Immunological Testing

Question 1

What are most immune based diagnostic/ lab assays based on?

Answer 1

AB Ag binding

Question 2

RECAP: what changes with repeated immunisation/ infection?

Answer 2

1. Ab titre changes in size from low to high magnitude
2. Isotope changes from IgM (low affinity to antigen) to IgG (high)
3. More rapid onset (recall of memory cells)
4. Longer duration response
5. Primary is initiated in local lymph nodes (Ag presented to naive lymphocytes) whereas secondary initiated in local lymphoid tissues (activation of primed lymphocytes)

Question 3

ELISA

1. What does it stand for?

Answer 3

Enzyme linked immunoSorbent Assay

Question 4

What does ELISA measure and what is good about using it?

Answer 4

1. Measures: Ag or Ab in an infection or hypersensitivity reaction
   Pros: quantifiable, quick, reproducible, inexpensive

Question 5

How do Elisa’s tests work? (colour ones)

Answer 5

1. Link enzyme to Ab
2. enzyme gives signal e.g. light (visible or uv).
3. Magnitude of light signal depends on the proportion of binding within the Elisa well - greater binding = more Ag Ab complexes

Question 6

Talk through steps of Elisa, e.g. canine distemper virus

Answer 6

Physical complex is: Antigen, test serum (Ab), detection Ab

1. Ag for CDV on well
2. Add animal serum to sample well
3. Relies on animal generating a kinetic immune response
4. If animal whose serum in well HAS generated CDV Ab (i.e got disease) then Ab will bind to Elisa well.
5. Those CDV -ve won’t bind
6. WASH well = remove unbound serum
7. Add canine ANTIBODY that detects all other canine Ab with an enzyme attached that gives signal (light)
8. WASH plate to remove unbound Canine Ab detection with enzyme
9. Stronger colour = stronger response

Question 7

What is a flaw of taking serum sample adn putting on ELISA plate?

Answer 7

doesn’t give accurate idea of titre/ concentration Ab as there is a finite capacity of binding with ELISA well = saturates assay

Question 8

What can you do to work out the Ab titre after doing an ELISA? e.g. 2 serum samples, same colour and want to see whihc has more Ab

Answer 8

END point titration

1. Keep diluting sample until can no longer measure Ab (no colour). It tells us which serum has the highest level of Ab

Question 9

Why could serum A and B samples have the same colour from ELISA test but after end point titration they have different Ab concs?

Answer 9

There reason why we cannot distinguish serum A form B at a single time point is because there is a finite capacity of binding within ELISA well = saturated assay

Question 10

When would we use ELISA testing on a healthy animal?

Answer 10

Also useful when measuring if vaccination has actually worked.
• Take serum sample and look for Ab in animal
• However need certain number Ab before animal is protected