EQUINE VETERINARY EDUCATION / AE / july 2022
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Serum sample a) No antibody present
Interaction
Compliment free to fix to red blood cells
Result Haemolysis occurs
Sample is seronegative Antibody present b)
Compliment fixed to antigen-antibody complexes
No haemolysis as no free complement.
Red blood cells sink to bottom of the well
Sample is seropositive
Fig 5: Complement fixation test; a) interaction in a negative sample, haemolysis occurs indicating that there are no antibodies to the virus present in the sample to form Ab-Ag complexes and bind complement, and free complement lyses red blood cells; b) interaction in a positive sample (the highest last dilution at which haemolysis is prevented).
Fluorochrome labelled secondary antibody conjugate
Antibody in sample
Advantages of VN • High sensitivity and very high specificity
Disadvantages of VN
• Intensive for laboratory staff, requiring viral cell culture facilities
Antigen Fig 6: Indirect fluorescence antibody test.
effect will be observed. When performing a viral neutralisation (VN) test (may also be referred to as a serum neutralisation or SN test), the specific test virus is added to serial dilutions of sample serum. The diluted serum–antigen mix is then added to the wells of plates containing preformed monolayers of a susceptible cell line. If antibodies are present in the sample serum, they will bind to and neutralise the virus. With no virus available, the cells will show no viral cytopathic effect as a result. The plate will be read to determine the highest dilution of serum at which cytopathic effect occurs, to define the sample serum’s antibody titre. Very occasionally, the sample serum added may cause a cytotoxic effect to the cells which appears similar to cell cytopathic effect, interfering with the reading of the test. For equine viral arteritis, this phenomenon may be correlated to the animal recently receiving an inactivated equine herpes vaccination, which is based on virus grown in the same cell culture type as that used in the VN test (Newton et al. 2004). The main utilisation of the VN test is for prebreeding and
pre-export equine viral arteritis (EVA) testing. An ELISA is usually initially run for EVA and this ELISA has a high sensitivity. If a sample is positive for EVA on the ELISA, a VN test will be performed for confirmation, as the VN test is a more specific test when compared to the EVA ELISA. If an animal is known to be seropositive or vaccinated against EVA, the VN test is used directly.
Interpretations of doubling dilution assays such as CFT, HI and VN serological testing methods
The antibody titre of a sample is obtained by testing doubling dilutions of the sample serum and reporting the highest dilution at which antibody was still detected. Result authorisation is usually performed by a veterinary surgeon, assisting the laboratory. Positive and negative phrasing in result interpretation is used with caution as a case could have a ‘negative’ serology result but still have the infection. The most common reason for this phenomenon is whether serological sampling is conducted in the acute phase of the disease prior to an immune response being detectable. A titre does not need to be zero for a result to be interpreted as ‘no evidence of infection’, and each test titre result will have its own levels for suspicion of infection. Test interpretation is easiest when acute and convalescent samples are taken (paired samples), usually 14 days apart, with adequate history included in the submission to aid the laboratory. In double dilution assays such as CFT and HI, seroconversion is considered confirmed if there is a fourfold or greater rise in titre between the acute and convalescent samples. If a case is seropositive or seroconversion is confirmed, agent detection methods may be advised to investigate the infectious status of the case and/or contacts. Although the first serum sample from a case may have been tested at the time of acquisition, it will, ideally, need
© 2021 EVJ Ltd
• Requires highly skilled laboratory staff • Slow diagnostic test, can take up to 3 days • High level of interlaboratory variation • Occasional interference from some sera causing cell cytotoxicity
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