Serological Assays for Virus Detection

ELISA and western blot analyses were carried out as previously described [41 (link)]. ELISA results were considered positive when the optical density (O.D.) values were at least 2.5 times the negative (healthy) control. The negative control O.D. range was 0.005–0.015. For western blot analyses, the leaf samples were compared at constant ratios of urea-SDS-β-mercaptoethanol lysis buffer and leaf weight. Accordingly, the increase in PepMV-IL in ToBRFV and PepMV-IL mixed infected plants was a quantitative comparison with PepMV-IL singly infected plants. The specific antisera prepared against purified virions of ToBRFV from Tm-2² allele-bearing tomato plants and PepMV-IL virions isolated on D. stramonium plants, as previously described [19 (link), 42 (link)], were used in the assays. Ponceau-S staining was conducted before or following the detection of the specific coat proteins, the latter identifying the viral coat proteins (CP) and RuBisCO on the membrane.

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Matzrafi M., Abu-Nassar J., Klap C., Shtarkman M., Smith E, & Dombrovsky A. (2023). Solanum elaeagnifolium and S. rostratum as potential hosts of the tomato brown rugose fruit virus. PLOS ONE, 18(3), e0282441.

Publication 2023

Allele Antisera Assays Buffer Elisa Leaf Membrane Mercaptoethanol Optical Plants Ponceau s Proteins Rubisco Stramonium Tomato Urea Viral coat proteins Virions Western blot analyses

Corresponding Organization : Hebrew University of Jerusalem

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Variable analysis

independent variables

Infection with ToBRFV and PepMV-IL

dependent variables

Optical density (O.D.) values in ELISA
Expression levels of viral coat proteins (CP) in western blot analysis

control variables

Ratio of urea-SDS-β-mercaptoethanol lysis buffer to leaf weight in western blot analysis
Negative (healthy) control samples in ELISA

controls

Negative control (healthy) samples in ELISA with O.D. range of 0.005–0.015
Specific antisera prepared against purified virions of ToBRFV from Tm-2^2 allele-bearing tomato plants and PepMV-IL virions isolated on D. stramonium plants, used in the assays

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