Prac 2

Question 1

Precipitation

Answer 1

Soluble / small (virus size)Atg + specific (at least bivalent)Ab + adequate circumstances
= Complex formation, precipitation
Phases • Liquid • Gel
Hours, days

Question 2

Precipitation v Agglutination

Answer 2

Agglutination
• Insoluble Atg • Big sized Atg • Minutes, hours
Precipitation
• Soluble Atg • Small sized Atg • Hours, days

Question 3

Precipitation in liquid phase

Answer 3

Tube precipitation tests
1. Mix the Atg & Ab = precipitate
2. Atg & Ab layered on each other =precipitation ring (disk)
Microtiter plates

Question 4

Ascoli’s thermoprecipitation test

Answer 4

Detection of Bacillus anthracis heat-stable cell wall Atg Cross reacts with other Bacillus spp.
Method
1. 5 ml physiological saline solution + approx. 2 g organ sample + acetic acid up to 1%
2.Boil for 5 minutes 3.Filter through filter paper after cooling down
4. Layer 1-2 drops of the Atg on top of the specific Ab
5. If positive, a precipitation ring is formed within 15 minutes, where the two layers meet

Question 5

Immuno-diffusion tests

Answer 5

1. Simple gel diffusion
   Layer the Atg solution on top of Ab agar containing
2. Radial gel diffusion (Mancini test)
   Atg in a well, Ab in the agar; Quantitative test
   The diameter of the ring is proportional to the cc.
   The relative concentration of the Atg can be determined, if the test is done parallel with known concentrations of a standard Atg
3. Double gel diffusion (Ouchterlony test)
   Atg and Ab in separate wells

Question 6

3. Double gel diffusion (Ouchterlony test)

Atg and Ab in separate wells

Answer 6

1. Add 1 g agar to 100 ml buffer
2. Boil in microwave oven
3. Measure 3-3 ml agar into Petri dishes, when it cooled down
4. Cut wells according to picture, once solidified
5. Make twofold dilutions of the test sample (Ab) in PBS: 50µl PBS + 50 µl sample, mix, transfer half of it into the next 50µl PBS and so on
6. Measure 20 µl Atg into the middle well and 20-20 µl of the diluted sample into the surrounding wells
7. Incubate on a level surface, in a humid chamber at 37 °C

Question 7

Complement fixation assays

Answer 7

The complement fixation assays utilize these characteristics of the system:

1. Immune complexes can activate the cascade (classical pathway);
2. The final product of the reaction (Membrane Attack Complex, MAC) causes cytolysis

Types:
Reaction volume
• Macro (in tubes)
• Micro (on microtiter plates)

Nature of the antibody
• Direct (complement-activating Ab)-Positive reaction: NO hemolysis; Negative reaction: hemolysis
• Indirect (non-complement-activating Ab)