EQUINE VETERINARY EDUCATION / AE / july 2022
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Disadvantages of ELISA
• Commonly requires two serum samples, taken 10-14 days apart, to assist interpretation and demonstrate a rising titre to a particular infection
• If a case is vaccinated with a nonmarker vaccine, no discrimination can be made between infected and vaccinated
• Some ELISAs, particularly those designed to be performed stable side, only give a positive or negative result, with no result quantification
Agar gel immunodiffusion
Agar gel immunodiffusion (AGID) utilises agar gel with a specific antigen at its centre, and serum samples and positive controls will be added in wells around the periphery of the agar (Gershwin 2008). If a sample contains antibodies, a precipitation line will form (Fig 2). An example of this is the Coggins test for equine infectious anaemia (EIA). An ELISA is usually run initially for EIA, unless there are export requirements for a Coggins test. This ELISA has high sensitivity, but low specificity, so if a sample is positive for EIA on the ELISA, it will be run on a Coggins test for confirmation, as the Coggins test provides a better specificity (Scicluna et al. 2013).
Haemagglutination inhibition assay
Some viruses (e.g. equine influenza virus) bind (haemagglutinate) red blood cells together, and this characteristic is utilised in the haemagglutination inhibition (HI) assay. Sample serum is added in quantified serial dilutions to wells on a plate, and a known titre of virus is then added, followed by red blood cells. If there are no specific antibodies present in the sample serum to the added virus, the virus will bind to the red blood cells, agglutinating them. If there are specific antibodies to the test virus present, they will bind to the virus and as a result, there will be no unbound virus to agglutinate red blood cells (Fig 3). The plate will be read to determine the highest dilution of serum where haemagglutination by nonbound virus starts to occur again, to define the samples antibody titre.
Advantages of HI • Quick and easy to perform
• Readily quantifiable for clear detection of a rising titre, when testing paired serum samples
a) C+ 1 AG C+ 3 C+ C+ 3
Fig 2: Agar gel immunodiffusion method. AG is equine infectious anaemia antigen, C+ is positive control sera. a) test sera 1, 2 and 3 are all positive. b) test sera 1, 2 and 3 are all negative.
2 1 AG C+ b) C+ 2
Disadvantages of HI
• Only detects antibodies to viruses that induce haemagglutination
• Will only detect antibodies to the particular viral subtype being tested for, or those strains which cross react with that subtype
Single radial haemolysis
Single radial haemolysis (SRH) is generally used in equine influenza research studies as it provides a continuous quantitative result when compared to HI’s dilution titre. SRH involves virus being incubated with red blood cells. Complement is added, and the mixture is set in agar gel. Sample serum is then added to wells cut out in the gel. If the serum sample contains specific antibodies, antigen–antibody complexes form and complement fixation occurs. This results in red blood cell lysis, and a zone of haemolysis will be seen on the agar gel (Fig 4). The size of the zone of haemolysis around the well is measured to provide a quantified result. If there are no antibodies in the sample serum, there are no antigen– antibodies to fix complement and no haemolysis zone is seen.
Advantages of SRH • Provides a quantitative result
• Poor differentiation of response to different strains of H3N8 EI virus, so therefore remains relevant despite viral evolution
Disadvantages of SRH • Specialist test requiring trained personnel to perform and read the test results
• Does not differentiate between antibody from natural infection and that from vaccination (not DIVA)
• Poor differentiation of response to different viral strains, so therefore does not provide information on viral strain typing
Complement fixation test
When performing a complement fixation test (CFT), the test virus is added to serial doubling dilutions of sample serum. Complement is then added, followed by antibody-coated red blood cells, which are the indicator for unbound complement in the absence of target anti-viral antibody. If the sample serum contains specific complement activating antibody, these will bind to the viral antigens and the formed antigen–antibody complex will fix the complement. There will be no free complement to cause red blood cell lysis. If there is no antibody present in the sample serum, there will be no formation of
antigen–antibody complexes and therefore no complement fixed. The complement will remain free, with the indicator stage involving the complement reacting with antibody-coated red blood cells, resulting in haemolysis (Fig 5). Some sample serum can demonstrate anti- complementary activity, interrupting interpretation of the test (false positives) which is often caused by samples containing high serum protein levels. Therefore, each sample serum is also tested for its anti-complementary activity to allow for this possibility and aid result interpretation. To detect whether this phenomenon is present in test sera, an additional well is included and the viral antigen is left out. This is to see
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