Haemagglutination Test

. Haemagglutination test

Introduction

All strains of Newcastle disease virus will agglutinate chicken red blood cells. This is the result of the haemagglutinin part of the haemagglutinin/neuraminidase viral protein binding to receptors on the membrane of red blood cells. The linking together of the red blood cells by the viral particles results in clumping. This clumping is known ashaemagglutination.

Haemagglutination is visible macroscopically and is the basis of haemagglutination tests to detect the presence of viral particles. The test does not discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells. Both can cause the agglutination of red blood cells.

Note that some other viruses and some bacteria willalso agglutinate chicken red blood cells. To demonstrate that the haemagglutinating agent is Newcastle disease virus, it is necessary to use a specific Newcastle disease virus antiserum to inhibit the haemagglutinating activity.

Substances that agglutinate red blood cells are referred to as haemagglutinins.

Note:

The abbreviation HA is used in this manual for haemagglutinin andhaemagglutination.

Two tests are described, the rapid test which takes one minute and the micro test which takes 45 minutes.

Figure 19: Principle of the haemagglutination test

Figure 19. - A. Negative control well (no haemagglutinin)

Figure 19 - B. Positive control well (contains haemagglutinin)

Red blood cell control in the haemagglutination test

Every time a haemagglutination test iscarried out, it is necessary to test the settling pattern of the suspension of red blood cells. This involves mixing diluent with red blood cells and allowing the cells to settle.

1. Dispense diluent.

2. Add red blood cells and mix by gently shaking.

3. Allow the red blood cells to settle and observe the pattern.

4. Observe if the cells have a normal settling pattern and there is noauto-agglutination. This will be a distinct button of cells in the micro test and an even suspension with no signs of clumping in the rapid test.

Note:

The diluent used for haemagglutination tests in this manual is PBS.

There should be no signs of haemolysis in the red blood cell suspension. If there are signs of haemolysis, a fresh suspension must be prepared.

There should not be anysign of auto-agglutination in the red blood cell control. If an agglutination pattern is observed, discard the suspension of red blood cells. Prepare a fresh suspension and test again.

Control allantoic fluid samples

Negative and positive control samples are tested in both the rapid and micro haemagglutination tests to ensure the validity of the test.

Negative control allantoic fluid isharvested from 14-day old embryonated eggs that have not been inoculated with Newcastle disease virus. It should always test negative for the presence of haemagglutinins. There should not be any sign of haemagglutination.

Positive control allantoic fluid is known to contain a high infectivity titre of Newcastle disease virus. It should always test positive for the presence of haemagglutinins.Haemagglutination should be visible.

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 bloodcells. 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...