S-ELISA

Checkerboard titration was done according to Harlow and Lane [37] to obtain the different dilution of antigen, sera, and conjugate. A ninety-six well, flat-bottomed ELISA plates were incubated overnight with 100 μl/well of hyperimmune sera from adult and nymphs diluted in carbonate buffer. After 3 times washings with PBS- Tween-20; 100 μl of diluted antigens of nymphs and adults at 10 μg/ml in PBS were incubated 37°C for 2 h. 100 μl/well of naturally infected and unknown sera were added with concentration 1:100 in PBS. Then, run the ELISA plate as indirect ELISA procedure mentioned above. The procedure followed according to Ana and Finlay [38], Rui et al. [39], and Dixit et al. [40].