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.

Highlights

  • ObjectivesWe aimed to produce monoclonal antibodies (MAbs) specific to A. citrulli using membrane protein extracts of A. citrulli as immunogens

  • Culture supernatants were simultaneously screened against fluorescently barcoded microbeads coupled to membrane protein complex (MPC) of five different A. citrulli strains and D. acidovorans

  • monoclonal antibodies (MAbs) 11E5, a positive control obtained in our previous study [18], is known to react with four A. citrulli strains but does not recognize Acidovorax citrulli (Ac) PSA or D. acidovorans

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Summary

Objectives

We aimed to produce MAbs specific to A. citrulli using membrane protein extracts of A. citrulli as immunogens. The aims of this study were to produce MAbs specific to A. citrulli strain PSA and to use these MAbs in the development of a new ELISA method that can detect all available A. citrulli strains

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Results
Conclusion

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