Abstract
Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC50) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.
Highlights
Rice false smut, caused by the ascomycete fungus Villosiclava virens (Nakata) Tanaka & Tanaka [1], is one of the most destructive rice (Oryza sativa L.)fungal disease in many rice-growing areas worldwide over the past few years [2,3,4,5]
We developed a rapid, sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) based on a monoclonal antibody against ustiloxin B, and it was evaluated for the analysis of ustiloxin B in the rice samples, including rice false smut balls and rice grains
When the concentration of the coating antigen (UB-BSA) was 0.25 μg/mL and the antisera were diluted with 0.01 M pH 7.5 phosphate buffer containing 0.9% NaCl (w/w), 0.1% Tween-20 (v/v) and 0.5% gelatin (w/v) (PBSTG) at 1:2000, the best inhibition by ustiloxin B standard solution at 2000 ng/mL was 80% for antisera from seven mice, and the titer of antisera was higher than 8000
Summary
Rice false smut, caused by the ascomycete fungus Villosiclava virens (Nakata) Tanaka & Tanaka (anamorph: Ustilaginoidea virens Takahashi) [1], is one of the most destructive rice (Oryza sativa L.). The rice false smut was previously regarded as a minor disease, but in recent years, the disease has become significant due to the heavy application of nitrogenous fertilizers and widespread cultivation of hybrid cultivars without high-level resistance sources in the existing rice germplasm [7,8], resulting in yield loss, rice grains, feed contamination and, even more important, generating mycotoxin poisoning of humans and animals [9,10,11] Both ustiloxin A and the crude fraction obtained from the water extract of rice false smut balls caused liver and kidney damage in mice [12]. It is necessary to establish a simple, rapid, low-cost and sensitive method for routine screening for monitoring purposes Immunochemical determinations, such as enzyme-linked immunosorbent assays (ELISAs), applying antigen-antibody interaction showing a specific response against a particular substance, bring the detection of mycotoxins many advantages. The results of ustiloxin B content in the samples determined by both icELISA and HPLC methods were compared
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