Immunology in the clinic and research lab

Question 1

What are immunoassays?

Answer 1

immuno
uses antibody-antigen interaction (one of which is “labelled” or “tagged” to allow its detection)

assays
measures (amount, concentration) of antibody or antigen
very sensitive and specific
hence, widely used in research and analytical labs

Immunoassays use polyclonal or monoclonal antibodies

Question 2

How are antibodies/antigens labelled?

Answer 2

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 3

What do we know about Solid phase immunoassays?

Answer 3

e.g. ELISA
Direct/Indirect
Often used to quantify an antibody

Sandwich (Capture)
Often used to quantify an antigen

Using these assays, the concentration of analyte (antibody or antigen) in the sample can be calculated by comparison to analyte standards of known concentration

Question 4

How does a direct ELISA work and what are its uses?

Answer 4

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 5

How does an indirect ELISA work?

Answer 5

antigen immobilised on solid support
primary antibody which binds to antigen is then added
Secondary antibody covalently attached to enzyme is subsequently added. Secondary antibody binds to Fc region of primary antibody
Enzyme substrate added, colour measured by absorbance

Secondary antibody is often polyclonal and so may bind to different epitopes on a primary antibody. This allows multiple secondary antibodies to bind to the same primary antibody thereby amplifying the signal and increasing the sensitivity of the test

Question 6

How does a sandwich (Capture) ELISA work?

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

What do we use an Elispot immunoassay to do?

Answer 7

measure level of cytokines

Question 8

What is involved in Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western Blotting immunoassaying?

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

Protein standards of known size and concentration (‘ladder’) – usually labelled to allow detection in western blot

SDS PAGE/Western blotting often used alongside ELISA

In WB, protein concentration can be measured by comparing intensity of band we are detecting to band from a protein standard of known concentration

If protein is degraded it may be more useful to use WB to calculate protein concentration, as some of degradation fragments may contribute to signal in ELISA if both coating and detecting antibody are able to bind to them

Question 9

What tests can be conducted to diagnose potential allergies?

Answer 9

Skin Prick Test: In allergic individual, IgE binds to allergen and via the Fc region of the antibody binds to receptors on mast cells. This causes mast cells to degranulate causing the release of mediators (e.g histamine) which causes reddening and swelling of skin

RAST (RadioAllergoSorbent Test) The suspected allergen is bound to an insoluble material and the patient’s serum is added. If the serum contains antibodies to the allergen, those antibodies will bind to the allergen. Radiolabeled anti-human IgE antibody is added where it binds to those IgE antibodies already bound to the insoluble material. The amount of radioactivity is proportional to the serum IgE for the allergen

Often fluorescence is used instead of radioactivity