20.E: Laboratory Analysis of the Immune Response (Exercises)
20.1: Practical Applications of Monoclonal and Polyclonal Antibodies
In addition to being crucial for our normal immune response, antibodies provide powerful tools for research and diagnostic purposes. The high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets, from drugs to serum proteins to microorganisms. With in vitro assays, antibodies can be used to precipitate soluble antigens, agglutinate cells, and neutralize drugs, toxins, and viruses.
Multiple Choice
For many uses in the laboratory, polyclonal antibodies work well, but for some types of assays, they lack sufficient ________ because they cross-react with inappropriate antigens.
- specificity
- sensitivity
- accuracy
- reactivity
- Answer
-
A
How are monoclonal antibodies produced?
- Antibody-producing B cells from a mouse are fused with myeloma cells and then the cells are grown in tissue culture.
- A mouse is injected with an antigen and then antibodies are harvested from its serum.
- They are produced by the human immune system as a natural response to an infection.
- They are produced by a mouse’s immune system as a natural response to an infection.
- Answer
-
A
Fill in the Blank
When we inject an animal with the same antigen a second time a few weeks after the first, ________ takes place, which means the antibodies produced after the second injection will on average bind the antigen more tightly.
- Answer
-
affinity maturation
When using mAbs to treat disease in humans, the mAbs must first be ________ by replacing the mouse constant region DNA with human constant region DNA.
- Answer
-
humanized
If we used normal mouse mAbs to treat human disease, multiple doses would cause the patient to respond with ________ against the mouse antibodies.
- Answer
-
neutralizing antibodies
A polyclonal response to an infection occurs because most antigens have multiple ________,
- Answer
-
epitopes
Short Answer
Describe two reasons why polyclonal antibodies are more likely to exhibit cross-reactivity than monoclonal antibodies.
Critical Thinking
Suppose you were screening produce in a grocery store for the presence of E. coli contamination. Would it be better to use a polyclonal anti- E. coli antiserum or a mAb against an E. coli membrane protein? Explain.
20.2: Detecting Antigen-Antibody Complexes in vitro
Laboratory tests to detect antibodies and antigens outside of the body (e.g., in a test tube) are called in vitro assays. When both antibodies and their corresponding antigens are present in a solution, we can often observe a precipitation reaction in which large complexes (lattices) form and settle out of solution.
Multiple Choice
The formation of ________ is a positive result in the VDRL test.
- flocculant
- precipitin
- coagulation
- a bright pink color
- Answer
-
A
The titer of a virus neutralization test is the highest dilution of patient serum
- in which there is no detectable viral DNA.
- in which there is no detectable viral protein.
- that completely blocks plaque formation.
- that reduces plaque formation by at least 50%.
- Answer
-
D
In the Ouchterlony assay, we see a sharp precipitin arc form between antigen and antiserum. Why does this arc remain visible for a long time?
- The antibody molecules are too large to diffuse through the agar.
- The precipitin lattice is too large to diffuse through the agar.
- Methanol, added once the arc forms, denatures the protein and blocks diffusion.
- The antigen molecules are chemically coupled to the gel matrix.
- Answer
-
B
Fill in the Blank
When slowly adding antigen to an antiserum, the amount of precipitin would gradually increase until reaching the ________; addition of more antigen after this point would actually decrease the amount of precipitin.
- Answer
-
equivalence zone or zone of equivalence
The radial immunodiffusion test quantifies antigen by mixing ________ into a gel and then allowing antigen to diffuse out from a well cut in the gel.
- Answer
-
antiserum
Short Answer
Explain why hemolysis in the complement fixation test is a negative test for infection.
What is meant by the term “neutralizing antibodies,” and how can we quantify this effect using the viral neutralization assay?
Critical Thinking
Both IgM and IgG antibodies can be used in precipitation reactions. However, one of these immunoglobulin classes will form precipitates at much lower concentrations than the other. Which class is this, and why is it so much more efficient in this regard?
20.3: Agglutination Assays
In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination. Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate.
Multiple Choice
We use antisera to distinguish between various ________ within a species of bacteria.
- isotypes
- serovars
- subspecies
- lines
- Answer
-
B
When using antisera to characterize bacteria, we will often link the antibodies to ________ to better visualize the agglutination.
- latex beads
- red blood cells
- other bacteria
- white blood cells
- Answer
-
A
The antibody screening test that is done along with pretransfusion blood typing is used to ensure that the recipient
- does not have a previously undetected bacterial or viral infection.
- is not immunocompromised.
- actually does have the blood type stated in the online chart.
- is not making antibodies against antigens outside the ABO or Rh systems.
- Answer
-
D
The direct Coombs’ test is designed to detect when people have a disease that causes them to
- have an excessively high fever.
- quit making antibodies.
- make too many red blood cells.
- produce antibodies that bind to their own red blood cells.
- Answer
-
D
Viral hemagglutination assays only work with certain types of viruses because
- the virus must be able to cross-link red blood cells directly.
- the virus must be able to lyse red blood cells.
- the virus must not be able to lyse red blood cells.
- other viruses are too dangerous to work with in a clinical lab setting.
- Answer
-
A
Fill in the Blank
In the major cross-match, we mix ________ with the donor red blood cells and look for agglutination.
- Answer
-
patient serum
Coombs’ reagent is an antiserum with antibodies that bind to human ________.
- Answer
-
immunoglobulins/antibodies and/or complement
Short Answer
Explain why the titer of a direct hemagglutination assay is the highest dilution that still causes hemagglutination, whereas in the viral hemagglutination inhibition assay, the titer is the highest dilution at which hemagglutination is not observed.
Why would a doctor order a direct Coombs’ test when a baby is born with jaundice?
Critical Thinking
When shortages of donated blood occur, O-negative blood may be given to patients, even if they have a different blood type. Why is this the case? If O-negative blood supplies were depleted, what would be the next-best choice for a patient with a different blood type in critical need of a transfusion? Explain your answers.
20.4: Enzyme Immunoassays (EIA) and Enzyme-Linked Immunosorbent Assays (ELISA)
Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen. Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular structures (immunocytochemistry). Direct ELISA is used to quantify an antigen in solution.
Multiple Choice
In an enzyme immunoassay, the enzyme
- is bound by the antibody’s antigen-binding site.
- is attached to the well of a microtiter plate.
- is conjugated to the suspect antigen.
- is bound to the constant region of the secondary antibody.
- Answer
-
D
When using an EIA to study microtubules or other structures inside a cell, we first chemically fix the cell and then treat the cells with alcohol. What is the purpose of this alcohol treatment?
- It makes holes in the cell membrane large enough for antibodies to pass.
- It makes the membrane sticky so antibodies will bind and be taken up by receptor-mediated endocytosis.
- It removes negative charges from the membrane, which would otherwise repulse the antibodies.
- It prevents nonspecific binding of the antibodies to the cell membrane.
- Answer
-
A
In a lateral-flow pregnancy test, you see a blue band form on the control line and no band form on the test line. This is probably a ________ test for pregnancy.
- positive
- false-positive
- false-negative
- negative
- Answer
-
D
When performing an FEIA, the fluorogen replaces the ________ that is used in an EIA.
- antigen
- chromogenic substrate
- enzyme
- secondary antibody
- Answer
-
B
Fill in the Blank
To detect antibodies against bacteria in the bloodstream using an EIA, we would run a(n) ________, which we would start by attaching antigen from the bacteria to the wells of a microtiter plate.
- Answer
-
indirect ELISA
Short Answer
Why is it important in a sandwich ELISA that the antigen has multiple epitopes? And why might it be advantageous to use polyclonal antisera rather than mAb in this assay?
The pregnancy test strip detects the presence of human chorionic gonadotrophin in urine. This hormone is initially produced by the fetus and later by the placenta. Why is the test strip preferred for this test rather than using either a direct or indirect ELISA with their more quantifiable results?
Critical Thinking
Label the primary and secondary antibodies, and discuss why the production of end product will be proportional to the amount of antigen.
20.5: Fluorescent Auto-Antibody Techniques
Rapid visualization of bacteria from a clinical sample such as a throat swab or sputum can be achieved through fluorescent antibody (FA) techniques that attach a fluorescent marker (fluorogen) to the constant region of an antibody, resulting in a reporter molecule that is quick to use, easy to see or measure, and able to bind to target markers with high specificity. We can also label cells, allowing us to precisely quantify particular subsets of cells or even purify them for further research.
Multiple Choice
Suppose you need to quantify the level of CD8 T cells in the blood of a patient recovering from influenza. You treat a sample of the patient’s white blood cells using a fluorescent mAb against CD8, pass the cells through a flow cytometer, and produce the histogram shown below. The area under the peak to the left (blue) is three times greater than the area of the peak on the right (red). What can you determine from these data?
- There are no detectable CD8 cells.
- There are three times as many CD4 cells than CD8 cells.
- There are three times as many CD8 cells than CD4 cells.
- CD8 cells make up about one-fourth of the total number of cells.
- Answer
-
D
In the data described in the previous question, the average fluorescence intensity of cells in the second (red) peak is about ________ that in the first (blue) peak.
- three times
- 100 times
- one-third
- 1000 times
- Answer
-
B
In a direct fluorescent antibody test, which of the following would we most likely be looking for using a fluorescently-labeled mAb?
- bacteria in a patient sample
- bacteria isolated from a patient and grown on agar plates
- antiserum from a patient smeared onto a glass slide
- antiserum from a patient that had bound to antigen-coated beads
- Answer
-
A
Fill in the Blank
In flow cytometry, cell subsets are labeled using a fluorescent antibody to a membrane protein. The fluorogen is activated by a(n) ________ as the cells pass by the detectors.
- Answer
-
laser
Fluorescence in a flow cytometer is measured by a detector set at an angle to the light source. There is also an in-line detector that can detect cell clumps or ________.
- Answer
-
fragments
Critical Thinking
A patient suspected of having syphilis is tested using both the VDRL test and IFA. The IFA test comes back positive, but the VDRL test is negative. What is the most likely reason for these results?
A clinician suspects that a patient with pneumonia may be infected by Legionella pneumophila . Briefly describe two reasons why a DFA test might be better for detecting this pathogen than standard bacteriology techniques.