Block A

Question 1

What is the primary function of antibodies in research, diagnostics, and treatment?

Answer 1

Antibodies specifically bind to antigens.

Question 2

What are the key characteristics needed when generating antibodies for use in assays?

Answer 2

High specificity and high avidity.

Question 3

What are the benefits of multiple immunisations when generating antibodies?

Answer 3

Multiple immunisations increase the amount and affinity of antibodies in the serum of immunised animals. They also result in class-switching, leading to more IgG/IgA.

Question 4

What is antiserum, and how is it produced?

Answer 4

Antiserum is blood serum containing high levels of specific antibodies. It’s collected from animals that have been immunologically primed.

Question 5

What are the advantages and disadvantages of using polyclonal antibodies?

Answer 5

Polyclonal antibodies are relatively cheap and easy to produce. However, serum contains many other proteins that may cause problems in sensitive assays, and polyclonal antibodies recognize multiple epitopes, with different classes and isotypes, and have the potential for contaminating antibodies.

Question 6

What are the advantages of using monoclonal antibodies?

Answer 6

Monoclonal antibodies can be produced in large quantities, bind to a very specific epitope of an antigen, are of the same Ig class and isotype, can be highly purified, and allow for in-depth analysis without batch variation.

Question 7

What is phage display in the context of monoclonal antibody production?

Answer 7

Phage display uses bacteriophage to screen and produce monoclonal antibodies, allowing rapid screening of many V region-expressing clones.

Question 8

What are nanobodies and what are their advantages?

Answer 8

Nanobodies are produced recombinantly, are small in size, have no heavy or light chains, and are highly stable. They are made through phage display using libraries from immunised camelids.

Question 9

How are antibodies typically detected in assays?

Answer 9

Often, antibodies require conjugation with an enzyme or fluorochrome to allow for detection.

Question 10

What are some common labels used for antibody detection?

Answer 10

Enzymes (e.g., horseradish peroxidase), radioactive molecules, and fluorescent molecules.

Question 11

What is serology?

Answer 11

Serology is the detection or quantification of antibodies made in response to microbial infections.

Question 12

What can the type of antibody (IgM vs IgG) tell you about an infection?

Answer 12

IgM is associated with a recent infection, while IgG is associated with a historic or chronic infection.

Question 13

Briefly describe the steps of an ELISA for detecting IgG.

Answer 13

1) Surface coated with antigen, 2) Add serum (contains IgG), 3) Add anti-IgG, 4) Add substrate, 5) Stop reaction and measure.

Question 14

Why are samples serially diluted in an ELISA?

Answer 14

To determine an endpoint, since the range of antibody concentrations in serum is large and not all of it will be within the linear portion of a graph of absorbance versus antibody concentration.

Question 15

How can the principle of the ELISA be adapted?

Answer 15

By changing the label on the antibodies or the plate, such as using fluorescence, radioactivity, or a microscope slide with a tissue section (immunohistochemistry).

Question 16

How can microbes be directly detected using antibodies?

Answer 16

Using antibodies that have been generated to them, which can be polyclonal or monoclonal.

Question 17

What is an ELISPOT assay?

Answer 17

It is based on ELISA, but allows for the quantification of cytokine-producing cells.

Question 18

What is fluorescence and how is it used in immunoassays?

Answer 18

Fluorescence is the emission of light from a substance that has absorbed radiation of another wavelength. Fluorochromes are conjugated onto antibodies to detect where those antibodies are bound.

Question 19

What are some tools that are used in research to detect fluorescence in immunoassays?

Answer 19

Fluorescent microscopes and flow cytometers are used.

Question 20

What does forward scatter measure in flow cytometry?

Answer 20

Forward scatter measures diffracted light, which is related to cell size.

Question 21

What does side scatter measure in flow cytometry?

Answer 21

Side scatter measures reflected light, which is related to cell granularity and complexity.

Question 22

What type of results are generated in flow cytometry?

Answer 22

Results are displayed as distribution histograms (single parameter studies) or 2D dot plots (multiple parameter studies).

Question 23

What are some examples of cell-based immunoassays?

Answer 23

Measuring cell proliferation, viability, and functions such as killing or cytokine secretion.

Question 24

How are white blood cells isolated from peripheral blood?

Answer 24

Using density centrifugation, such as Ficoll-Histopaque.

Question 25

How can specific cell types be isolated?

Answer 25

Using antibodies and magnets.

Question 26

How can the number of cells be quantified?

Answer 26

Using a hemocytometer.

Question 27

How can cell proliferation be measured?

Answer 27

Enzymatic approaches (e.g., MTT assay), radioactive approaches (e.g., H-Thymidine incorporation), and fluorescent approaches (e.g., CFSE labeling).

Question 28

How can cell death be measured?

Answer 28

Using vital dyes like Trypan Blue or Propidium Iodide.