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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.


Related in: MedlinePlus

HPLC analysis of ustiloxin B in rice false smut balls. (A) HPLC profile of the reference ustiloxin B; (B) HPLC profile of the water extract of rice false smut balls from Linyi, Shandong (0.52 mg/g); (C) HPLC profile of the water extract of rice grains from Donggang, Liaoning of China (<10 µg/g).
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toxins-07-03481-f003: HPLC analysis of ustiloxin B in rice false smut balls. (A) HPLC profile of the reference ustiloxin B; (B) HPLC profile of the water extract of rice false smut balls from Linyi, Shandong (0.52 mg/g); (C) HPLC profile of the water extract of rice grains from Donggang, Liaoning of China (<10 µg/g).

Mentions: For rice false smut ball samples, 0.1 g of powdered sample was extracted with ultrapure water three times (3 × 1.5 mL, 30 min each time) in an ultrasonic bath at room temperature, followed by centrifugation at 6710× g for 15 min. The supernatant extracts were combined and concentrated by a vacuum freeze dryer to dryness, and the residue was dissolved in 1 mL of ultrapure water in a test tube. The extracted solution was divided into two aliquots. One aliquot (100 µL) was diluted 2000-fold with PBSTG, followed by analysis with icELISA in triplicate. The other aliquot (900 µL) of concentrated solution was then filtered through a filter (pore size, 0.22 μm) and analyzed by HPLC. Figure 3 shows the HPLC analysis of ustiloxin B in rice false smut balls and rice grains. The conditions of HPLC were the same as described above (Section 3.5.3.). The retention time of ustiloxin B was 9.17 min. The HPLC calibration curves of ustiloxin B showed a good linearity, Y = 41,981.5058X − 19,568.6954, R2 = 1.0000, where Y is the peak area of analyte and X is the concentration (μg/mL) of analyte.


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)

HPLC analysis of ustiloxin B in rice false smut balls. (A) HPLC profile of the reference ustiloxin B; (B) HPLC profile of the water extract of rice false smut balls from Linyi, Shandong (0.52 mg/g); (C) HPLC profile of the water extract of rice grains from Donggang, Liaoning of China (<10 µg/g).
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-03481-f003: HPLC analysis of ustiloxin B in rice false smut balls. (A) HPLC profile of the reference ustiloxin B; (B) HPLC profile of the water extract of rice false smut balls from Linyi, Shandong (0.52 mg/g); (C) HPLC profile of the water extract of rice grains from Donggang, Liaoning of China (<10 µg/g).
Mentions: For rice false smut ball samples, 0.1 g of powdered sample was extracted with ultrapure water three times (3 × 1.5 mL, 30 min each time) in an ultrasonic bath at room temperature, followed by centrifugation at 6710× g for 15 min. The supernatant extracts were combined and concentrated by a vacuum freeze dryer to dryness, and the residue was dissolved in 1 mL of ultrapure water in a test tube. The extracted solution was divided into two aliquots. One aliquot (100 µL) was diluted 2000-fold with PBSTG, followed by analysis with icELISA in triplicate. The other aliquot (900 µL) of concentrated solution was then filtered through a filter (pore size, 0.22 μm) and analyzed by HPLC. Figure 3 shows the HPLC analysis of ustiloxin B in rice false smut balls and rice grains. The conditions of HPLC were the same as described above (Section 3.5.3.). The retention time of ustiloxin B was 9.17 min. The HPLC calibration curves of ustiloxin B showed a good linearity, Y = 41,981.5058X − 19,568.6954, R2 = 1.0000, where Y is the peak area of analyte and X is the concentration (μg/mL) of analyte.

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