HAEMAGGLUTINATION TEST (HA-TEST)

A specific form of agglutination (clumping of cells) that involves red blood cells (RBCs). It
has two common uses in the laboratory: blood typing and the quantification of virus
dilutions.

Materials/Equipments required:

1. RBC from SPF chickens.

2. Killed Antigens
3. Pipettes
4. Microtitre plates
5. Microtitre plate sealer
6. Multichannel pipettes
7. PBS 0.01 M (pH 7.0 - 7.2)
8. Disinfectants
9. Fridge
10. Syringe 5 – 10ml
11. Needles
12. Incubator
Preparation of 1% chicken red blood cell (RBC) suspension

A total of 5 ml of chicken blood was collected aseptically in a disposable syringe

containing 1 ml of sodium citrate (4% solution) as anticoagulant. The blood was
centrifuged at @1500 rpm for 15 minutes and the plasma and buffy coat was removed
with a pipette. After washing three times with phosphate buffered saline (PBS), 1%
suspension in PBS was prepared for HI test. To prepare 1 % RBC suspension, 1mL
packed RBCs is dissolved in 99mL of PBS.

Haemagglutination Test (HA TEST)

Principle: Some HA-bearing viruses agglutinate RBCs in vitro.

RBCs + Viruses Haemagglutination.

Procedure:

1. Add 50 µL PBS to all wells (1-12) of micro-titration plate.

2. Add 50µL antigen to 1st well and make two fold dilution up to 11th well.
3. Add 50µL 1% RBC suspension to all wells (1-12)
4. Incubate the plate at 37 0C for 30 minutes.
5. Record the results
6. Button formation is negative and clumped formation is positive result.
7. Calculate 4HAU of the virus

Calculation of 4HAU

If titer of the virus in HA test is up to the 10th well

Then at 10th well titer is 1024
So 4HAU = 1024/4
= 256
It means we will add 1mL of the virus and 255mL PBS that will be 4HAU of the virus.

Frequently Asked Questions (FAQs):

1. What is agglutination?
A clumping of bacteria or red blood cells when held together by antibodies.
2. What is haemagglutination?
The clumping together of red blood cells.
3. What is basic principle of agglutinations tests?
The Antigens and Antibodies are very specific in their reaction.
4. What is meant by 1 HA unit?
One HA unit in the haemagglutinin titration is the minimum amount of virus that
will cause complete agglutination of the red blood cells. The last well that shows
complete agglutination is the well that contains one HA unit
5. Why do we use 4HA unit of virus for HI test?
To maintain the required concentration of the virus that is able to agglutinate the
RBCs.
6. Which viruses give HA test?
Newcastle disease virus (NDV), Adenovirus III, Avian Influenza Virus (AIV),
IBV, Mycoplasma (Bacterium), measles virus, Mumps virus etc.