Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Himananto O

PROVIDER: S-EPMC7451559 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Himananto Orawan O Yoohat Kirana K Danwisetkanjana Kannawat K Kumpoosiri Mallika M Rukpratanporn Sombat S Theppawong Yada Y Phuengwas Sudtida S Makornwattana Manlika M Charlermroj Ratthaphol R Karoonuthaisiri Nitsara N Thummabenjapone Petcharat P Kositcharoenkul Nuttima N Gajanandana Oraprapai O

PloS one 20200827 8

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 ...[more]

PMID: 32853255

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Project description:Acidovorax citrulli causes bacterial fruit blotch (BFB) of cucurbits, a disease that threatens the cucurbit industry worldwide. Despite the economic importance of BFB, little is known about pathogenicity and fitness strategies of the bacterium. We have observed the phenomenon of phenotypic variation in A. citrulli. Here we report the characterization of phenotypic variants (PVs) of two strains, M6 and 7a1, isolated from melon and watermelon, respectively. Phenotypic variation was observed following growth in rich medium, as well as upon isolation of bacteria from inoculated plants or exposure to several stresses, including heat, salt and acidic conditions. When grown on nutrient agar, all PV colonies possessed a translucent appearance, in contrast to parental strain colonies that were opaque. After 72 h, PV colonies were bigger than parental colonies, and had a fuzzy appearance relative to parental strain colonies that are relatively smooth. A. citrulli colonies are generally surrounded by haloes detectable by the naked eye. These haloes are formed by type IV pilus (T4P)-mediated twitching motility that occurs at the edge of the colony. No twitching haloes could be detected around colonies of both M6 and 7a1 PVs, and microscopy observations confirmed that indeed the PVs did not perform twitching motility. In agreement with these results, transmission electron microscopy revealed that M6 and 7a1 PVs do not produce T4P under tested conditions. PVs also differed from their parental strain in swimming motility and biofilm formation, and interestingly, all assessed variants were less virulent than their corresponding parental strains in seed transmission assays. Slight alterations could be detected in some DNA fingerprinting profiles of 7a1 variants relative to the parental strain, while no differences at all could be seen among M6 variants and parental strain, suggesting that, at least in the latter, phenotypic variation is mediated by slight genetic and/or epigenetic alterations.

Dataset Information

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

Publications

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

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