Page 348 - The Veterinary Laboratory and Field Manual 3rd Edition

Serology and immunology 317

Haemagglutination inhibition test Ha tESt
Preparation of chicken RBCs (1.0%)
This test is usually performed using a 96 well
microtitre plate (Figure 6.9a and b). In this test, 1 Collect chicken RBCs in an equal volume of
each well used will contain equal volumes and Alselver’s solution at least 3 days before the
strengths of antigen, an equal volume of 1% test.
RBCs and the same volume of doubling dilutions 2 Wash RBCs three times in PBS (pH 7.2 to 7.4
of test serum (antibody). Haemagglutinating isotonic saline).
properties of the virus are blocked by antibody 3 Mix 1.0 ml of packed RBCs with 99.0 ml of
binding to the virus. Blocking takes place up to PBS (1% VV).
the dilution of serum (antibody) where there is
no longer sufficient antibody to combine with, Twofold dilutions of a 0.025 ml (25 µl) volume
and eliminate the virus. At this dilution virus of antigen are made in sterile PBS (pH 7.0 to
will be free to agglutinate RBCs. 7.4). To do this, place 0.025 ml of PBS in each
Protocols for HA and HI tests for Newcastle well, 0.025 ml of virus solution is added to the
disease (NCD) in poultry follow. first well, mixed, and 0.025 ml is taken to the

Figure 6.9 (a) Photograph of a plate
used to perform the HI test (note
that this is not the same test plate
as that shown in Figure 6.9b). Where
there is a ‘button’ of red cells, this
indicates that agglutination has not
taken place. The HI test is used for
the detection of antibodies against,
for example, influenza virus. The
test sera are treated to eliminate any
non-specific haemagglutinins and (a)
inhibitors and are two-fold diluted
with PBS in 96 well micro titre
plate. Antigen (in this case influenza (b)
virus) is then added. Column 1–10
HI titres = 64, 128, 128, 32, < 4,
16, 32, 16, 16, 32 respectively.
See also Plate 31. Photo: Dr Davor
Ojkic, Animal Health Laboratory,
University of Guelph, Canada. (b)
Diagrammatic representation of the
haemagglutination inhibition test.
Rows F, G and H contain reagent
controls. The button of RBCs
indicates no agglutination. See also
Plate 32. Source: M. Sarjoon Abdul-
Cader, University of Calgary, Canada.

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