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Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains.

Fu X, Wang A, Wang X, Lin F, He L, Lai D, Liu Y, Li QX, Zhou L, Wang B - Toxins (Basel) (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China. xiaoxiaofu@cau.edu.cn.

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.

No MeSH data available.


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Chemical structures of ustiloxins.
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toxins-07-03481-f001: Chemical structures of ustiloxins.

Mentions: 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.) fungal disease in many rice-growing areas worldwide over the past few years [2,3,4,5]. This fungus infects the rice filament and transforms individual grains of the panicle into greenish balls (namely rice false smut balls) covered by powdery dark-green chlamydospores [4,6]. 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]. The cytotoxic activity of the ustiloxins has been approved to be antimitotic by inhibition of the microtubule assembly and cell skeleton formation [13]. Two kinds of mycotoxins, namely ustiloxins and ustilaginoidins, have been isolated and identified from rice false smut balls and false smut pathogen [10,14,15]. The ustiloxin family, consisting of ustiloxins A, B, C, D and F (Figure 1), belongs to the cyclopeptides containing a 13-membered cyclic core structure with a phenol ether linkage, and ustiloxin A is the most toxic and predominant among them, followed by ustiloxin B [9,16,17,18]. It has been reported that ustiloxins had antimitotic activity by inhibiting microtubule assembly and cell skeleton formation of plant and animal cells [13,19,20]. The crude water extract of rice false smut balls was found to cause necrosis of the liver and kidney in mice quite similar to that observed in lupinosis caused by phomopsin A, a mycotoxin produced by Phomopsis leptostromiformis [12,21]. Meanwhile, ustiloxins functioned as the phytotoxins by inhibiting the radicle and plumule growth during seed germination of rice, wheat and maize, inducing an abnormal swelling of the seeding roots and resulting in the growth reduction, necrotic and dead frond tissue to duckweed (Lemna pausicostata) [5,9,16]. The content of ustiloxin A in rice grains from rice false smut disease regions ranged from 0.0073 to 4.33 µg/g [22]; while ustiloxins A and B content was much higher in rice false smut balls, ranging from 0.13 to 1.08 mg/g [18,23]. Therefore, both rice false smut balls and false smut pathogen-contaminated rice food and forage have created concerns for food and feed safety [10].


Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains.

Fu X, Wang A, Wang X, Lin F, He L, Lai D, Liu Y, Li QX, Zhou L, Wang B - Toxins (Basel) (2015)

Chemical structures of ustiloxins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591656&req=5

toxins-07-03481-f001: Chemical structures of ustiloxins.
Mentions: 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.) fungal disease in many rice-growing areas worldwide over the past few years [2,3,4,5]. This fungus infects the rice filament and transforms individual grains of the panicle into greenish balls (namely rice false smut balls) covered by powdery dark-green chlamydospores [4,6]. 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]. The cytotoxic activity of the ustiloxins has been approved to be antimitotic by inhibition of the microtubule assembly and cell skeleton formation [13]. Two kinds of mycotoxins, namely ustiloxins and ustilaginoidins, have been isolated and identified from rice false smut balls and false smut pathogen [10,14,15]. The ustiloxin family, consisting of ustiloxins A, B, C, D and F (Figure 1), belongs to the cyclopeptides containing a 13-membered cyclic core structure with a phenol ether linkage, and ustiloxin A is the most toxic and predominant among them, followed by ustiloxin B [9,16,17,18]. It has been reported that ustiloxins had antimitotic activity by inhibiting microtubule assembly and cell skeleton formation of plant and animal cells [13,19,20]. The crude water extract of rice false smut balls was found to cause necrosis of the liver and kidney in mice quite similar to that observed in lupinosis caused by phomopsin A, a mycotoxin produced by Phomopsis leptostromiformis [12,21]. Meanwhile, ustiloxins functioned as the phytotoxins by inhibiting the radicle and plumule growth during seed germination of rice, wheat and maize, inducing an abnormal swelling of the seeding roots and resulting in the growth reduction, necrotic and dead frond tissue to duckweed (Lemna pausicostata) [5,9,16]. The content of ustiloxin A in rice grains from rice false smut disease regions ranged from 0.0073 to 4.33 µg/g [22]; while ustiloxins A and B content was much higher in rice false smut balls, ranging from 0.13 to 1.08 mg/g [18,23]. Therefore, both rice false smut balls and false smut pathogen-contaminated rice food and forage have created concerns for food and feed safety [10].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China. xiaoxiaofu@cau.edu.cn.

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.

No MeSH data available.


Related in: MedlinePlus