This study determined the effect of sample mishandling on the performance of ELISAs for detection of antibodies against infectious bronchitis virus (IBV), avian encephalomyelitis virus (AEV) and chicken anaemia virus (CAV) in the serum of chickens. The effects of five different sample mishandling treatments were assessed: heat treatment, repetitive freezing and thawing and three levels of severity of haemolysis. These mishandling treatments simulated different conditions that might occur during routine blood collection, transport or storage in a clinical practice setting. Each mishandling treatment was experimentally applied under laboratory conditions and then samples were assayed for antibodies against IBV, AEV and CAV using commercial ELISA kits. Severe haemolysis had the most consistent detrimental effect on ELISA performance, producing results that were significantly different from the reference standard in all three ELISAs, although the direction of the effect varied (less positive for the IBV and CAV assays; more positive for the AEV assay). Moderate levels of haemolysis had a similar, but less consistent, effect to that of severe haemolysis, producing results that were significantly different from the reference standard only for the IBV (less positive) and AEV (more positive) ELISAs. Repetitive freeze-thawing also produced a significant effect on ELISA results for IBV (less positive) and AEV (more positive). The IBV ELISA appeared to be most susceptible to the effects of serum maltreatment. The findings from this study suggest that unpredictable variation in the results of ELISAs can occur due to different sample mishandling treatments.
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