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.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.
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.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?
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.
Why would antisera be recognized as a foreign antigen?
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?
31. 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.
Pentadecacatechol is an example of a small molecule that can act as an ______________.
Explain why pentadecacatechol triggers an immune response when exposure is through the skin.
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:
suffering from multiple sclerosis. Serum samples from
each patient ( A – E) are in wells 1 – 12.
Sample wells numbered from 1 - 12 contain:
serum from different patients.
serum samples tested against different
types of antigens.
antibodies against different types of antigens.
serially diluted serum from different patients.
B. Which patient has the highest titer of anti-MBP antibodies? (A, B, C, D or E)
8. This diagram shows the steps in the production of monoclonal antibodies.
Identify the components labeled A – E.
Describe the process that is needed to go from step C to step E
9. 1Match the following technologies with their appropriate application.
MethodologyWould be best method for:
___ Elisa A. showing the presence of an intracellular pathogen.
___ Western blot B. showing the presence of a specific antigen in serum.
___ Flow cytometery C. separating a particular antigen for a tissue homogenate.
___ Immunofluorescence D. quantifying the titer of serum antibody.
___ Immunoprecipitation 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.
Which blood fraction will be most useful for distinguishing these individuals. Why?
Which sample is most probably from the person who is: (Explain your selections)
Recently infected _____:
With plasmacytoma _____:
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.
What type of methodology was used to stain the T-cells in this image?
What might be some target surface proteins on T-cells that could be used as T-cell specific target antigens?
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)
The data shown in lane 1 were obtained using which antibody based technology?
Why does lane ‘L’ contain many different types of proteins, lane 1 contains very few, and lane 2 none?
What is the approximate MW of the SV40 Large T antigen?
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?