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 a...

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Main Authors: Orawan Himananto, Kirana Yoohat, Kannawat Danwisetkanjana, Mallika Kumpoosiri, Sombat Rukpratanporn, Yada Theppawong, Sudtida Phuengwas, Manlika Makornwattana, Ratthaphol Charlermroj, Nitsara Karoonuthaisiri, Petcharat Thummabenjapone, Nuttima Kositcharoenkul, Oraprapai Gajanandana
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2020-01-01
Series:PLoS ONE
Online Access:https://doi.org/10.1371/journal.pone.0237940
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author Orawan Himananto
Kirana Yoohat
Kannawat Danwisetkanjana
Mallika Kumpoosiri
Sombat Rukpratanporn
Yada Theppawong
Sudtida Phuengwas
Manlika Makornwattana
Ratthaphol Charlermroj
Nitsara Karoonuthaisiri
Petcharat Thummabenjapone
Nuttima Kositcharoenkul
Oraprapai Gajanandana
author_facet Orawan Himananto
Kirana Yoohat
Kannawat Danwisetkanjana
Mallika Kumpoosiri
Sombat Rukpratanporn
Yada Theppawong
Sudtida Phuengwas
Manlika Makornwattana
Ratthaphol Charlermroj
Nitsara Karoonuthaisiri
Petcharat Thummabenjapone
Nuttima Kositcharoenkul
Oraprapai Gajanandana
author_sort Orawan Himananto
collection DOAJ
description 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.
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spelling doaj.art-87adac8b8aa4461aa629e0f58d6f2a892022-12-21T18:33:45ZengPublic Library of Science (PLoS)PLoS ONE1932-62032020-01-01158e023794010.1371/journal.pone.0237940Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.Orawan HimanantoKirana YoohatKannawat DanwisetkanjanaMallika KumpoosiriSombat RukpratanpornYada TheppawongSudtida PhuengwasManlika MakornwattanaRatthaphol CharlermrojNitsara KaroonuthaisiriPetcharat ThummabenjaponeNuttima KositcharoenkulOraprapai GajanandanaAcidovorax 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.https://doi.org/10.1371/journal.pone.0237940
spellingShingle Orawan Himananto
Kirana Yoohat
Kannawat Danwisetkanjana
Mallika Kumpoosiri
Sombat Rukpratanporn
Yada Theppawong
Sudtida Phuengwas
Manlika Makornwattana
Ratthaphol Charlermroj
Nitsara Karoonuthaisiri
Petcharat Thummabenjapone
Nuttima Kositcharoenkul
Oraprapai Gajanandana
Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.
PLoS ONE
title Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.
title_full Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.
title_fullStr Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.
title_full_unstemmed Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.
title_short Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.
title_sort double antibody pairs sandwich elisa daps elisa detects acidovorax citrulli serotypes with broad coverage
url https://doi.org/10.1371/journal.pone.0237940
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