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}