Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms

Nguyen Xuan Vu; Natcha Pruksametanan; Witsanu Srila; Watcharin Yuttavanichakul; Kamonluck Teamtisong; Neung Teaumroong; Nantakorn Boonkerd; Panlada Tittabutr; Montarop Yamabhai

doi:10.1371/journal.pone.0179983

Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms

PLoS One. 2017 Jun 27;12(6):e0179983. doi: 10.1371/journal.pone.0179983. eCollection 2017.

Authors

Nguyen Xuan Vu^{1

2}, Natcha Pruksametanan¹, Witsanu Srila¹, Watcharin Yuttavanichakul², Kamonluck Teamtisong³, Neung Teaumroong², Nantakorn Boonkerd², Panlada Tittabutr², Montarop Yamabhai¹

Affiliations

¹ Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
² Biofertilizer & Plant-Microbe Interaction Research Unit, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
³ The Center for Scientific and Technological Equipment, Suranaree University of Technology, Nakhon Ratchasima, Thailand.

Abstract

A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

MeSH terms

Animals
Bradyrhizobium / isolation & purification*
Enzyme-Linked Immunosorbent Assay
Fertilizers / microbiology*
Rabbits
Single-Chain Antibodies*
Soil Microbiology

Substances

Fertilizers
Single-Chain Antibodies

Grants and funding

This work was supported by Suranaree University of Technology, the SUT-PhD Scholarship Program for ASEAN, and by the Office of the Higher Education Commission under the NRU project of Thailand. It was also supported by the National Research Council of Thailand and by the Agricultural Research Development Agency (Public Organization), Thailand CRP5507010890. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.