脱氧雪腐镰刀菌烯醇ELISA定量检测试剂盒的研制

研制了脱氧雪腐镰刀菌烯醇ELISA试剂盒,用于检测谷物粮食中的脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)。在抗DON单克隆抗体的基础上,用间接竞争酶联免疫吸附检测试剂盒各项指标。结果为:试剂盒最低检测浓度为20ng/mL,检测线性范围为50～400ng/mL,IC50103ng/mL。平均加标回收率>80%,批内变异系数小于10%,批间变异系数<15%。用试剂盒测定盲样,其结果与德国拜发试剂盒及免疫亲和柱-高效液相色谱检测结果无显著性差异。说明试剂盒指标符合相关技术要求,具有快速、灵敏、准确、方便等特点。     

脱氧雪腐镰刀烯醇时间分辨免疫法的建立

目的：利用抗脱氧雪腐镰刀烯醇多克隆抗体及其人工抗原,采用时间分辨免疫荧光技术建立间接竞争脱氧雪腐镰刀烯醇时间分辨免疫检测方法(DON-TRFIA)。方法：以DON人工抗原(DON-BSA)包被微孔板,与DON标准或样品中的DON共同竞争结合抗DON多克隆抗体,然后用稀土离子Eu3+标记羊抗兔抗体进行示踪检测,并对建立DON-TRFIA进行方法学的考核。结果：该方法的灵敏度为0.01ng/ml,检测范围为0.01～100ng/ml,IC50为4.84ng/ml,批内、批间变异率均小于10%。不同样品添加回收实验表明小麦、玉米、啤酒、面包回收率分别为89.41%～123.2%、115.6%～123.6%、80.35%～118%、87%～92.45%。玉米样品检测结果表明DON-TRFIA与商品化DON-ELISA试剂盒结果高度相关,具有较好的一致性。结论：DON-TRFIA是目前已见报道文献中最灵敏的DON免疫检测法,具有很好的应用前景。     

超导体包被免疫荧光试纸法同时测定玉米中脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮适用性研究

为了能同时快速测定玉米中脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和玉米赤霉烯酮(Zearalenone,ZEN)两种毒素,采用基于超导体包被的免疫荧光快速定量检测技术,检测了多个玉米样品,结合加标回收率、稳定性、检出限、精密度等指标,并与液相色谱法的检测结果进行比较分析,评估方法的适用性。结果表明,超导体包被的免疫荧光试纸法检测DON含量在100～2 500μg/kg,检测ZEN含量在5～200μg/kg的范围内线性良好。DON在500～2 000μg/kg添加水平范围内,回收率为103.72%～108.17%,ZEN在50～150μg/kg添加水平范围内,回收率为97.81%～111.27%。该方法于液相色谱法检测结果对比,DON相对偏差在3.72%～8.17%,ZEN相对偏差在2.19%～11.27%,均低于液相色谱法允许偏差23%和15%,超导体包被的免疫荧光试纸法是一种有效、实用、快速、定量的分析方法,能满足同时检测玉米中DON和ZEN的要求。     

Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method is described for simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T2 and HT2-toxin in cereals. One-step extraction using solvent mixtures of acetonitrile:water:acetic acid (79:20:1) without any clean-up was employed for extraction of these mycotoxins from cereals. The mean recoveries of mycotoxins in spiked cereals ranged from 76.8% to 108.4%. Limits of detection (LOD) and quantification (LOQ) ranged 0.01–20 and 0.02–40 ng/g, respectively. The developed method has been applied for the determination of mycotoxins in 100 cereal samples collected from Malaysian markets. A total of 77 cereal samples (77%) contaminated with at least one of these mycotoxins. Occurrence of mycotoxins in commercial cereal samples were 70%, 40%, 25%, 36%, 19%, 13%, 16, and 16% for aflatoxins, OTA, ZEA, DON, FB₁, FB₂, T2 and HT2-toxin, respectively. The results demonstrated that the procedure was suitable for the determination of mycotoxins in cereals and could be implemented for the routine analysis.     

呕吐毒素时间分辨荧光免疫层析法的建立与评价

构建并评价了呕吐毒素(Deoxynivalenol,DON)时间分辨荧光免疫层析法(TRFIA)。以Eu3+螯合物的纳米微球为荧光探针标记抗DON单抗,采用竞争抑制建立免疫层析定量方法,优化了微球与抗体标记量,检测的环境温度、反应时间、加样体积及缓冲体系;研究了方法的灵敏度、精密度及准确度。结果表明,每100 μL荧光微球结合纯单抗50 μg,最佳划膜浓度为1.0 mg/mL,最佳测定条件为室温(25±2)℃,10 min,加样体积100 μL,PBS (0.01 mol/L,pH7.2)的缓冲体系,方法的灵敏度为0.25 ng/mL,线性范围为0.5~25.0 ng/mL,玉米、小麦阴性样本(加标浓度200、500、1000、2000 μg/kg)平均加标回收率为91.4%~109.3%,6种典型样本经TRFIA与免疫亲和净化-高效液相色谱法(IAC-HPLC)同时测定DON含量,相关系数r达0.9754。TRFIA具有灵敏度高、速度快、定量准等技术特点,适用于谷物及制品中DON的快速定量。     

A new solid phase extraction clean-up method for the determination of 12 type A and B trichothecenes in cereals and cereal-based food by LC-MS/MS

A new reliable and cost-efficient solid phase extraction-based clean-up method for the determination of 12 type A and B trichothecenes [deoxynivalenol (DON), nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenon-X, T-2 toxin, HT-2 toxin, neosolaniol, monoacetoxy-scirpenol, diacetoxyscirpenol, T-2 triol and T-2 tetraol] in cereals and cereal-based food is presented. Furthermore, the suitability for the simultaneous determination of zearalenone is examined. Toxins were extracted from cereal samples using ACN/water (80/20, v/v), purified by means of a new Bond Elut Mycotoxin column and analyzed via liquid chromatography-electrospray ionization tandem mass spectrometry. Limits of detection were calculated for the matrix wheat and ranged from 0.3 to 5 ng/g, depending on the toxin. Average recovery rates for the tested compounds in seven cereal-based matrices have been determined ranging from 65 to 104%. The relative standard deviations of the complete method ranged from 2.67 (DON, wheat) to 20.0% (T-2 toxin, oats).     

Optical waveguide lightmode spectroscopy technique–based immunosensor development for deoxynivalenol determination in wheat samples

Contamination by deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium species, occurs in cereals worldwide; therefore, efforts have been made toward the development of rapid and sensitive methods for the detection of this compound. In our investigation, optical waveguide lightmode spectroscopy (OWLS) technique has been applied to label-free detection of DON in both competitive and in direct immunoassay formats using DON-specific polyclonal antibodies. After immobilizing the antibody or the antigen conjugate for the direct or indirect measurement, the sensor chip was used in a flow-injection analyzer system. The direct method was found to result in an unstable sensor response and sensitivity insufficient to determine DON in different grains. In contrast, a competitive immunosensor format provided reproducible quantitative detection in the sub-ppt range. For competitive sensor investigation with the sensitized chip, first the optimal dilution rate of polyclonal antibodies was determined. For the measurements, antibody stock solution was diluted to 8 μg mL⁻¹. During the competitive measurement, standard solutions were mixed with the antibodies at the appropriate concentration, and the mixture was incubated for 1 min and injected into the OWLS system. The sensitive detection range of the competitive detection method was between 0.01 and 50 ng mL⁻¹. After the establishment of the indirect method, spiked wheat flour samples were investigated. Results obtained with spiked samples showed that OWLS detection has a potential for quick determination of DON in wheat samples.     

QuEChERS-HPLC-MS/MS法检测谷类杂粮制品中4种真菌毒素

建立了一种快速、高效的QuEChERS-高效液相色谱-质谱联用法（QuEChERS-HPLC-MS/MS）测定谷类杂粮制品中脱氧雪腐镰刀菌烯醇（DON）、3-乙酰脱氧雪腐镰刀菌烯醇（3-ADON）、15-乙酰脱氧雪腐镰刀菌烯醇（15-ADON）和玉米赤霉烯酮（ZON）共4种真菌毒素。样品前处理采用乙腈-水溶剂提取,经Florisil+C18+无水硫酸镁净化后检测。以0.10%甲酸-乙腈作为流动相,在质谱检测器的多反应监测模式下进行分析。结果表明,4种真菌毒素在各自的线性范围内线性关系良好,相关系数R2均大于0.999,回收率在85.1%～102.0%,相对标准偏差（RSD）为2.11%～6.22%。该方法具有前处理简单、净化效果好、灵敏度高和检测速度快的优点,适用于谷类杂粮制品中DON、3-ADON、15-ADON 和ZON的分析和定量检测。     

Development of a direct competitive enzyme-linked immunoassay for carbofuran in vegetables

Three ELISA formats, antigen coated, antibody coated and the second antibody coated for the determination of carbofuran were investigated with conjugations including hapten–BSA, hapten–OVA, hapten–HRP and anticarbofuran IgG–HRP. Results showed that the second antibody-coated method of ELISA had a better performance in the establishment of standard curves and detection of carbofuran residue in vegetables samples. The sensitivity for detection, the I₅₀ value was 36.1 ng/ml at a practical working concentration range from 3.44 to 380.1 ng/ml and the limit of detection for carbofuran was 3.44 ng/ml. The average recoveries of determination for carbofuran spiked in cabbage, lettuce, carrot, winter fragrant-flowered garlic, bamboo shoot and green soy bean were 85.24%, 101.8%, 103.6%, 90.52%, 106.9% and 94.08%, respectively. Additional analyses confirmed that the results given by the ELISA method was in agreement with those of the gas chromatography (GC) method.     

脱氧雪腐镰刀菌烯醇酶联免疫检测方法的建立

应用抗脱氧雪腐镰刀菌烯醇单克隆抗体12D1建立了竞争间接ELISA方法,用于检测小麦、玉米中的脱氧雪腐镰刀菌烯醇(DON),最低检出限为20ng/mL,检测范围为20￣460ng/mL。用蒸馏水提取掺合DON(250￣4000ng/g)的小麦样品,回收率为82.1%￣96.6%,变异系数为0.6%￣7.1%。     

Detection of <Emphasis Type="Italic">Clostridium botulinum</Emphasis> neurotoxins A and B in milk by ELISA and immuno-PCR at higher sensitivity than mouse bio-assay

Using commercially available antibodies and toxoids as templates, an ELISA, immuno-quantitative PCR (iqPCR), and multiplex immuno-PCR (iPCR) were developed for detection of Clostridium botulinum neurotoxins A and B. The obtained sensitivities for ELISA ranged from 1 ng/ml in PBS + 1% BSA to 15 and 10 ng/ml in skimmed milk for botulinum neurotoxins (BoNT)/A and BoNT/B, respectively. In semi-fat milk, the limit of detection (LOD) for both toxoids was 30 ng/ml. Quantitative immuno-PCR (iqPCR) had an LOD of 4.5–9 pg/reaction for BoNT/A in both PBS and semi-fat milk, while this was 18.5–37 pg/reaction for BoNT/B in PBS + 1% BSA and semi-fat milk, respectively. The sensitivities of ELISA and iqPCR were improved to 0.5 ng/ml and 3.75 pg/ml (0.2 pg/reaction) in semi-fat milk, respectively, when toxoid of BoNT/A was substituted with actual toxin. Multiplex iPCR with both toxoids run in the same reaction was able to distinguish presence/absence of tested BoNT/A and BoNT/B at 25 pg/reaction. The tested system offers a realistic alternative with much better sensitivity to the standard mouse assay.     

Analysis and occurrence of deoxynivalenol (DON) in cocoa

The primary objective of this study was to establish a current situation assessment of the possible occurrence of deoxynivalenol in cocoa and cocoa products. Since there was no analytic method for determining DON in cocoa and cocoa products, a special method was developed. The applicability and consistency of the method was confirmed by performing recovery assays on various cocoa products. A special post-column derivatisation procedure was used to increase selectivity and raise sensitivity by a factor of 80. The method’s limit of detection (LOD) was thereby reduced to 7 μg/kg; the limit of quantification (LOQ) was 14 μg/kg. The method was used to test 230 samples for possible DON content, ranging from cocoa beans to cocoa bean shells, nibs, cocoa liquor and cocoa powders through to finished cocoa-based products. In the case of cocoa beans and cocoa bean shells, DON content close to the detection limit was only determined in isolated cases. No DON content was detected in nibs, cocoa liquor, cocoa powders and finished cocoa-based products. Analogous to ochratoxin A and aflatoxins, the results show DON is more likely to be found in cocoa bean shells. Separation of shells during cocoa processing can reduce potential DON contents. Since no DON was determined in the fractions relevant for chocolate production, these assays show it does not represent a considerable issue for the cocoa and chocolate industry.     

Development of a homogeneous immunoassay based on the AlphaLISA method for the detection of chloramphenicol in milk, honey and eggs

BACKGROUND: A homogenous light‐induced chemiluminescence immunoassay was developed using AlphaLISA technology for the detection of chloramphenicol (CAP). This technology is based on two different kinds of bead, namely light‐sensitive donor beads and beads containing chemiluminescers, also called acceptor beads. A competitive CAP AlphaLISA method was established using artificial antigen‐coated acceptor beads, polyclonal antibodies, biotinylated goat anti‐rabbit IgG and streptavidin‐coated donor beads. RESULTS: The sensitivity of detection was 0.0086 ng mL^?1 and the working range was from 0.0096 to 25 ng mL^?1. The intra‐ and inter‐assay coefficients of variation were both below 10%. The average recovery rates at spiked levels of 0.05–10 ng mL^?1 were 103.2, 108.4 and 91.6% for milk, honey and eggs respectively. The data obtained from the samples showed good correlation with ELISA results. CONCLUSION: The CAP AlphaLISA method is highly sensitive, specific and rapid and is suitable for screening large quantities of samples. Copyright © 2012 Society of Chemical Industry     

Simultaneous detection of 12 mycotoxins in cereals using RP-HPLC-PDA-FLD with PHRED and a post-column derivatization system

A new method for the simultaneous quantification of 12 mycotoxins was developed and optimized using reverse phase high performance liquid chromatography (RP-HPLC) with a photodiode array (PDA) and fluorescence detector (FLD), a photochemical reactor for enhanced detection (PHRED) and post-column derivatization. The mycotoxins included aflatoxins (AFB(1), AFB(2), AFG(1), and AFG(2)), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB(1), FB(2), and FB(3)), T-2 and HT-2 toxins. A double sample extraction with a phosphate-buffered saline solution (PBS) and methanol was used for co-extraction of mycotoxins, and a multifunctional immunoaffinity column was used for cleanup. Optimum conditions for separation of the mycotoxins were obtained to separate 12 mycotoxins in FLD and PDA chromatograms with a high resolution. The method gave recoveries in the range 72-111% when applied to spiked corn samples. The limits of detection (LOD) were 0.025 ng/g for AFB(1) and AFG(1), 0.012 ng/g for AFB(2) and AFG(2), 0.2 ng/g for OTA, 1.5 ng/g for ZEA, 6.2 ng/g for FB(1), FB(3) and HT-2 toxin, 9.4 ng/g for FB(2) and T-2 toxin, and 18.7 ng/g for DON. In addition, the limits of quantification (LOQ) ranged from 0.04 ng/g for AFB(2) and AFG(2) to 62 ng/g for DON. The method was successfully applied to the determination of these mycotoxins in 45 cereal samples obtained from the Malaysian market. The results indicated that the method can be applied for the multi-mycotoxin determination of cereals.     

Stability of the mycotoxin deoxynivalenol (DON) during the production of flour-based foods and wheat flake cereal

Deoxynivalenol (DON) is a mycotoxin found in cereal grains and cereal-based foods. DON concentrations in finished products are reduced under some processing conditions, but not others. DON concentrations in flour, wheat and selected foods made from them under commercially relevant conditions were compared by GC with electron capture detection. Average concentrations (n = 9/item) in cookies, crackers and pretzels ranged from 61% (cookies) to 111% (pretzels) compared with flour (100% = 0.46 μg g?1). Lesser amounts were found in donuts and bread: their respective DON concentrations were 44% and 30% that of flour. Mass balance estimates for DON (μg g?1 flour equivalents) ranged from 50% (bread = 0.23 μg g?1 flour equivalents) to 120% (donuts), indicating that dilution by recipe ingredients contributed to DON reductions in bread and accounted for all of the apparent reduction in donuts. Mass balance estimates averaged 76% (crackers) to 107% (pretzels) for the other flour products. DON concentrations were higher in cereal flakes (0.55 μg g?1 in the finished product and 0.58 μmg g?1 on a mass balance basis) than in wheat (0.40 μg g?1), suggesting that DON concentrations might increase during processing of wheat cereals under some conditions. In summary, DON concentrations of finished food products were reduced ≥ 50% only in bread and donuts. Reduction in bread resulted from a combination of DON ‘loss’ and dilution by recipe ingredients whereas the reduction in donuts was due entirely to dilution. These results are further evidence of DON stability during the preparation of popular flour or wheat-based products.     

Stability of the mycotoxin deoxynivalenol (DON) during the production of flour-based foods and wheat flake cereal

Deoxynivalenol (DON) is a mycotoxin found in cereal grains and cereal-based foods. DON concentrations in finished products are reduced under some processing conditions, but not others. DON concentrations in flour, wheat and selected foods made from them under commercially relevant conditions were compared by GC with electron capture detection. Average concentrations (n?=?9/item) in cookies, crackers and pretzels ranged from 61% (cookies) to 111% (pretzels) compared with flour (100%?=?0.46?µg?g^?1). Lesser amounts were found in donuts and bread: their respective DON concentrations were 44% and 30% that of flour. Mass balance estimates for DON (µg?g^?1 flour equivalents) ranged from 50% (bread?=?0.23?µg?g^?1 flour equivalents) to 120% (donuts), indicating that dilution by recipe ingredients contributed to DON reductions in bread and accounted for all of the apparent reduction in donuts. Mass balance estimates averaged 76% (crackers) to 107% (pretzels) for the other flour products. DON concentrations were higher in cereal flakes (0.55?µg?g^?1 in the finished product and 0.58?µg?g^?1 on a mass balance basis) than in wheat (0.40?µg?g^?1), suggesting that DON concentrations might increase during processing of wheat cereals under some conditions. In summary, DON concentrations of finished food products were reduced?≥50% only in bread and donuts. Reduction in bread resulted from a combination of DON ‘loss’ and dilution by recipe ingredients whereas the reduction in donuts was due entirely to dilution. These results are further evidence of DON stability during the preparation of popular flour or wheat-based products.     

Occurrence of deoxynivalenol (vomitoxin) in processed cereals and pulses in Turkey

The aim was to investigate the occurrence of deoxynivalenol (DON) in cereal and pulse products in Turkey. DON was detected using high-performance liquid chromatography (HPLC) with ultraviolet detection at 220 nm and positive results greater or equal to 0.60 ppm were confirmed by thin layer chromatography (TLC). An acetonitrile-water (21:4 v/v) extract of the sample was cleaned up on a column packed with alumina-Celite-charcoal (0.35 + 0.25 + 0.40 g). The detection limits for DON were 3 ng/injection (0.10 ppm) and 50 ng/spot (0.60 ppm) for HPLC and TLC, respectively. Eighty-three commercially available cereal and pulse product samples collected from markets and street bazaars were analysed. The recovery rates for boiled, pounded wheat and rice spiked with added DON (1 ppm) were 80.9% (SD 8.37, n =5) and 72.3% (3.85, n =5), respectively. DON was detected in six (8.82%) of 68 cereal and in none of 15 pulse products. The maximum detected amount was 2.67 ppm in a corn flour sample.     

脱氧雪腐镰刀菌烯醇(DON)直接竞争ELISA方法的建立

利用前期制备得到的DON酶标抗原(辣根过氧化物酶标记)以及抗DON抗体,建立检测食品中DON含量的直接竞争ELISA方法。该方法的检测范围1～100ng/mL,灵敏度达0.56ng/mL,半数抑制浓度IC50为10ng/mL;与DON类似物T-2毒素的交叉反应率为12%;玉米淀粉样品回收率在80.2%～91.1%之间,平均批间变异<15%,平均批内变异<3%。     

Determination of Zearalanol and Its Analog Zearalanone in Muscle Tissues by Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA)

A homogenous light-induced chemiluminescence immunoassay (AlphaLISA) method was established for the determination of residues of zearalanol and its analog zearalanone in muscle tissue samples. AlphaLISA is a bead-based proximity assay. When donor and acceptor beads proximity, a cascade of chemical reactions begin. The end result is a greatly amplified signal that contributes to the detection sensitivity down to the attomole level. Compared with other methods, the AlphaLISA has characteristics of homogeneity, being free of cleaning, high sensitivity. The method showed a linear relationship in the range of 0.01–4 ng/mL (R ²?>?0.99); the sensitivity of the assay was 0.066 ng/mL. Cross-reactivity rate of zearalanol was 100 % and zearalanone was 82.1 %, and other compounds were not more than 40 %. The average recovery rates of Zearalanol at spiked levels of 1–4 ng/mL were 96.3 to 105.0 and 91.7 to 100.5 % for pork and bovine muscles; the intra-day precision ranged from 2.6 to 9.8 %, and the inter-day precision ranged from 8.7 to 17.5 %. These results indicated that the proposed method was successfully applied in the analysis of zearalanol and its analog zearalanone in muscle tissues.     

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography-tandem mass spectrometry

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS), allowed unambiguous identification of the selected trichothecenes at low µg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis® HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep® #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5-4.0 µg kg^-1 for NIV, 2.8-5.3 µg kg^-1 for DON, 0.4-1.7 µg kg^-1 for HT-2 and 0.4-1.0 µg kg^-1 for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 µg kg^-1, ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.