Biol 430
Question Bank
Antigens and Technologies
Self-Test Questions
A. Monoclonal Abs
A.1 If you have two test tubes, one containing monoclonal antibodies against a particular
antigen, and the other containing polyclonal antibodies against the same antigen, how are the
two samples different in regards to how they will interact with the antigen?
A.2 Explain the roles of spleen cells, myeloma cells and hybridoma cells in the process of
making monoclonal antibodies.
A.3 Explain why use of monoclonal antibodies may be better than polyclonal antibodies is
certain clinical and research applications. When used as the primary antibody in Western
blotting or immunohistology procedures, why might polyclonal antibodies give a better result
than monoclonal antibodies?
B. Elisa
B.1 Which techniques are most appropriate for quantifying an antigen in a sample? Which
techniques are most appropriate for quantifying an antibody in a sample?
B.2 The indirect method requires use of a ‘secondary’ antibody, whereas the direct
methodology does not. Explain the difference.
B.3 Explain the need for serial dilution of the sample when performing an Elisa.
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Antigens and Technologies
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C. Western Blotting
C.1 Why must
1. electrophoresis of the protein sample, and
2. transfer to nitrocellulose
precede the actual probing of the sample with an antibody?
C.2 What is the function of the enzyme bound to the secondary antibody?
D. Immunohistology
D.1 Explain the similarities and differences between western blotting and immunohistology.
D.2 Immunohistochemistry requires a conjugated enzyme, whereas immunofluorescence
does not. Explain the difference.
E. Immunoprecipitation & affinity chromatography
E.1 Describe the basic steps that would be followed for each of these procedures.
E.2 What are some treatments that could be used to disrupt the antibody-antigen complexes?
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Additional questions
1. Antisera is produced by inoculating animals (such as sheep) with a toxin (such as the tetanus
toxin) and then purifying the antibodies produced. The antisera can then be administered to treat
a person suffering acute symptoms of the toxin. Unfortunately, adverse reactions sometimes
occur since humans may react immunologically against the anitibody as a foreign substance.
A. Why would antisera be recognized as a foreign antigen?
B. How might transgenic animals that produce human antibodies (such as those
described in the question of the Antibodies Question Bank) help to eliminate such
reactions?
2. An investigator wanted to produce rabbit antiserum specific for mouse IgG, so she injected a
rabbit with purified mouse IgG. However, to her dismay, the antiserum reacted with all isotypes
of mouse antibodies. Why? How could she modify the procedure to obtain IgG specific serum?
3. Oils on the leaves of poison ivy contain a small molecule called a pentadecacatechol. These
molecules can be shown to be non-antigenic on their own, but can trigger an immune response
when bound to proteins of the skin.
A. Pentadecacatechol is an example of a small molecule that can act as an
______________.
B. Explain why pentadecacatechol triggers an immune response when exposure is
through the skin.
4. Antibody – antigen interactions might involve all types of chemical bonding, except which
one? Explain.
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Antigens and Technologies
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5. Certain oncogenes, such as RAS, code for proteins that differ only by one amino acid from the
normal form of the protein. Suppose that you wished to look for the presence of specific forms
of RAS on tumor cells from several patients using Western blotting. Would use of monoclonal
antibodies or polyclonal antibodies against the mutant RAS be most appropriate? Explain.
6. The figures below show lymphocyte analysis from patients with normal and abnormal blood
cell counts.
A. The results shown in this diagram were produced by which methodology?
_______________________
B. The cell markers used in this analysis
were CD19 for B-cells and CD3 for T-cells.
(The scales on the x-axis and on the y-axis
are not shown.) These results most
indicative of:
a. a T-cell malignancy
b. lymphoma
c. monoclonal antibodies
d. a B-cell deficiency
Explain.
7. MBP is a self-antigen against which the immune system
reacts in people with the autoimmune disorder multiple
sclerosis. The microtitration plate shown here presents
Elisa analysis of anti-MBP antibodies in patients
suffering from multiple sclerosis. Serum samples from
each patient ( A – E) are in wells 1 – 12.
A. Sample wells numbered from 1 - 12 contain:
a. serum from different patients.
b. serum samples tested against different
types of antigens.
c. antibodies against different types of antigens.
d. serially diluted serum from different patients.
Explain.
B. Which patient has the highest titer of anti-MBP antibodies? (A, B, C, D or E)
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Antigens and Technologies
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8. This diagram shows the steps in the
production of monoclonal antibodies.
A. Identify the components
labeled A – E.
B. Describe the process that is
needed to go from step C to
step E
9. Match the following technologies with their appropriate application.
Methodology
___ Elisa
___ Western blot
___ Flow cytometery
___ Immunofluorescence
___ Immunoprecipitation
Would be best method for:
A. showing the presence of an intracellular pathogen.
B. showing the presence of a specific antigen in serum.
C. separating a particular antigen for a tissue homogenate.
D. quantifying the titer of serum antibody.
E. quantifying and separating different cell types.
10. This figure shows the results of electrophoresis of
serum proteins from three people. One person is a
normal, healthy individual; one has recently been
subject to a severe bacterial infection; and one suffers
from a monoclonal producing plasmacytoma.
A. Which blood fraction will be most useful for
distinguishing these individuals. Why?
B. Which sample is most probably from the
person who is:
Healthy
_____:
Recently infected
(Explain your selections)
_____:
With plasmacytoma _____:
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11. The image to the right shows a tissue with a
colorectal tumor (stained blue with hematoxylin)
and T-cells (stained brown) in the connective tissue
around it.
A. What type of methodology was used to stain
the T-cells in this image?
B. What might be some target surface proteins
on T-cells that could be used as T-cell
specific target antigens?
C. How was the brown color created?
From Galon, et al. 2006. Type, Density, and Location of Immune Cells Within Human Colorectal Tumors Predict
Clinical Outcome. Science 313 (5795), 1960.
12. The figure to the right shows several samples subjected
to protein gel electrophoresis, and stained for total protein.
Lane ‘L’ is a homogenate of cultured cells infected with the
SV40 virus, and lane ‘M’ is a sample that contains
molecular weight marker proteins that range from 205 to 29
Kd. Lane 1 contains protein obtained when the
homogenate was treated with antibodies against the SV40
‘Large T antigen’ (the antigen is indicated by the arrow).
Lane 2 was treated as for lane 1 but without the antibody.
(Image is from www.miltenyibiotec.com Protein A and MACS Protein
G MicroBeads.aspx)
A. The data shown in lane 1 were obtained using which
antibody based technology?
B. Why does lane ‘L’ contain many different types of
proteins, lane 1 contains very few, and lane 2 none?
C. What is the approximate MW of the SV40 Large T antigen?
D. Notice that several other bands appear in lane 1. What are three possible explanations for
the presence of multiple peptides in the sample run in lane 1?
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13. Some questions about antigenicity, etc….
A. Explain the statement “All immunogens are antigens, but not all antigens are
imunogens.”
B. Collagen, is a filamentous protein with a repetitive structure, composed of a sequence of
three repeating amino acids. In general, collagen has very low antigenicity. Why would
this be expected?
C. The structure of collagen differs very little between species. Thus, would collagen from
rabbits be more immunogenic in rats than rat collagen? Explain.
D. Suppose that you were able to prepare IgG, IgA and IgM antibodies that all recognized
the same epitope in collagen. Order the antibodies from lowest (1) avidity to highest (2)
avidity toward collagen:
Monomeric IgG _____
Dimeric IgA
_____
Pentameric IgM _____
Explain your ranking.
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