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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

Inhibition curves of ustiloxin A/B in icELISA format (each value represents the mean of triplicate ± standard deviations. B0 and B are the absorbance values at 492 nm in the absence and presence of ustiloxin A/B, respectively). (A) Inhibition curve of ustiloxin A/B in icELISA format based on 2D3G5. The cross-reactivity with ustiloxin B was 4.1% for mAb 2D3G5, as previously reported [23]. (B) Inhibition curve of ustiloxin A/B in icELISA format based on mAb 1B5A10. The cross-reactivity with ustiloxin A was 13.9% for mAb IB5A10.
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toxins-07-03481-f004: Inhibition curves of ustiloxin A/B in icELISA format (each value represents the mean of triplicate ± standard deviations. B0 and B are the absorbance values at 492 nm in the absence and presence of ustiloxin A/B, respectively). (A) Inhibition curve of ustiloxin A/B in icELISA format based on 2D3G5. The cross-reactivity with ustiloxin B was 4.1% for mAb 2D3G5, as previously reported [23]. (B) Inhibition curve of ustiloxin A/B in icELISA format based on mAb 1B5A10. The cross-reactivity with ustiloxin A was 13.9% for mAb IB5A10.

Mentions: Both ustiloxins A and B are the predominant ustiloxins in rice false smut balls and rice grains [9,18]. As ustiloxins A and B are available at present, the specificity of mAb 1B5A10 against ustiloxins A and B was evaluated. The structure of ustiloxin B is the most similar to ustiloxin A among the five known ustiloxins. There is a minor difference with two methyl groups at the C-24 position between ustiloxins A and B (Figure 1). In the preparation of hapten-protein conjugates, ustiloxin B was conjugated with carrier proteins via –NH2 at the C-5ʹ position with the glutaraldehyde method. In general, there is some correlation between the position conjugated to the carrier protein and the recognition of epitopes on the hapten by the prepared antibodies. The epitopes distant from the site of conjugation tend to be well recognized by antibodies, whereas epitopes neighboring the coupling site tend to be less well recognized. Although a structural difference between ustiloxins A (HR-ESI-MS, m/z 674.26859 [M + H]+) and B (HR-ESI-MS, m/z 646.23751 [M + H]+) exists on the opposite side of the conjugation site, the high molecular weight of the cyclopeptide ustiloxins might affect the specificity of mAb 1B5A10, resulting in worse recognition [31,32]. The IC50 values of ustiloxins A and B were 122.6 and 17.1 ng/mL, respectively. There was still 13.9% cross-reactivity with ustiloxin A relative to ustiloxin B (Figure A1). Ustiloxins C, D and F are structurally very different from ustiloxins A and B (Figure 1) and are less abundant than ustiloxins A and B. Therefore, ustiloxins C, D and F presumably have minor cross-reactivities and may not interfere with the developed icELISA, which, however, needs to be verified.


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)

Inhibition curves of ustiloxin A/B in icELISA format (each value represents the mean of triplicate ± standard deviations. B0 and B are the absorbance values at 492 nm in the absence and presence of ustiloxin A/B, respectively). (A) Inhibition curve of ustiloxin A/B in icELISA format based on 2D3G5. The cross-reactivity with ustiloxin B was 4.1% for mAb 2D3G5, as previously reported [23]. (B) Inhibition curve of ustiloxin A/B in icELISA format based on mAb 1B5A10. The cross-reactivity with ustiloxin A was 13.9% for mAb IB5A10.
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toxins-07-03481-f004: Inhibition curves of ustiloxin A/B in icELISA format (each value represents the mean of triplicate ± standard deviations. B0 and B are the absorbance values at 492 nm in the absence and presence of ustiloxin A/B, respectively). (A) Inhibition curve of ustiloxin A/B in icELISA format based on 2D3G5. The cross-reactivity with ustiloxin B was 4.1% for mAb 2D3G5, as previously reported [23]. (B) Inhibition curve of ustiloxin A/B in icELISA format based on mAb 1B5A10. The cross-reactivity with ustiloxin A was 13.9% for mAb IB5A10.
Mentions: Both ustiloxins A and B are the predominant ustiloxins in rice false smut balls and rice grains [9,18]. As ustiloxins A and B are available at present, the specificity of mAb 1B5A10 against ustiloxins A and B was evaluated. The structure of ustiloxin B is the most similar to ustiloxin A among the five known ustiloxins. There is a minor difference with two methyl groups at the C-24 position between ustiloxins A and B (Figure 1). In the preparation of hapten-protein conjugates, ustiloxin B was conjugated with carrier proteins via –NH2 at the C-5ʹ position with the glutaraldehyde method. In general, there is some correlation between the position conjugated to the carrier protein and the recognition of epitopes on the hapten by the prepared antibodies. The epitopes distant from the site of conjugation tend to be well recognized by antibodies, whereas epitopes neighboring the coupling site tend to be less well recognized. Although a structural difference between ustiloxins A (HR-ESI-MS, m/z 674.26859 [M + H]+) and B (HR-ESI-MS, m/z 646.23751 [M + H]+) exists on the opposite side of the conjugation site, the high molecular weight of the cyclopeptide ustiloxins might affect the specificity of mAb 1B5A10, resulting in worse recognition [31,32]. The IC50 values of ustiloxins A and B were 122.6 and 17.1 ng/mL, respectively. There was still 13.9% cross-reactivity with ustiloxin A relative to ustiloxin B (Figure A1). Ustiloxins C, D and F are structurally very different from ustiloxins A and B (Figure 1) and are less abundant than ustiloxins A and B. Therefore, ustiloxins C, D and F presumably have minor cross-reactivities and may not interfere with the developed icELISA, which, however, needs to be verified.

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