1. Agglutination tests
HA & HI

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. Examples Slide Agglutination Test Plate Agglutination Test
Tube Agglutination Test
Passive Agglutination Test
Microscopic Agglutination Test
Haemagglutination test (HAT)

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

5. Slide Agglutination Test

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
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

7. Tube Agglutination Test

8. Tube Agglutination Test
No agglutination
1/10
1/20
1/40
1/80
1/160
1/320
Neg. ctrl
In this case, the titre is 1/40

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.

10. Quantitative Micro Hemagglutination Test (HA)
Haemagglutination Test (HA)

11. Haemagglutination

12. Haemagglutination
RBC

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
Comparison of Mycoplasma synoviae hemagglutinating antigens by the hemagglutination-inhibition test
Authors: T H Vardaman, J H Drott
Impact factor: 1.07, Cited half life: >10.0, Immediacy index: 0.29
Journal: Avian Diseases
Mycoplasma synoviae (MS) strains were isolated from the trachea of hens from MS-positive broiler breeder flocks having progeny condemnations due to airsacculitis. Hemagglutinating (HA) antigens were made from several strains. The HA antigen made from the 95th medium passage of MS FMT strain was compared with that of the standard MS WVU 1853 strain by the hemagglutination-inhibition (HI) test. Sixty-six sera from 10 MS-positive flocks, 4 Mycoplasma gallisepticum (MG)-positive sera, and 7 normal sera were used in the HI test. The geometric mean (GM) HI titer with MS WVU 1853 antigen was on 66 sera from 10 MS-positive flocks, whereas the GM HI titer with MS FMT antigen was

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

15. Equivalence point: (suitable proportion between the virus particles and RBCs)

16. Negative control well (only RBCs+ buffer) (no haemagglutinin)
Positive control well (contains haemagglutinin)

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
The HA titer of this virus in this row is 256 or 28
(1:256 dilution contains (1 HA unit) (one haemagglutinating unit)

18. Example of readings

19. mix with red blood cells
Hemagglutination test: method
1:8
1:2
1:2
1:2
1:2
1:2
virus
serial dilution 8 16 32 64
128
256
mix with red blood cells
side view
top view
Titer = 32 HA units/ml
One HA unit :minimum amount of virus that causes complete agglutination of RBCs

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. 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 HI buffer.

22. WHAT DO WE NEED?

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

24. WASHING RBCs

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. 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. 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.

28. Well – done !!

29. HEMAGGLUTINATION INHIBITION TEST (HI)
VIRUSE SERUM

30. In the absence of anti-virus antibodies
Erythrocytes
Virus
Virus agglutination of erythrocytes

31. In the presence of anti-virus antibodies
Erythrocytes
Anti-virus antibodies
Virus
Viruses unable to bind to
the erythrocytes

33. Purpose: To quantitate serum antibody to a specific avian antigen
Procedure:
A constant amount of haemagglutinating (HA) antigen is added to each well in a microtiter plate.
The test serum is then placed in the first well and serially diluted.
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.
HI NEGATIVE wells will have a diffuse sheet of agglutinated RBCs covering the bottom.
HI POSITIVE wells will have a well circumscribed button of unagglutinated       RBCs

34. Antibody Titer
Is the lowest concentration of antibodies against a particular antigen.
Figure 18.6

36. Readings The end point is the well with the lowest
concentration of the serum where a clear button is seen.
The antibody titer in this row will be 512 (29).
(the lowest concentration of Abs which inhibit HA caused by the virus )