"Microbiology/Immunology Immunoassays" MARY

Question 1

The heavy chain and light chain contain 3 each of what component of the variable region?

Answer 1

Hypervariable regions

Question 2

Define antibody avidity.

Answer 2

The strength of binding of multivalent antiserum to multivalent antigen

Question 3

Why do antisera usually contain several Ab populations?

Answer 3

In may cases, the immunizing agent is not pure, so there are antigens present in addition to the antigen of interest. (Cross-reactivity)

Question 4

Name two common techniques employed to reduce cross-reactivity in a sample.

Answer 4

Absorption

Affinity chromatography

Question 5

Explain the preparation of monoclonal antibodies.

Answer 5

1. Immunize an animal (mice)
2. Isolate spleen cells from the immunized animal
3. Fuse the spleen cells to plasmacytoma tumor cells
4. Select for only those cells that are hybrids of tumor cells and cells
5. Clone the hybridomas so that each single cells grows up independently
6. Select the individual clone with the specificity that you are interested in.

Question 6

Why use tumor cells in the preparation on monoclonal Abs?

Answer 6

The unlimited growth potential of tumor cells means that you can clone the B cells of interest.

Question 7

What is the major advantage to using monoclonal Abs over over immune research techniques?

Answer 7

No heterogeneity - one B cell type produced (“mono”clonal)

Question 8

What three types of MABs were employed to reduce the human response to the Abs being administered?

Answer 8

1. Chimeric - constant regions are human, variable regions are mouse
2. Humanized - Only the points of contact with the antigen were mouse
3. Human monoclonals - generally completely human

Question 9

Explain the process of ELISA.

Answer 9

1. Coat plastic wells with Ab you are interested in so the Ab sticks
2. Incubate solutions with different amounts of the antigen that will attach to the Ab.
3. Wash the well, and the unbound Ag will be washed away
4. Add second Ab to Ag coupled to enzyme that you can test for, and measure the enzyme (ie color density)

Question 10

How is immunofluorescence different from ELISA?

Answer 10

1. Use tissue or actual cells
2. Instead of colored enzyme, second Ab has a UV-reactive molecule attached so it can fluoresce under UV light
3. Can identify a specific cell or cellular structure, or a pathogen

Question 11

Explain flow cytometry.

Answer 11

Flow cytometry uses a FACS, fluorescence activated cell sorter, a way to automate the process of immunofluorescence. Separates cells by both size and immunofluorescence and allows different ways to plot them on a graph.

Question 12

T/F: Western immunoblotting provides both qualitative and quantitative results.

Answer 12

True, can analyze both the amount and the molecular weight of the antigen, and different types of antigen that may be present.