Correction to Antibody Array in a Multiwell Plate Format for the Sensitive and Multiplexed Detection of Important Plant Pathogens

Correction to Antibody Array in a Multiwell Plate Format for the Sensitive and Multiplexed Detection of Important Plant Pathogen

Selection of blocking buffers.

<p>A mixture of antibody-coated microsphere, MPC react to <i>Acidovorax avenae</i> subsp. <i>citrulli</i> (Aac), 1B4 specific to chilli vein-banding mottle virus (CVbMV), 2D6 specific to watermelon silver mottle virus (WSMoV) and 5E7 specific to melon yellow spot virus (MYSV), was tested with a single antigen and no pathogen using (A) 1% skimmed milk, (B) 1% casein or (C) 1% bovine serum albumin (BSA) as the blocking agent. Mixture of RPE-labeled antibodies, MPC, 1G8, A3 and 5E7, were used as a detecting system for Aac, CVbMV, WSMoV and MYSV, respectively. Y-axis is median fluorescent intensity (MFI). Each dataset was plotted as a mean of duplicates ± standard deviation.</p

Multiplex detection of four plant pathogens.

<p><i>Acidovorax avenae</i> subsp. <i>citrulli</i> (Aac) (10⁸ CFU mL<b>⁻</b>¹), chilli vein-banding mottle virus (CVbMV) (0.2 µg mL<b>⁻</b>¹), watermelon silver mottle virus (WSMoV) (5 µg mL<b>⁻</b>¹), melon yellow spot virus (MYSV) (10 µg mL<b>⁻</b>¹) and mixed pathogens (10⁸ CFU mL<b>⁻</b>¹) Aac, 0.2 µg mL<b>⁻</b>¹ CVbMV, 5 µg mL<b>⁻</b>¹ WSMoV and 10 µg mL<b>⁻</b>¹ MYSV) in (A) 1% casein in PBST and (B) artificially spiked healthy watermelon leaf extract were tested using immuno microsphere. Antibody (MPC, 1B4, 2D6 and 5E7) coated microspheres were used to detect Aac, CVbMV, WSMoV and MYSV, respectively. Normalized signal (Y-axis) is a ratio of signal obtained from pathogen detection in the samples to the signal obtained when no pathogen was present. Each dataset was plotted as a mean of duplicates ± standard deviation.</p

Selection of antibody pairs for the multiplex detection using a microsphere immunoassay.

<p>The detection of (A) <i>Acidovorax avenae</i> subsp. <i>citrulli</i> (Aac) and (B) no antigen using eight antibodies-coated microsphere and R-Phycoerythrin (RPE) labeled antibodies, including 11E5 antibody. The detection of (C) Aac, (D) chilli vein-banding mottle virus (CVbMV), (E) watermelon silver mottle virus (WSMoV), (F) melon yellow spot virus (MYSV) detection and (G) no antigen with seven antibodies- coated microsphere and RPE-labeled antibodies without using 11E5 antibody. X-axis is antibody-coated microsphere and y-axis is median fluorescent intensity (MFI) from each RPE-labeled antibody. (H) Summary of selected antibody pair sets for the detection of the four plant pathogens.</p

Selection of antibody pairs for the multiplex detection.

<p><i>Acidovorax avenae</i> subsp. <i>citrulli</i> (Aac or A), potyvirus (P), watermelon silver mottle virus (WSMoV or W), capsicum chlorosis virus (CaCV or C) and melon yellow spot virus detection (MYSV or M) were used for finding the proper antibody pairs. Note: RPE is R-Phycoerythrin.</p

Effects of assay time on sensitivity of detection.

<p>The different concentrations of <i>Acidovorax avenae</i> subsp. <i>citrulli</i> (Aac) (A), recombinant coat protein (CP) of chilli vein-banding mottle virus (CVbMV) (B), recombinant nucleocapsid protein (NP) of watermelon silver mottle virus (WSMoV) (C) and melon yellow spot virus (MYSV) (D) were detected in the microsphere immunoassay using four incubation times: 15 min (circle), 30 min (square), 45 min (triangle) and 60 min (diamond). Y-axis is a median fluorescent intensity (MFI). Each data point was plotted as a mean of duplicates ± standard deviation. (E) Comparison of sensitivity of detection between microsphere immunoassay (four different incubation times) and sandwich ELISA (60 min incubation only) with the same sets of antibodies.</p

Scheme of magnetic microsphere immunoassay.

<p>(A) The specific antibody-coated microspheres were mixed samples and incubated. (B) The unbound antigens were washed and removed by using magnetic separator. (C) The cocktail of RPE-labeled antibodies was added and incubated. (D) The unbound RPE-labeled antibodies were washed and removed by using magnetic separator before signals acquired by Luminex machine.</p

Antibodies used in the study.

<p>Antibodies used in the study.</p