Page 349 - The Veterinary Laboratory and Field Manual 3rd Edition

318 Susan C. Cork, M. Faizal Abdul Careem and M. Sarjoon Abdul-Cader

second well and double diluting is continued to in the fridge and leave for 60 min at 4°C. The
the last well from which 0.025 ml is discarded. HI titre is the highest dilution of serum caus-
Then 0.025 ml of 1% v/v of washed chicken RBC ing complete inhibition of agglutination. This is
is added to each well and gently mixed. assessed by tilting the plate and observing the
The test is carried out in a 96 well (U-shaped highest dilution of serum at which the button
bottom) microtitre plate and the results are of red cells ‘streams’ at the same rate as the
recorded after 60 min. Agglutinated cells will be positive control RBC.
prevented from settling. The test should be read Both the HA and HI tests can usually be done
at the highest dilution giving complete aggluti- at room temperature unless this is extremely
nation. This is assessed by tilting the plate and high, in which case the reagents should be used
the highest dilution where no ‘streaming’ occurs directly from a refrigerator and the plates placed
is the endpoint and represents one HA unit. at 4ºC during the test. Titres may be expressed
Some technicians have difficulty understand- as a dilution, for example, 1 : 8 or 1 : 256; as
ing this titration and its conclusions. Imagine the reciprocal of the dilution, that is, 8 or 256,
that neat virus solution has 100 agglutinating or as log of the reciprocal, for example, 3 or 8.
2
units, then a 1 : 2 dilution will have 50, and a Alternatively, as mentioned previously, the titre
1 : 4 will have 25 and so on. A point will be can be represented as the highest dilution that
reached when there is less than 1 unit in the inhibits haemagglutination multiplied by the
diluted virus (in this case 1 : 128) and at this number of HA units used.
point the RBC will no longer agglutinate. In
other words, it takes at least 1 unit to agglu- rEaGEntS
tinate RBC. In this example, a 1 : 64 dilution Diluent: Sterile isotonic saline (0.85%)
was the last dilution to have at least 1 unit. If preferably use PBS to ensure a pH of 7.2
we take this dilution as 1 unit then 1 : 32 must to 7.4.
be 2 and 1 : 16 must be 4 HA units. 1 : 16 is the
dilution of antigen (virus) which must be used RBC (2–3 ml blood); ideally, these should be
in subsequent HI tests. The reason why this step collected into an equal volume of Alselver’s
is necessary is that different preparations of viral solution from not less than four (2–4-week old)
antigen may have different levels of reactivity, specific pathogen free (SPF) chickens. The cells
that is, the titre is variable and must be stan- should be washed not less than three times in
dardized so that test results can be compared sterile isotonic saline followed by centrifugation
between test batches and between laboratories.
at 1000 g for 5 min. The washed cells should
be diluted to a 1.0% suspension which can be
HI tESt determined photometrically or volumetrically.
Test and control sera are diluted to 1 : 2 in PBS The working suspension can be used for up to
and inactivated at 56°C for 30 min (to remove 36 hours if stored at 4ºC prior to use. Blood
complement). Twofold dilutions of 25 µl cells can be collected from birds known to be
amounts of serum to be tested are made in PBS. free of NCD but it may be difficult to interpret
Then 25 µl of diluted virus (4 HAU) is added to the results if they have been vaccinated.
each well. After a reaction time of 15 to 30 min,
25 µl of 1% v/v washed chicken RBC are added
to each well. After gentle mixing the plate is left
for 45 min at room temperature (22–24°C). If
the ambient temperature is high, put the plate

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