Hemagglutination (HA) Assay Protocol
The hemagglutination assay is a method for titering influenza viruses based on their ability to
attach to molecules present on the surface of red blood cells. A viral suspension may agglutinate
the red blood cells, thus preventing them from settling out of suspension. By serially diluting a
virus in a 96-well plate and adding a consistent amount of red blood cells, an estimation of the
amount of virus present can be made.
Equipment and Materials Required
• Certified Biological Safety Cabinet
• Tabletop centrifuge with appropriate fittings
• Inverted microscope (optional)
• 15 ml conical tubes
• Disposable pipettes – 1 ml, 5 ml, 10 ml
• Micropipette and sterile disposable aerosol resistant tips – 160 µl
• PBS
• Turkey red blood cells in Alsevers solution purchased from a supplier such as Lampire
Biological Products
• Round-bottomed 96-well dish
RBC preparation:
1. 4 ml of blood is pipetted into a 15 ml conical and topped off with PBS.
2. Spin in tabletop centrifuge at 800 rpm for 10 minutes.
3. Aspirate the supernatant without disturbing the blood cells.
4. Add 12 ml PBS and mix by inverting – do not vortex.
5. Spin at 800 rpm for 5 minutes and repeat wash two more times.
6. Aspirate supernatant after final wash and add enough PBS to make a 10% solution of red
blood cells. This solution is useable for one week.
7. Make a final working solution of 0.5% RBCs in PBS.
Rapid haemagglutination test
This test can determine the presence of a haemagglutinating agent in one minute. If testing many
samples at the same time, it is necessary to test the negative and positive control samples only
once.
Materials
Clean glass microscope slide or a clean white ceramic tile.
10 percent suspension of washed chicken red blood cells. See Section 8.
Micropipette and tips, glass Pasteur pipette or a wire loop.
PBS.
Negative and positive control allantoic fluid samples.
Sample to be tested for the presence of Newcastle disease virus, for example allantoic
fluid.
Method
1. Place 4 separate drops of 10 percent chicken red blood cells onto a glass slide or a white tile.
2. To each drop of blood, add one drop of the control and test samples as follows. Use separate
tips, pipettes or a flamed loop to dispense each sample.
Drop 1 PBS
Drop 2 Negative control allantoic fluid (no haemagglutinin)
Drop 3 Positive control allantoic fluid (contains haemagglutinin)
Drop 4 Unknown sample to be tested
3. Mix by rotating the slide or tile for one minute.
4. Observe and record results. Compare results of the test samples with the control samples.
Results
Agglutinated red blood cells in suspension have a clumped appearance distinct from non-
agglutinated red blood cells.
The red blood cells mixed with the positive control allantoic fluid will clump within one
minute.
The red blood cells mixed with the PBS and negative control allantoic fluid remain as an
even suspension and do not clump.
Judge the results of the test sample by comparison with the positive and negative controls.
The PBS and negative allantoic fluid controls are used to detect clumping of the red
blood cells in the absence of virus. This is unlikely to occur. If it does occur, the test is
invalid.
Micro haemagglutination test in a V-bottom microwell plate
This method is convenient when testing allantoic fluid from a large number of embryonated eggs
for the presence or absence of haemagglutinin. A 1 percent solution of red blood cells is used.
The cells settle faster in V-bottom plates and there is a better contrast between positive and
negative results than observed in U-bottom plates. The method for preparing eggs for and
harvesting of allantoic fluid for this test is described in detail in Section 9.
Materials
Inoculated eggs, chilled for at least 2 hours, preferably overnight
Negative and positive control samples
V-bottom microwell plate and lid
Micropipette and tips to measure 50 µL
1 percent suspension of red blood cells
70 percent alcohol solution
Cotton wool
Forceps and/or small scissors
Absolute alcohol
Discard tray
Microwell plate recording sheet. See Appendix 9
Method
1. Fill in the details of samples being tested on a recording sheet. Samples and controls will be
distributed into the wells as indicated on this sheet.
2. Use a micropipette to remove 50 mL of allantoic fluid from each egg and dispense into a well
of the microwell plate. Use a separate tip for each sample.
3. Include negative and positive control allantoic fluid samples on one of the plates.
4. Dispense 50 mL of PBS into two wells. These wells will be the red blood cells controls for
auto-agglutination.
5. Add 25 mL of 1 percent red blood cells to each well.
6. Gently tap sides of the plate to mix. Place a cover on the plate.
7. Allow the plate to stand for 45 minutes at room temperature.
8. Observe and record the results.
Results
The settling patterns of single and agglutinated red blood cells are different. Single cells roll
down the sides of the V-bottom well and settle as a sharp button. Agglutinated cells do not roll
down the sides of the well to form a button. Instead they settle as a diffuse film.
Negative HA result = a sharp button
Positive HA result = a diffuse film
Red blood cell control = a sharp button
Mark the HA results on microwell recording sheet.
