2. 2
Agglutination tests
• Antibodies can agglutinate multivalent
particulate antigens, such as Red Blood
Cells (RBCs) or bacteria
• Some viruses also have the ability to
agglutinate with RBCs.
• This behavior is called agglutination.
• Serological tests based on agglutination
are usually more sensitive than those
based on precipitation
3. 3
Examples
• Slide Agglutination Test
• Plate Agglutination Test
• Tube Agglutination Test
• Passive Agglutination Test
• Microscopic Agglutination Test
• Haemagglutination test (HAT)
4. 4
Slide Agglutination Test
• Used for serotyping (e.g. Salmonella)
• Antigen: isolated Salmonella in suspension
• Antibody: specific antisera against Salmonella
• Place test Salmonella in a drop of saline on a
slide
• Add a drop of antiserum, mix and rock slide for
approx 1 minute
• Examine for agglutination
6. 6
Tube Agglutination Test
• Also known as the standard agglutination test or serum
agglutination test (SAT)
• Test serum is diluted in a series of tubes (doubling
dilutions)
• Constant defined amount of antigen is then added to
each tube and tubes incubated for ~20h @37°C
• Particular antigen clumps at the bottom of the test tube
• Test is read at 50% agglutination
• Quantitative
• Confirmatory test for ELISA reactors
• Example: Brucellosis screening
9. 9
Passive Agglutination Test
• Converting a precipitating test to an
agglutinating test
• Chemically link soluble antigen to inert
particles such as LATEX or RBC
• Addition of specific antibody will cause the
particles to agglutinate
• Reverse PAT: antibody linked to LATEX
e.g. Lancefield grouping in Streptococci.
13. 13
Viral Haemagglutination
• Some viruses and microbes contain proteins
which bind to erythrocytes (red blood cells)
causing them to clump together
• NDV
• Adenovirus III
• AIV
• IBV
• Mycoplasma
14. 14
Viral Hemagglutination
• the attachment of viral particles by their receptor sites
to more than 1 cell.
• As more and more cells become attached in this
manner agglutination becomes visible
16. Negative control well (only RBCs+ buffer) (no
haemagglutinin)
Positive control well (contains haemagglutinin)
17. 17
Readings The results
• Titer: The maximum dilution that gives
visible agglutination.
• The end point: is the well with the lowest
concentration of the virus where there is
haemagglutination
2 4 8 16 32 64 128 256 512 1024 2048 4096
The HA titer of this virus in this row is 256 or 28
(1:256 dilution contains (1 HA unit) (one
haemagglutinating unit)
19. Titer = 32 HA units/ml
Hemagglutination test: method
1:8
1:2 1:2
1:2
1:2
1:2
8 16 32 64 128 256
virus
serial dilution
mix with red
blood cells
side view
top view
One HA unit :minimum amount of virus that causes
complete agglutination of RBCs
20. 20
CALCULATIONS
• For HI systems with different HA units:
(4 HA units for NDV ,8 HA unit for AIV, 4
or 8 for adenovirus group III).
• divide the virus titer on the needed HA
unit.
21. 21
CALCULATIONS
• If the titer is 32, How can you get 4 HA
units?
• 1/32 contain 1 HA
• 1/32 * 4 = 4/32 = 1/8.
• 1/8 dilution contain 4 HA units.
• So 1/8 = (1/ 1+7)
– 10 wells * 50μl/well = 500 μl total volume.
• 500/8= 62.5 μl virus + 437.5 HI buffer.
23. 23
PROCEDURE
(CONTROLs)
• Always run four control rows:
_ Positive: Contains antibodies against the specific virus
_ Negative: Contains no antibodies against the specific virus
_ Antigen
_ RBCs
25. 25
Why do we have to wash
RBCs?
• To obtain pure RBCs and to get rid
from any other blood components
such as WBCs, immune complexes,
and Abs
26. 26
Washing process
• Take place 4-5 time .
• Until get clear solution above the RBCs
after centrifugation .
• Using PBS or normal saline .
Note :(avoid using water to wash RBCs
because it will definitely lead to RBCs lyses)
27. 27
Procedure
• Obtain blood samples in tubes, spin at 1500
RPM for 5 minutes.
• Draw off the supernatant using Pasteur pipette.
• Add 2ml PBS to each tube and move to a clean
test tube.
• Centrifuge again. Each time draw off the
washing solution and add 10 ml PBS until the
solution above the RBCs layer becomes clear.
33. 33
• Purpose: To quantitate serum antibody to a specific
avian antigen
Procedure:
1. A constant amount of haemagglutinating (HA) antigen is
added to each well in a microtiter plate.
2. The test serum is then placed in the first well and
serially diluted.
3. The plates are incubated for one hour and then chicken
RBCs are added to each well. If antibody is present in
the test serum the RBCs will not agglutinate with the HA
antigen.
1. HI NEGATIVE wells will have a diffuse sheet of agglutinated
RBCs covering the bottom .
2. HI POSITIVE wells will have a well circumscribed button of
unagglutinated RBCs
34. 34
Antibody Titer
• Is the lowest
concentration of
antibodies against
a particular
antigen.
Figure 18.6
36. 36
Readings
• The end point is the well with the lowest
concentration of the serum where a clear
button is seen.
2 4 8 16 32 64 128 256 512 1024 2048 4096
The antibody titer in this row will be 512 (29).
(the lowest concentration of Abs which inhibit HA caused
by the virus )
