Page 347 - The Veterinary Laboratory and Field Manual 3rd Edition

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

Figure 6.8(c) Agar gel immunodiffusion is a qualitative test used for the detection of antibodies against
influenza A virus routinely. A semi-solid agar gel base is used to cut the wells as indicated in the Figure
6.8(a). The central well is allocated for placing the antigen of interest and the well numbers 1, 3 and 5 are
used for placing the positive serum to the antigen of interest (a). The well numbers 2, 4 and 6 have been
used for the serum samples to be evaluated. Within 48 hours of placing the sera and antigen precipitin
lines are identifiable. The left image shows the standard avian influenza antigen, serum and a prepared
agar gel base. The right image shows the antigen–antibody reactivity as visualized by precipitin lines. Well
number 2 = weak positive, well number 4 = negative and well number 6 = strongly positive. Photos: Dr
Davor Ojkic, Animal Health Laboratory, University of Guelph, Canada.

oblique electric light may assist with the reading haemagglutinating virus in cell cultures or in
of opaque lines. Positive and negative controls faecal emulsions (for example, parvovirus in
should be included in each test. Occasionally a dog faeces) can be carried out by demonstrat-
haze forms around the well due to lipids or other ing haemagglutination with appropriate RBC.
material in which case the test may need to be Haemagglutinating properties of viruses are
repeated with a fresh sample. most often used in diagnostic laboratories to
detect and titrate antibodies by using the HI.
However, before the HI test can be carried out,
Haemagglutination tests the haemagglutinating strength of the virus must
be determined. This is done in order to standard-
HA is the agglutination of RBC, which can ize the test so that the HI test is carried out in
be caused by chemical agents or viruses. different laboratories using a uniform strength of
Haemagglutination inhibition (HI) is the inhibi- antigen. Doubling dilutions of antigen are mixed
tion of HA, for example by using hyperimmune with washed RBC and allowed time to aggluti-
serum specific to a haemagglutinating virus to pre- nate, the dilution at which agglutination stops
vent haemagglutination occurring. The property is considered to contain 1 haemagglutinating
of some viruses to agglutinate the RBC of poul- unit (1 HAU). By counting back two doubling
try and certain other animals is used in a range of dilutions the dilution containing 4 HAU is deter-
systems to detect viral antigen and also to titrate mined and this is the strength of antigen usually
specific antibody. The agglutination of RBCs is used in the HI test. Note that there are a number
accomplished by the binding of proteins on the of methods published for HI tests, here we will
outer coat (envelope) of the virus with receptor outline a method that we have used.
sites on the red cells. Presumptive tests to detect

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