Abstract
Ustiloxins are a group of mycotoxins produced by rice false smut pathogen. Previous studies have shown that the false smut balls contain six types of ustiloxins, and these toxins are toxic to living organisms. Thus, immunoassay for on-site monitoring of ustiloxins in rice is urgently required. The current immunoassays are only for detecting single ustiloxin, and they cannot meet the demand for synchronous and rapid detection of the group toxins. Therefore, this study designed and synthesized a generic antigen with ustiloxin G as material based on the common structure of the mycotoxins. Ustiloxin G was conjugated to two carrier proteins including bovine serum albumin (BSA) and ovalbvmin (OVA) by carbon diimide method. The mice were immunized with ustiloxin-G-BSA to generate the antibody serum, which was further purified to obtain the generic antibody against ustiloxins. The conjugated ustiloxin G-OVA and generic antibodies were used for establishing the enzyme-linked immunosorbent assay (ELISA) for ustiloxin detection and optimizing experiment conditions. The characterization of the antibody showed that the semi-inhibitory concentrations (IC50) of ustiloxin A, B, and G were 0.53, 0.34, and 0.06 µg/mL, respectively, and that their corresponding cross-reactivities were 11.9%, 18.4%, and 100%, respectively. To increase ELISA detection efficiency, generic antibody was combined with magnetic beads to obtain sensitive and class-specific immune-magnetic beads. Based on these immuno-magnetic beads, a high-efficiency enzyme-linked immunoassay method was developed for ustiloxin detection, whose sensitivity to ustiloxin A, B, and G was improved to 0.15 µg/mL, 0.14 µg/mL, and 0.04 µg/mL, respectively. The method accuracy was evaluated by spiking ustiloxin G as standard, and the spiked samples were tested by the immune-magnetic bead-based ELISA. The result showed the ustiloxin G recoveries ranged from 101.9% to 116.4% and were accepted by a standard HPLC method, indicating that our developed method would be promising for on-site monitoring of ustiloxins in rice.
Highlights
Key Contribution: We provided a new, sensitive and generic immunoassay which can be used for on-site monitoring ustiloxins in rice
The result revealed that the generic antigen was suitable for the preparation of generic antibodies against ustiloxins
With the generic antigen and antibody, a sensitive and class-specific immuno-magnetic bead-based enzymelinked immunoassay was developed for ustiloxin detection, and its sensitivity to ustiloxin
Summary
Rice false smut (RFS) has become an emerging and economically important fungal disease in most rice-producing countries recently [1,2,3,4,5,6]. Since the 1970s, the harm of RFS has been widely reported in the major planting areas in China. After the 1980s, with the change of farming methods and the large-scale promotion of hybrid rice varieties, RFS has exhibited an increasing trend year by year, and currently, it harms nearly one third of the rice cultivation area [7,8,9]. Of all the known toxins produced by RFS pathogen, ustiloxins are the most widely concerned
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