Download Antibodies and Antigens

Biol 430
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Antibodies
1. In this diagram of an antibody molecule
identify the components labeled as:
A. _________
B. _________
C. _________
D. _________
E. _________
F. _________
G. _________
H. _________
I. _________
J. _________
2. This Figure shows the genetic
recombination of Ig Heavy Chain
genes and subsequent processing of
the transcript in a mature B-cell.
1. Identify the genetic elements
identified as
A1:
A2:
A3:
A4:
2. Briefly describe what is happening in the steps labeled as ‘B’.
3. What is happening in step ‘C’?
4. What are the two products shown as ‘D’
Biol 430
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Antibodies
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3. Some questions about regulation of gene segment rearrangements…
A. Through the regulatory process called __________________, rearrangement and
expression of antibody genes is restricted to a single locus at a time. Why is this essential
for the proper functioning of mature B-cells?
B. How many chances does a B-cell have to make a functional heavy chain gene? Explain.
C. How many chances to make a functional light chain gene? Explain.
4. Using the following estimates of the number of antibody gene segments in mice, and the
following questions:
H chain
V
D
J
κ light chain
γ light chain
Estimated number of segments
200
300
3
15
0
0
4
4
3
A. What is the number of possible VDJ combinations for the heavy, kappa and lambda
chains?
B. What is the total number of possible of heavy and light chain combinations? Explain.
C. What are three other mechanisms that will contribute to the total diversity of
antibodies produced?
D. Why will only a small percentage of the B-cells be released from the bone marrow?
What will happen to the rest?
Biol 430
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Antibodies
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5. The figure to the right shows the steps of a
classic experiment performed by Tonegawa and
Hozumi in 1976. In this experiment DNA from
normal mouse embryo cells and from a
plasmacytoma were used. A plasmacytoma
(another name for a myeloma) is a malignancy in
which there is a proliferation of a single plasma
cell clone which produces a single type of
antibody.
DNA from the two cell types were cleaved
with a DNA restriction enzyme, the DNA
fragments were separated by size using
electrophoresis and transferred to nitrocellulose.
The nitrocellulose was then probed with radiolabeled RNA complementary to the heavy chain
rearranged constant region of the
plasmacytoma. The lower diagram shows how
the probe would bind to a DNA fragment
containing the embryonic constant region gene.
The autoradiogram shows that the probe bound
to a single DNA fragment from embryonic cells,
but to two fragments from the plasmacytoma.
A. Why does a RNA probe bind to only
certain DNA fragments?
B. Why is the DNA fragments transferred to nitrocellulose after separation by gel
electrophoresis?
C. Explain why the probe binds to one DNA fragment from embryonic cells but two DNA
fragments from the plasmacytoma cells.
D. Was HC allele rearrangement successful? Explain.
Biol 430
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Antibodies
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6. In 1994, Lonberg et al. (Nature 368:856) reported the creation of transgenic mice that
produced human antibodies. They started with a strain of mouse in which the heavy chain and
light chain loci had been ‘knocked-out’ (made non-functional). They then introduced into
embryos from these mice genes for the heavy and light chains from humans; transgenic mice
were then grown from these embryos. When exposed to antigens, these mice produce human
antibodies against the antigens.
A. Why was it necessary to start with Ig knock-out mice?
B. The antibodies produced from the transgenic mice are ‘humanized’ antibodies; how
do these differ from the antibodies from normal mice?
C. Suppose the transgenic mice were inoculated with a human protein; would they
respond to that protein differently than normal mice? Explain.
7. Match each of the following terms with its best description.
Term
Description:
a. ___ IgA
1. DNA sequences that flank variable region gene segments
b. ___ RAG
2. new bases added to a gene segment junction
c. ___ IgM
3. most abundant form of antibody found in the blood
d. ___ JDV
4. antibody isotype of uncertain function
e. ___ IgE
5. enzyme required for antibody gene segment recombination
f. ___ mu (μ)
6. blood protein fraction containing antibodies
g. ___ IgG
7. arrangement of heavy and light chains in secreted IgM
h. ___ JV
8. one of the light chain constant regions
i. ___ RSS
9. form of antibody common in breast milk and digestive tract
j. ___ IgD
10. example of a Ig heavy chain
k. ___ P-nucleotides 11. gene segments found in the heavy chain variable region
l. ___ Lambda (λ)
12. antibody class associated with hypersensitivities
m. ___ γ-globulin
13. gene segments found in the light chain variable region
n. ___ Igα/Igβ
14. additional proteins found in B-cell receptor
o. ___ H10L10
15. Ig class of the B-cell receptor
Biol 430
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Antibodies
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8. It is possible to produce antisera (antibodies) against other antibodies or parts of antibodies.
Generally this is done by injecting the antibodies (or molecular parts thereof) from one animal
species into a different species, which then reacts against the antigen as a foreign substance.
Suppose antisera were produced against various molecular components (listed at the top of this
table); indicate whether each type of antisera would contain antibodies (+) or not (--) against the
molecules listed in the left hand column.
Antisera produced against_____________
γ
κ
IgG
IgG
μ
Chain
Chain
Fab
Fc
Chain
gamma chain
kappa chain
whole IgG
IgM Fc fragment
IgM Fab Fragment
9. As an enthusiastic young investigator you have isolated a protein that you believe is a new
type of human immunoglobin (“IgF”), and you undertake a series of experiments to test this
hypothesis.
A. You find that the protein possesses Ig domains. Is this enough to confirm that this is
a type of immunoglobin? Explain.
B. You test the ability of the protein to bind to various types of bacteria, but see no
reaction. Is this enough to confirm that this is not an immunoglobin? Explain.
C. If you were to prepare antisera against human Gamma heavy chain and Kappa light
chain, which antisera would contain antibodies that would react with “IgF” if it is
indeed an immunoglobin? Explain.
10. Complete the table below pertaining to the properties of IgG and its various subcomponents.
Indicate if the molecule (or part) has the property (+) or not (– ).
Property
Can bind antigen
Bivalent antigen binding
Binds to Fc receptors
Activates complement in
presence of antigen
Has V domains
Has C domains
Biol 430
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whole H
IgG chain
λ
chain
Fab
Antibodies
F(ab)2
Fc
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11. IgG antibodies occur in several subclasses, although the particular effector functions of each
subclass are not well understood. The clinical response to antibody-antigen complex is mediated
by receptors that bind to the Fc region of the antibody; there are several types of receptors, some
(such as FcγRIV and FcγRIII) lead to effector response activation, whereas other receptors (such
as FcγRIIB) cause inhibition. To better understand how subclasses regulate immune responses,
Nimmerjan and Ravetch (2005) studied binding of IgG subclasses to different receptors.
For their investigation, an Fab region that recognizes an antigen called TA99 was grafted
onto Fc regions for the different IgG subclasses. In one experiment, the affinity of each Fc +
TA99 for different receptors was measured. Since the presence of the carbohydrate fucose in the
Fc oligosaccharide had been previously reported to influence receptor affinity, antibodies with
and without fucose present were compared. Some of the results are presented in the table below.
IgG subclass
IgG1 (+fucose)
IgG1 (- fucose)
IgG2a (+ fucose)
IgG2a (- fucose)
IgG2b (+ fucose)
IgG2b (- fucose)
Binding affinity for receptors
FcγRIV or FcγRIII
FcγRIIB
0.3
3.3
0.5
1.3
29
0.4
310
3.3
17
2.2
200
10
A/I ratio
_____
_____
_____
_____
_____
______
A. Explain the nomenclature used in naming the receptors, such as “FcγRIV”.
B. Calculate the ratio of activator receptor to inhibitor receptor affinity (A/I ratio) for
each type of antibody.
C. Which subclass(es) would appear to activate immune responses? Which would
appear to inhibit response? Explain.
D. What is the effect of fucose removal? Does it increase affinity for activating or
inhibiting receptors? Explain.
E. The results suggest two ways in which the immunological response to antigen+IgG
can be regulated; explain.
(Nimmerjah F, Ravetch JV.2005. Divergent immunoglobin G subclass activity through selective Fc receptor
binding. Science 310:1510-1512.)
Biol 430
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Antibodies
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12. The properties of, and the effector responses to, different antibody isotypes is exquisitely
adapted to its particular immunological role. In the table below, identify the isotype of the
antibodies being described, and then explain how those properties relate to the immunological
function.
Type
Properties
Pentameric
structure
_____ Low
affinity
Activates
complement
Relation of Properties to Immunological Function
Dimeric
structure
_____ Large quantities
secreted
Doesn’t activate
complement
High numbers in
serum and lymph
_____ Activates
complement
Triggers ADCC
and opsonization
Respond to
eukaryotic antigens
_____ Triggers
Mast cell ADCC
Associated with
mucosyl tissues
Some of these questions have been adapted from Goldsby et al (2003) Immunology, ed 5.
Biol 430
Question Bank
Antibodies
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