Novel and Highly Specific Monoclonal Antibody to Acidovorax citrulli and Development of ELISA-Based Detection in Cucurbit Leaves and Seed

Orawan Himananto; Petcharat Thummabenjapone; Plearnpis Luxananil; Mallika Kumpoosiri; Ratchanee Hongprayoon; Wichai Kositratana; Oraprapai Gajanandana

doi:10.1094/PDIS-12-10-0889

Novel and Highly Specific Monoclonal Antibody to Acidovorax citrulli and Development of ELISA-Based Detection in Cucurbit Leaves and Seed

Plant Dis. 2011 Sep;95(9):1172-1178. doi: 10.1094/PDIS-12-10-0889.

Authors

Orawan Himananto¹, Petcharat Thummabenjapone², Plearnpis Luxananil³, Mallika Kumpoosiri³, Ratchanee Hongprayoon⁴, Wichai Kositratana⁴, Oraprapai Gajanandana³

Affiliations

¹ Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand; Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand; and National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand.
² Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE); and Agricultural Biotechnology Research Center for Sustainable Economy, Khon Kaen University, Khon Kaen 40002, Thailand.
³ National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand.
⁴ Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand; Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand; and Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University.

PMID: 30732068
DOI: 10.1094/PDIS-12-10-0889

Abstract

A novel monoclonal antibody (MAb) specific to the seedborne bacterium Acidovorax citrulli was produced. MAb 11E5 reacted specifically with 19 strains of A. citrulli but not with three closely related bacteria in the family Comamonadaceae (i.e., A. facilis, Comamonas acidovorans, and C. testosteroni) and another seven phytopathogenic bacteria. Moreover, this MAb detected a strain of A. citrulli that was not detected by a commercial enzyme-linked immunosorbent assay (ELISA)-based kit and a commercial immunochromatographic strip test. In Western blot analysis, MAb 11E5 reacted with an A. citrulli protein of a molecular mass >170 kDa. MAb 11E5 was employed to develop two sandwich ELISA systems: MAb captured-sandwich ELISA (MC-sELISA) and polyclonal antibody captured-sandwich ELISA (PC-sELISA). MC-sELISA was 10 times more sensitive than PC-sELISA for detection of A. citrulli in cucurbit leaf and seed extracts. The detection limit of the MC-sELISA was 5 × 10⁴ CFU/ml. Detection of A. citrulli in naturally infected cucurbit leaves, fruit, and seed was also feasible using MC-sELISA. The newly established MCsELISA provides another alternative for specific detection of A. citrulli in cucurbits and can be applied for routine field inspection.