Evaluation of short reaction times for optimising das-ELISA schedule for three flexuous potato viruses*

A simple and rapid protocol of alkaline phosphatase - based das-ELISA was standardized to detect three common flexuous potato viruses, viz. PVX, S and Y, from either potato sprouts/leaves or other systemically infected hosts. In all, five different combinations of varying incubation periods were tried, viz. 1 to 3 h at 37°C for each of coating, sample loading and over night probing at +4°C. Samples were extracted in the PBS+T soln. with 2% PVP+1M urea+0.2% eggalbumin and also diluted 1:10, 1:100, 1:1000 with the same. It was possible to detect all the three flexuous potato viruses after 1 h incubation at 37°C at each step but the A₁₀ values were significantly higher with longer incubation periods (either 2 or 3 h at each step); 1 or 2h coating coupled with 3 h each for trapping and probing and the controls, i.e. 2-day standard schedule. Detection of PVX and PVS was possible in a short substrate reaction of 15 min. while it required almost 30 min for PVY. The short incubation protocol is helpful in rapid assay requiring only about half a working day for 90×3=270 samples being handled by one technician.