2.2 Indirect ELISA
Antibodies specific for MYXV were detected by a commercial indirect ELISA (iELISA) (Civtest® Cuni Mixomatosis, Hipra, Girona, Spain), following manufacturer’s instructions. Antigens consisted of MYXV Sanarelli strain and protein G-horseradish peroxidase was used as conjugate. Briefly, serum samples were added to the plate (100 µl/well) at a dilution of 1:40 in dilution solution and incubated for 20 min at 37oC. After three washes with 300 µl washing solution/well, protein G-horseradish peroxidase conjugate was added (100 µl/well) and incubated for 20 min at 37oC. The plate was washed three times and 3,3’,5,5’-Tetramethylbenzidine (TMB) was added (100 µl/well) as liquid substrate and incubated for 10 min at room temperature. The reaction was stopped with 100 µl/well of H2SO4 and the optical density (OD) measured in a spectrophotometer at 450 nm. Positive and negative controls and samples were tested in duplicate in each plate. Results were expressed as Relative Index 10 (RI10) using the formula:
\begin{equation} RI10=\left[\frac{Mean\ OD\ sample-Mean\ OD\ negative\ control}{Mean\ OD\ positive\ control-Mean\ OD\ negative\ control}\right]\times 10\nonumber \\ \end{equation}