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.

Significant Sensitivity Improvement for Camera-Based Lateral Flow Immunoassay Readers.

Recent developments in smartphone-based strip readers have further improved the performances of lateral flow test kits. Most smartphone cameras encode an unaltered and nonlinear power-law transfer function that maps the light intensity to a pixel value; this poses some limitations for camera-based strip readers. For faint-color test lines which are almost as white such as with nitrocellulose pads, the slope of the transfer function is low. Therefore, it is difficult to differentiate between the faint test lines and the white background. We show that by manually setting the camera exposure time-instead of using the automatic settings-to the high-slope region of the transfer function, the reader's sensitivity can be improved. We found that the sensitivity and the limit of detection of the Acidovorax avenae subsp. citrulli (Aac) test kit were enhanced up to 3-fold and 5-fold, respectively, when using the readers at the optimal camera settings, compared to the automatic mode settings. This simple technique can be readily applied to any existing camera-based colorimetric strip reader to significantly improve its performance.

Fluorescein Isothiocyanate Labeling Antigen-Based Immunoassay Strip for Rapid Detection of Acidovorax citrulli.

A simple and fast immunoassay strip to detect Acidovorax citrulli (Ac) using fluorescein isothiocyanate as a marker was developed. Fluorescein isothiocyanate (FITC) was added to sample culture medium for bacteria incubation, and the bacteria could emit a yellow-green fluorescence under ultraviolet light and become a fluorescent probe. This immunofluorescence strip (IFS) was based on the binding between fluorescent bacteria and the unlabeled monoclonal antibody (McAb) immobilized on the test area in nitrocellulose membrane. The detection limit of the strip was 106 CFU/ml with a result that could be observed within 10 min. The IFS could detect eight strains of Ac and display no cross-reactions with 30 other pathogenic strains. The detection results would not be affected by impurities in plant or unknown microorganisms in natural field samples and were consistent with PCR results, indicating that the IFS has high accuracy. This is the first report of using only one unlabeled McAb to develop a direct-type immunofluorescence strip for the rapid detection of Ac. The IFS reduced detection time and simplified operation procedures compared with the traditional enzyme-linked immunosorbent assay (ELISA) and PCR methods. In addition, this simple and inexpensive method will play a significant role in monitoring plant pathogens on field detection.

Glycan moiety of flagellin in Acidovorax avenae K1 prevents the recognition by rice that causes the induction of immune responses.

Abstract Recognition of pathogen-associated molecular patterns (PAMPs) such as flagellin, a main component of the bacterial flagellum, constitutes the first layer of plant immunity and is referred to as PAMP-triggered immunity (PTI). The rice avirulent N1141 strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, induces PTI including H2O2 generation, while flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. Mass spectrometry analyses revealed that total 1,600-Da and 2,150-Da of glycan residues were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin, suggesting that the glycan in K1 flagellin prevent epitope recognition in rice. We identified three genes in K1 flagella operon, which regulate structural modification of glycan in K1 flagellin. The immature glycan-attached flagellin from three genes deletion mutant, KΔ3FG, induced H2O2 generation in cultured rice cells, whereas the K1 mature-type flagellin did not cause a detectable increase in H2O2. The data indicate that the immature glycan of flagellin from KΔ3FG cannot prevent the epitope recognition in rice.

Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.

Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation.

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin.

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H2O generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H2O2 generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹78, Ser¹8³, Ser²¹², and Thr³5¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹78 and Ser¹8³ in K1 flagellin prevent epitope recognition in rice.

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody.

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.

Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice.

Plants have sensitive perception systems that recognize various pathogen-derived molecules. We previously reported that rice detects flagellin from a rice-incompatible strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, which induces subsequent immune responses involving cell death. The mechanism of flagellin perception in rice, however, has remained obscure. In this study, we found that flg22, a peptide derived from the flagellin N-terminus, induced weak immune responses without cell death in cultured rice cells. To elucidate the mechanism by which flg22 induced signaling in rice, we characterized OsFLS2, the rice ortholog of AtFLS2, which mediates flg22 perception. Heterologous expression of OsFLS2 functions in Arabidopsis, showing the conservation of the flg22 signaling pathway across divergent plant taxa. OsFLS2-overexpressing rice cultured cells generated stronger immune responses with the induction of cell death following stimulation with flg22 and flagellin. However, examination of the growth rate of the compatible strain in inoculated OsFLS2-overexpressing rice could not confirm bacterial growth suppression compared with wild-type rice. These results suggest that rice possesses a conserved flagellin perception system utilizing the FLS2 receptor which, when upregulated, hardly affects resistance against compatible A. avenae.