Specificity determination.

<p>Specificity of the 11E5/MPC, 2D6/MYSV6, and 5E7/MYSV6 antibody pairs was tested for Ac, WSMoV, and MYSV, respectively. The antibody pairs were tested against bacteria (Ac, SQB, Pf, and DAc) and viral (TYLCV) protein standards (n = 3). The data shows averaged S/N values with error bars representing standard deviations. The dotted horizontal line indicates the threshold (or the cutoff value) of S/N = 2 (twice of values obtained from negative controls).</p

Coating and blocking buffer selection.

<p>A. Different coating buffers (Optibind A-L) were tested for maximum surface binding of 11E5, 2D6, and 5E7 (n = 3). B. Blocking buffers (2% BSA, 3% skim milk, 1% casein, and Optiblock solution) were tested for Ac, WSMoV, and MYSV detection (n = 3). An ideal blocking buffer resulted in highest S/N ratios. Error bars indicate ± standard deviations.</p

Study of repetitive sample loading.

<p>Effect of repetitive sample loading on assay dynamic range was investigated, using Ac as a model. The plot indicates that multiple loading helps increase assay sensitivity (n = 3). Error bars indicate ± standard deviations.</p

Schematic workflow depicting sequential molecular binding events of the sandwich ELISA.

<p>Within each reaction chamber, the capture antibody is adsorbed on the reactive surface followed by surface passivation by a blocking buffer. Upon target binding to the capture antibody, alkaline phosphates (AP)-tagged detection antibody specific to the antigen is added. Addition of fluorescent substrate (PNPP or <i>p</i>-nitrophenyl phosphate for the traditional well format, and Attophos for the micofluidic format) activated by AP generates detectable fluorescent signal, indicating successful binding events.</p

Sensitivity determination of the microfluidic platform.

<p>Comparison of assay dynamic range for Ac (A), WSMoV (B), and MYSV (C) detection between protein standards and spiked plant extracts (n = 3) by the microfluidic platform. Error bars indicate ± standard deviations.</p

Optimization of antibody concentration.

<p>Nine different conditions for each disease panel were tested on the microfluidic platform using combinations of three concentrations of capture Ab (11E5, 2D6, and 5E7) and three concentrations of detection Ab (MPC-AP, MYSV6-AP). Panel A–C show results for Ac, WSMoV, and MYSV detection, respectively (n = 4). The S/N ratios were plotted for each of the conditions tested. Error bars indicate ± standard deviations.</p

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