Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection

Antibodies are are very important materials for diagnostics. A rapid and simple hybridoma screening method will help in delivering specific monoclonal antibodies. In this study, we systematically developed the first antibody array to screen for bacteria-specific monoclonal antibodies using Listeria monocytogenes as a bacteria model. The antibody array was developed to expedite the hybridoma screening process by printing hybridoma supernatants on a glass slide coated with an antigen of interest. This screening method is based on the binding ability of supernatants to the coated antigen. The bound supernatants were detected by a fluorescently labeled anti-mouse immunoglobulin. Conditions (slide types, coating, spotting, and blocking buffers) for antibody array construction were optimized. To demonstrate its usefulness, antibody array was used to screen a sample set of 96 hybridoma supernatants in comparison to ELISA. Most of the positive results identified by ELISA and antibody array methods were in agreement except for those with low signals that were undetectable by antibody array. Hybridoma supernatants were further characterized with surface plasmon resonance to obtain additional data on the characteristics of each selected clone. While the antibody array was slightly less sensitive than ELISA, a much faster and lower cost procedure to screen clones against multiple antigens has been demonstrated

Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections

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

Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection

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

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