Dot-ELISA is a modified ELISA technique based on sample-dotted nitrocellulose membranes. The Dot-ELISA procedure was carried out as described previously [40 (link)]. Briefly, 2 µL of maize leaf crude extract samples were pipetted evenly onto the nitrocellulose membranes (GE Healthcare, Bucks, UK) and incubated for 10 min at room temperature (RT), and a noninfected and a known MCMV-infected maize leaf crude extract were used as the negative and positive controls, respectively. Then, the membranes were blocked with PBST (a PBS solution containing 0.05% Tween-20) containing 5% skimmed milk powder for 30 min, followed by 1 h of incubation in a diluted anti-MCMV mAb solution at RT. After washing three times with PBST, alkaline phosphatase (AP)-conjugated goat anti-mouse immunoglobulin G (IgG) diluted in PBS containing 5% skimmed milk powder was added and incubated for another 1 h at RT. Afterward, the color reaction was created by adding a combination of NBT/BCIP (nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate) substrate solutions for 15 min at RT.
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