Antigen & antibody reaction

(serum assay)
agglutination assay
ELISA
precipitation assay
complement fixation test
neutralization assay
 Precipitation/Immunodiffusion

Antigens and antibodies diffuse in the semi-

solid agar and form a precipitation line.
Radial Immunodiffusion
Double Immunodiffusion
Rocket immunoelectrophoresis
Coutercurrent immunoelectrophoresis (CIE)
Radial Immunodiffusion
Double Immunodiffusion
identity

nonidentity

Partial
identity
Rocket immunoelectrophoresis
Countercurrent immunoelectrophoresis
Countercurrent immunoelectrophoresis
Double Immunodiffusion
 Materials & Equipment
Powdered agar 1 bottle
N S 1
narrow neck flask: one for each group
Balance 1
Electric stove 1
Fridge
Gloves 1
Ear washing bulbs 4
Pipettes 4
Agar slide: one slide for each student
Perforator
 Materials & Equipment

Antibody (anti-serum)
Antigens
Positive control (+)
Negative control (-)
Sample 1
Sample 2
 Protocols

1. Prepare 1% agar solution 100ml

(1)Calculate required weight of agar powder
(2)Weigh agar powder
(3)Make 1% solution by mixing agar with N S***

***: 20ml NS first---powder second---remained

80ml NS at last
 Protocols

2. Heating up the agar solution

(1)Put the flask onto the stove and heating up
(2)Move away the flask every 30 seconds to
prevent overheat
(3) When small bubbles rising from the bottom,
its nearly boiling. To move away from stove.
No more bubbles, move onto the stove again.
Repeat this process for three times.
(4)Heating is over.
 Protocols
3. Making an agar slide
(1)To aspirate agar solution from flask using pipette
(2)The amount is 3.5ml.
(3)Let the solution fall down onto the slide slowly,
with the control of your finger (index finger)

*** leaving spaces between slides. If there is water

or agar solution around the slide, move this slide
to another place.
If the agar solution is spilled when making agar
slide, wash and dry it; then make again.
 The slides with agar solution cannot
be moved until they turn into white in
color about 5 minutes later.

Put all slides into fridge to promote their

solidification
 30 minutes later
 Perforating and application of samples
Try not to apply too much solutions into little wells
To make sure no spill

1 +

Ab

2 -
 18-24 hours later

Check the precipitation line between the wells

1 +

Ab

2 -
 Report 1 : results report

Positive control:
Negative control:
Sample 1:
Sample 2:
OR
To show the result with diagram
Conclusion:
Report 2 : protocol of double immunodiffusion

Concise
Key steps included
Things with more attention
Report 3 : diagrams of radial, rocket and
countercurrent
complement fixation test

MAC erythrocyte
complement fixation test
Neutralization assay
Neutralization assay
 Important Notice

Next time (one week later), one experiment left.

Practical Exam; contributing to final marks 10%