (Recombination signal
sequences) Mechanism is controlled by RAG-1 and
RAG-2 proteins and an enzyme TdT If any of these
proteins is defective no mature B cells can
form nor T cells
8
p. 112
9
Junctional flexibility contributes to diversity
p. 115
But not all rearrangements are productive
10
B cells are diploid and contain chromosomes from
both parents However, heavy chain genes are
rearranged from only one chromosome, as are light
chain genes. Therefore, any one B cell will
contain one VH and one VL (antigen
specificity) How? Allelic exclusion
(Yancopoulos and Alt, 1986)
11
Model for allelic exclusion
p. 116
12
Generation of antibody diversity (why are there
so many possible antigen combining sites?)
13
Multiple germline gene segments In human
germline 51 VH, 27 D, 6 JH 40 V?, 5 J ? 30 V
?, 4 J?
14
Combinatorial V-J and V-D-J joining 57 V X 27 D
X 6 J 8262 possible combinations for VDJ
joining 40 V X 5J 200 possible V? 120 possible
V? 8262 X (200120) 2.64 X 106 possible
combinations Without taking into account other
sources of diversity
15
Junctional flexibility in V-J or V-D-J
junction Additional nucleotides added at
junctions (P or N addition), if a
single-stranded region is created during the
joining process Somatic hypermutation mutations
occur AFTER rearrangement tends to occur in CDR
regions affects antigen affinity (tends to
increase) affinity maturation occurs in B
but not T cells
16
Class switching After antigen stimulation
heavy-chain DNA can rearrange so VDJ joins to any
isotype Cytokines help determine the
isotype IgG2a or IgG3 (mice) IFN-? IgM IL-2,
IL-4, IL-5 IgE IL-4
17
p. 122
18
Membrane-bound or secreted?
Alternative splicing, p. 124
19
Mature B cells express both mIgM and mIgD No
switch site between C? and C? The VDJC?C?
contains 4 polyadenylation sites mIgM or mIgD
can be generated depending on which
polyadenylation site is used
20
Regulatory elements of transcription Promoters E
nhancers Gene silencers Gene rearrangement
brings enhancers close to the promoter they
influence
21
Why arent Igs produced in B cells? In T cells a
protein may bind to the ?-enhancer and prevent
V-J joining Arrangement of immunoglobulin genes
(and formation from exons) and
greatly facilitated formation of
genetically engineered antibodies
22
What is a monoclonal antibody? Derived from a
single clone and specific for a single
epitope 1975- Kohler and Milstein developed
the hybridoma technique for developing monoclona
l antibodies

23
p. 99
24
Behavior of monoclonal vs polyclonal
antibodies Monoclonal antibodies tend to have
high affinity Polyclonal antiserum will have
mixture of low and high affinity
antibodies Avidity vs affinity Antibodies can
be cross-reactive (source of some autoimmune
disorders)
25
Genetically-engineered monoclonal antibodies are
widely produced Advantages over hybridoma
technology can choose isotype as well as
specificity Can be expressed in a variety of
host cells non-lymphoid mammalian
cells bacteria (antibody fragments) plants yeas
t Mutations of interest can be introduced
26
Therapeutic applications Cancer
treatment Imaging Immunotoxins Catalytic
antibodies? Research applications Structure-fun
ction analysis Recombinant antibodies Humanized
antibodies
27
p. 129
28
Immunological assays Antigen-antibody
interactions Antibody-based assays
29
Affinity and specificity for antigen Assays
detect presence of antigen or antibody diagnosis
monitoring humoral immune response therapeutics
analysis of interesting molecules
30
Nature of antigen-antibody interaction
p. 150
31
Affinity- strength of interaction between
one Ag-binding site and one epitope Measured by
various methods including equilibrium dialysis,
competition assays, microchips (e.g.,
Biacor) Association constants can be calculated
32
Avidity Multiple interactions between antibody
and antigen IgM (with 10 antigen binding sites
per molecule) tends to have low affinity but
high avidity May be more biologically
significant measure than affinity
33
Cross-reactivity if two antigens share an
epitope an antibody recognizes an
unrelated, but chemically similar, epitope
p. 153
34
Cross-reactivity between microbes and
human tissues can lead to disease Streptococcus
M and myocardium. JA? Vaccines- cowpox and
smallpox
35
Precipitation- classic demonstration of
antibody-antigen interaction Antibody and
soluble antigen aggregate to form a visible
precipitate Antibody must be bivalent (Fabs
wont work) Antigen must be multivalent
36
p. 154
37
Precipitation reactions in fluids (p. 154)
Precipitation reaction can be seen
38
Precipitation can also be seen in gels Antibody
and antigen diffuse toward each other and
precipitate where there is equivalence Radial
immunodiffusion Double diffusion Immunoelectrophor
esis
39
p. 155
40
Agglutination- reaction between antibody
and particulate antigen Presence of excess
antibody can inhibit agglutination (prozone
effect) Each antibody competes for epitopes
if it binds one, it cannot link one antigen to
another Some antibodies bind but do not
agglutinate Epitope density or availability
41
Hemagglutination- red blood cells
Well 10- sheep RBC only control 1 through 9-
serial twofold dilutions of anti-SRBC
p. 158
42
Bacterial agglutination If a patient has a
bacterial infection, the patient will produce
specific antibodies to that bacterium Serum can
be titered with bacterial agglutination reactions
Dilutions of serum are tested (usually
twofold) example 1256 dilution shows
agglutination but 1512 does not Titer is 256
43
Titer can be monitored over time
should decrease as patient recovers Antisera
are used to type bacteria, too (against surface
antigens, flagellar antigens, etc.) Example E.
coli O157H7
44
Passive agglutination Soluble antigens dont
agglutinate But if you stick them onto something
else (like a latex bead or, historically, a
RBC) you can obtain an agglutination
reaction Using beads increases sensitivity
45
Agglutination inhibition
p. 159
Current tests are ELISAs
46
Agglutination inhibition assays Can test for the
presence of substances in fluids (e.g., drugs in
urine) How?
Rubella test is an agglutination inhibition
assay rubella virus causes hemagglutination
47
ELISA- enzyme-linked immunosorbent assay Based
on RIA Nearly as sensitive Cheaper and
safer Many detection systems have been
developed Many variations of the assay have been
developed
48
All ELISAs use an antibody conjugated with
an enzyme that turns a colorless substrate into
a colored product Direct- detects antigens using
a single labeled antibody against that
antigen Relatively few applications and
permutations
49
Indirect ELISA detects antibodies (prior
exposure to antigen, e.g., HIV)
p. 162
50
Sandwich assay- detection of antigen
51
Competitive ELISA- less reaction indicates more
antigen in pre-incubation mixture
52
Chemiluminescence ELISA technique is the same,
detection system is different Luminol, hydrogen
peroxide and horseradish peroxidase react to
emit light Detection system is more sensitive
than the enzyme substrates
53
ELISPOT- can detect secreting cells
p. 163
54
The ELISA is very versatile
Enzyme-conjugated Sheep anti-goat Ab
substrate
Goat anti-human C1q
E
Complement (human)
Antibody (mouse Fab, Human Fc)
antigen
55
All IgG1 antibodies were identical except for
carbohydrate in CH2
56
Western blotting
p. 164
57
Immunofluorescence Antibodies can be labeled
with fluorescent dye Can localize binding sites
on cells Dyes Fluorescein, rhodamine,
phycoerythrin can be conjugated to Fc region of
Ab (so antigen binding is unaffected) Absorb at
one wavelength and emit at another
58
Direct and indirect immunofluorescence
p. 166
59
p. 166
60
Versatile technique Differentiate T cell
subsets Detecting Ag-Ab complexes Localization
of target molecules in tissue (variation
immunohistochemical staining)
61
Flow cytometry is quantitative FACS-
fluoresence-activated cell sorter Analyze cell
populations Sort cells with different features
into different containers (e.g., T and B cells
cells that are producing a cell-surface
marker from those that are not)
62
p. 167
63
Uses for flow cytometry Percentage of a total
population of cells Measuring antigen density
within a population of cells Multiple
antibodies can be used to assess several cell
surface antigens simultaneously Clinical
analysis (tumor characterization)
64
Animal models depends on the purpose Mouse
strains- inbred syngeneic- genetically
identical congenic-differ at one locus immune
response regulation nude mice SCID (and
SCID/hu) mice
65
Introduction of cloned genes into mice (and
other animals) Transgenic animals acquire a
trait Knockout animals lose a trait
66
p. 585
67
Building a knockout gene
p. 586
68
(No Transcript)
69
p
p. 584 Techniques are useful for studying gene
expression in living animals
70
Cell culture gene transfection hybridoma
technology
71
Summary The structure of antibodies enables
them to recognize and bind antigen and
to perform appropriate effector functions in
response The exquisite specificity and effector
activity of antibodies makes them very
usefuL in research and diagnostics
72
The organization of immunoglobulin genes allows
for the formation of over 10 billion antigen
specificities Various in vivo and in vitro
experimental systems have provided
significant insights into the immune
response and its regulation