DAS-ELISA was carried out as described by Clark and Adams with some minor modifications, using a commercially available PVY IgG and the alkaline phosphatase-conjugated PVY IgG. Polystyrene microtiter plates were coated for 3 h at 34°C, with 200 μl per well of IgG coating, in 50 mM carbonate buffer, pH 9.6. The plates were then incubated for 1 h at 34°C with PBS (10 mM phosphate buffer, pH 7.2, 0.8% NaCl and 0.02% KCl). After that, the plates were washed three times using washing buffer (0.8% NaCl, pH 7.2 and 0.05% Tween 20). The infection-free (control) and PVY-infected potato leaf samples were ground in ten volumes (w/v) of PBS buffer pH 7.2, containing 0.2% polyvinyl pyrrolidone and 2% of egg albumin (Sigma A5253).The infected preparations were serially diluted (fivefold dilution) at the same buffer. Aliquots of 195 μl of prepared samples were added to each well, and the plates were incubated overnight at 4°C. Plates were then washed three times with washing buffer, incubated for 4 h at 37°C, with 190 μl per well of alkaline phosphatase-conjugated PVY IgG diluted in sample buffer, washed again, and incubated lastly for 90 min, with p-nitrophenylphosphate (1 mg/ml), in 10% diethanolamine, pH 9.8. Data were expressed and recorded using Multiskan at A405 nm.
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Citation: Almasi MA, Dehabadi SH (2013) Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y. J Plant Pathol Microb 4: 188
Last date updated on November, 2024