L29 - Immunology in the Clinic and Research Lab

Question 1

What is the Fab and Fc region of the Antibody?

Answer 1

Fab = Fragment Antigen Binding

(antibody repertiore, affinity and avidity)

Fc = Fragment Crystallisable

(interacts with different elements of the immune system like ADCC, ADCP, CDC and Pharmacokinetics

Question 2

What is a Hybridoma?

Answer 2

Antibody producing B-cells are isolated from mice after immunising the mice with specific antigen. They are then fused with myeloma cells to form hybrid cells.

Hybrid cells selected via HAT (myeloma cells die as they cannot make nucleotides. B-cells die from short life span. Only Hybridomas grow.

Question 3

What are Immunosassays?

Answer 3

a procedure for detecting and measuring proteins/antibodies/antigens by tagging them

very sensitive and specific
can use polyclonal or monoclonal antibodies?

Question 4

Describe the labels for Immunoassays?

Answer 4

originally radioactive
radioimmunoassay (RIA)
commonly now enzyme e.g. horseradish peroxidase or alkaline phosphatase -usually detected by coloured product (colorimetric)
enzyme-linked immunosorbent assay (ELISA)
other alternatives are luminescent

Question 5

Describe the direct ELISA and its uses?

Answer 5

antigen immobilised on solid support

test antibody solution covalently linked to enzyme (e.g. Horseradish peroxidase or alkaline phosphatase for colorimetric ELISA) added

Enzyme substrate added, coloured product produced which can be measured by absorbance

USES:

• screen hybridoma supernatants
• detect exposure to infectious agent

Question 6

Describe the Sandwich ELISA?

Answer 6

Antigens may be present in low concentration. Because antibodies have high affinity for antigen this technique can concentrate the antigen

need two antibodies reacting with different epitopes on the antigen

one antibody immobilised on solid support

test antigen solution added, incubated and non-bound removed by washing

bound antigen detected by incubation with the other antibody, which has been labelled, and non-bound removed by washing

Question 7

Describe the Elispot Immunoassay?

Answer 7

TO DETECT CYTOKINES:

• Cytokine specific antibodies are bound to the surface of a plastic well.
• Activated T-cells are added to the well. These T-cells are a mixture of different effector functions
• Cytokine secreted by some activated T-cells is captured by the bound antibody
• The captured cytokine is revealed by
  a cytokine-specific antibody, which is coupled to an enzyme, giving rise to a spot of insoluble coloured precipitate

Question 8

What can SDS PAGE be used for?

Answer 8

1) Can be used to detect antigens or antibodies
2) Used to measure size of the protein being analysed
3) Can be used to calculate protein concentration
4) May show if protein has been degraded

Question 9

What is MHC typing and what is it used for?

Answer 9

MHC alleles of donor and recipient are identified by PCR

Needed for transplant compatibility

Question 10

How is Immunofluorescence used to detect autoantibodies

Answer 10

Immunofluorescence: serum added to human cell line. Then probed with fluorochrome-labelled anti-immunoglobulin antibody. Visualised by fluorescence microscopy