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Coating buffer

Manufactured by Agdia

Coating buffer is a laboratory reagent used to prepare surfaces for immunoassay testing. It is a solution that helps to bind and stabilize target analytes on a solid support, creating a surface suitable for subsequent detection steps.

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3 protocols using Coating buffer

Indirect ELISA analyses were performed on plant leaves using laboratory-produced specific antibodies against ToMV or ToBRFV (a kind gift from Dr. A. Dombrovsky, ARO, Rishon LeZion, Israel) as previously described [30 (link),31 (link)]. In the analysis, two discs, 1 cm in diameter, taken from the 4th and the 5th true leaf represented each plant. Samples were taken 30 days after inoculation, ground in coating buffer (Agdia) and incubated for 3 h at 37 °C with 1:5000 dilution of the specific laboratory-produced antiserum (anti-ToBRFV or anti-ToMV). Detection was carried out by incubating the samples with AP-conjugated goat anti-rabbit (IgG) (Sigma, Steinheim, Germany) for 3 h at 37 °C. P-nitro phenyl phosphate (Sigma) substrate was used at a concentration of 0.6 mg/mL. The developing color was recorded by Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham MA, USA) at 405 nm and 620 nm. Optical density (OD) values of a minimum ratio of three times the value of the negative, non-infected controls were considered positive.
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Leaf samples, as well as fruit pericarp and washed seeds (24 h in water), were subjected to indirect ELISA test, principally as previously described [32 (link)]. Samples were ground in coating buffer (Agdia) at a ratio of ~600 µL/µg sample and incubated with 1:5000 dilution of specific antibodies against ToBRFV [9 (link)] in PBS for 3 h at 37 °C. For detection, alkaline phosphatase (AP)-conjugated goat antirabbit IgG (Sigma, Steinheim, Germany, 1:5000 in PBS) and p-nitro phenyl phosphate substrate (Sigma, 0.6 mg/mL) were used. Data were recorded at 405 nm and 620 nm, and O.D. values that were 2.5 times the value of the negative controls were considered positive.
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Tomato fruit samples (calyx, pericarp) and mechanically inoculated tomato plants (leaves) seven days post disease symptom development, were subjected to indirect ELISA test [42 (link)]. Samples were ground in coating buffer (Agdia) and were incubated for 3 h at 37 °C with 1:5000 dilution of the ToBRFV antiserum or the PepMV antiserum. Detection was carried out by incubating the samples with AP-conjugated goat anti-rabbit (IgG) (Sigma, Steinheim, Germany) for 3 h at 37 °C. P-nitro phenyl phosphate (Sigma) substrate was used at a concentration of 0.6 mg/mL. The developing color was recorded by ELISA reader (Thermo Fisher Multiskan FC) at 405 nm and 620 nm. O.D. values of a minimum ratio of three times the value of the negative (healthy) controls were considered positive.
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