A2 Tests

Question 1

Ouchterlony test

Answer 1

both antigen and antibody allowed to diffuse in to the gel. lines of precipitation measured in gel matrix
lines determine if antigens are the same or different

Question 2

Name role of

1. Enzymes
2. Radiation
3. Fluorescence

Answer 2

1. Catalyzes reaction, often resulting in colour change
2. can link/incorporate radioactivity emitters with the Ab - detect radiation of e.g. alpha, beta or gamma
3. avoids hazards of radiation BUT is expensive

Question 3

Explain Serology

- what does it determine

Answer 3

Determines if antibodies have been produced in response to infection
~can determine strain of microbe (peptide ELISA)
Tells if infection recent or not: IgM/ IgG

Question 4

Describes steps of ELISA e.g. for IgG

2. describe sandwich ELISA

Answer 4

Step 1: surface coated with Antigen
Step 2: Add serum (contains IgG) 
Step 3: Add anti-IgG 
Step 4: Add substrate 
Step 5: Stop reaction and measure

2. same as above but first coated with anti-pathogen antibody

Question 5

Why are samples serially diluted?

Answer 5

So that the endpoint can be determined
results become
- more accurate

Question 6

Describe ELISPOT

Answer 6

Based on ELISA but for quantification of cytokine producing cells

1. cytokine specific antibodies are bound to well surface
2. Activated Tc added to well.
3. Cytokine secreted by some activated Tc is captured by bound antibody
4. Captured cytokine is revealed by a 2nd cytokine specific antibody (which is coupled to an enzyme) Gives rise to a spot of insoluble colored precipitate

Often used to measure frequency of cells producing certain cytokine

Question 7

Describe fluorescence

Answer 7

Fluorescence is the stimulated emission of light from a substance which has absorbed radiation (light) of another wavelength.

Useful for immunoassays by conjugating fluorochromes onto antibodies

Question 8

Describe fluorescence microscopy

Answer 8

Stain with enzyme-linked of fluorescence-linked antibodies to detect the cells or molecules of interest
Visualize under light microscope to see color changes (enzymes) or with laser-scanning fluorescence microscope

Question 9

Describe Flow cytometry

Answer 9

1. cells labelled w/ fluorescent antibodies
2. fluorescence detected by PMT and sent to computer
   - Gate towards cell of interest

photomultiplier tubes (PMT)

Question 10

Flow cytometry

1. what does forward scatter measure
2. side scatter

disadvant to flow cytometry

Answer 10

1. Diffracted Light: (shadow of the cell) & related to cell size
2. Reflected light: (light reflecting from cellular components) & related to cell granularity and complexity

Dis: doesn’t reveal location of cells/ how they are interacting
Solution: whole body imaging of Ag specific cells?