Optimization of Dot-ELISA
Upon optimization, checkerboard titration revealed the optimal antigen concentration, serum and conjugate dilution at 188 ng/dot, 1:800 and 1:4000 respectively for clear distinction between positive and negative serum samples (Fig. 2 ). The serum and conjugate dilution were selected to attain an acceptable well defined dots with less background/noise signal on strips at the minimum concentration of rHyC antigen. The antigen volume was selected at 2 µL to obtain appreciable size of dot for better visual observation of results.