Development of polyclonal antibodies-based serological methods and a DIG-labelled DNA probe-based molecular method for detection of the Vicia cryptic virus-M in field plants

Development of polyclonal antibodies-based serological methods for detection of the rehmannia mosaic virus in field plants

“…Recombinant proteins expressed by each of the two clones were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie brilliant blue (CBB) staining. The clone with the highest expression level of recombinant His-CP ReMV protein was selected for large-scale expression and purification, according to a previously described method (Zhang et al, 2022).…”

“…Sodium sulfate solution (50, 33, and 30%) was used to precipitate immunoglobulin IgG. After a series of purification procedures (Zhang et al, 2022), the polyclonal antibody against CP ReMV (PAb-CP ReMV ) with a relatively high degree of purity was obtained.…”

“…For western blotting, a 0.1 g sample was frozen in liquid nitrogen and ground quickly. Total protein was extracted using protein isolation buffer as previously described (Zhang et al, 2022). In the first antibody incubation step, the antiserum against His-CP ReMV was diluted 10 3 -fold (Figure 2D), and serially diluted (10 3 , 2 × 10 3 , 5 × 10 3 , 10 4 , and 2 × 10 4 ; Figure 2D), whereas the PAb-CP ReMV was diluted 10 3 -fold and 2 × 10 4 -fold (Figures 3C, 4, respectively).…”

“…For immuno-dot blotting, leaves were ground, and total protein was extracted and treated as previously described, with minor revisions (Zhang et al, 2022). In the first antibody incubation step, the antiserum was diluted for 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , and 10 6 -fold in Figure 2C, whereas the dilution was 10 5 -fold (Figures 4,5A,respectively).…”

“…Centrifugation was performed at 12,000 g for 10 min. The supernatant was collected, and ∼100 µL was loaded into a 96-well ELISA plate (100 µL/well), covered with silver paper, then incubated at 37 • C for 4 h, as previously described with minor revisions (Zhang et al, 2022). In the first antibody incubation step, the antiserum was diluted to 2.0 × 10 2 , 10 3 , 4 × 10 3 , 1.6 × 10 4 , 6.4 × 10 4 , 2.56 × 10 5 , and 1.024 × 10 6 -fold (Figure 2B).…”