基于CdSe阳离子交换的玉米赤霉烯酮新型荧光免疫检测方法的建立及应用

作为镰刀属真菌的次级代谢产物,玉米赤霉烯酮(zearalenone,ZEN)具有强烈的生殖毒性和免疫毒性,严重威胁动物和人类健康。本研究通过采用羧基修饰的CdSe水溶性量子点(quantum dots,QDs)标记ZEN单克隆抗体,并基于CdSe阳离子交换信号增强原理,建立了ZEN新型荧光免疫检测方法(CdSe QDs-FLISA),检测下限(IC₁₀)和半数抑制率(IC₅₀)分别为0.006 ng/mL和0.17 ng/mL,检测区间(IC₂₀-IC₈₀)为0.01-0.45 ng/mL。与ZEN的结构类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol and β-zearalanol)交叉反应性依次为22.3%、13.1%、6.2%、1.6%和3.9%,与农产品中其他真菌毒素如黄曲霉毒素B₁ (AFB₁)、赭曲霉毒素A (OTA)、呕吐毒素(DON)和伏马毒素B₁ (FB₁)几乎不存在交叉反应。该方法在玉米样本中加标回收率较高,且实际样本中ZEN的定量检测结果与LC-MS/MS一致性较好。本研究建立的CdSe QDs-FLISA操作简单,可实现对样本中ZEN的快速定量检测与分析,便于基层单位推广使用,具有较好的应用前景,同时也可为其他病原微生物检测技术的开发提供参考。     

C肽与胰岛素双标记时间分辨荧光免疫分析法的建立及初步临床应用

建立钐(Sm3+)标记检测C肽(C-peptide)及铕(Eu3+)标记检测胰岛素(insulin)的双标记时间分辨荧光免疫分析法(TRFIA),并初步尝试检测人血C肽以及胰岛素含量.将抗C肽单克隆抗体(Biodesign No.E54094M)与抗insulin单克隆抗体(BiodesignNo.E86306M)混合包被96孔板,然后用Sm3+标记抗C肽单克隆抗体(Medix No.9103),Eu3+标记抗insulin单克隆抗体(Biodesign No.E86802M),运用双抗体夹心一步法建立C肽/胰岛素双标记时间分辨免疫荧光分析法.结果显示:C肽分析灵敏度为0.2μg/L,线性范围为0.5～22μg/L,平均回收率达到99.6%,分析内和分析间变异系数分别为4.6%～6.0%和5.1%～7.6%.胰岛素分析灵敏度为0.8 mU/L,线性范围为3.6～180 mU/L,平均回收率为99.4%,分析内和分析间变异系数分别为3.7%～6.0%和5.1%～8.0%.C肽/胰岛素双标记检测试剂与PerkinElmer公司对应的单标记进口试剂盒分别同时测定血清样本200份,检测结果高度相关,具有较好的一致性,相关系数分别为0.98与0.99.总之,自建C肽/胰岛素双标记时间分辨荧光免疫分析方法的性能均可以达到临床检测要求,有望替代现有国内外较为昂贵的单标记试剂,可用于胰岛素分泌不足导致的糖尿病诊断及糖尿病的大规模普查筛选.     

胃蛋白酶原双标记时间分辨荧光免疫分析及其初步临床应用

采用钐(Sm3 )标记抗胃蛋白酶原Ⅰ单克隆抗体(PG Ⅰ)及铕(Eu3 )标记抗胃蛋白酶原Ⅱ单克隆抗体(PGⅡ),建立了双标记时间分辨荧光免疫分析法(TRFIA),同时检测人血清PGⅠ和PGⅡ.将抗PG Ⅰ单克隆抗体8003#、抗PGⅡ单克隆抗体8101#以一定比例共包被于96孔微孔板上,用Sm3 标记抗PGⅠ单抗8016#、Eu3 标记抗PGⅡ单抗8102#,采用双抗体夹心法建立PGⅠ/PGⅡ的双标记TRFLA.标准曲线由TRFLA检测仪自带的Log-LogB函数处理.PGI可测范围为0.2～300μg/L,批内和批间变异系数(CV%)分别为5.2%和8.1%,平均回收率为96.9%;PGⅡ可测范围为0.05～55μg/L,批内和批间变异系数(CV%)分别为7.1%和11.7%,平均回收率为103.7%;双标记PGⅠ/PGⅡ-TRFLA与PGⅠ-ELISA法、PGⅡ-ELISA法的测定结果高度相关,具有较好的一致性,相关系数分别为0.9426和0.9396.检测300例健康人员的PGⅠ为(157.3±51.0)μg/L,PGⅡ为(10.6±5.9)μg/L,PGⅠ/pGⅡ比值为(14.8±4.3)μg/L.PGⅠ的正常参考值范围在55.3～259.3μg/L之间,PGⅡ正常参考值为<23μg/L,PGⅠPGⅡ>6.双标记PGI/PGⅡ-TRFLA方法的研究在国内外尚未见报道,是一种灵敏、简便、快速、经济并可用于大批量样品筛查的方法,有利于各种胃病的大规模普查筛选及患者病程监测.     

玉米赤霉烯酮单克隆抗体酶联免疫测定方法的建立及初步应用

用ZEN—BSA人工抗原免疫BALB／c鼠,经融合、筛选和克隆化得到可稳定分泌抗ZEN单克隆抗体的杂交瘤细胞株ZEN—lC6。zEN一lC6属IgG₁,纯化腹水抗体效价为10^-5,与5种衍生物的交叉反应系数为0.16～1.20％。用ZEN一1C6建立了检测食品(玉米、小麦、大米)中玉米赤霉烯酮的CIEIA法。该法检测纯毒素的线性范围为5～1000ng／ml,最低检出浓度为0．1ng／ml,平均回收率为84．0～105．5％。用该法测定了81份样品,均有ZEN毒素检出。     

阿特拉津高灵敏酶联免疫吸附检测法的建立

目的：建立高灵敏度的阿特拉津酶联免疫吸附检测法。方法：将间接竞争ELISA进行条件优化以提高检测灵敏度,包括包被抗原与一抗的最佳工作浓度筛选、选择一抗的最佳稀释度对包被抗原进行细化筛选、不同有机溶剂对竞争结合反应的影响、酶标二抗稀释度筛选等。用建立的酶联免疫检测法检测实际样品,再与高效液相色谱法（HPLC）检测进行比较。结果：利用优化后条件建立了阿特拉津间接竞争ELISA检测曲线,标准曲线的相关系数R²=0.9958,相关性较好。另由此标准曲线可得LOD （最低检出限）为1.972 ng/ml。用于检测实际样品,回收率在80%-120%之间。当添加样品浓度为（0~6） ng/ml时,该法的检测灵敏度高于HPLC。结论：新建立的阿特拉津ELISA特异性好、精密度高,可代替大型仪器用于阿特拉津实际样品检测。     

霍乱疫苗中残余霍乱毒素检测方法的建立及验证

采用间接酶联免疫法,即用神经节苷脂包被,加入待检样品,再加入兔抗霍乱毒素B亚单位抗体,用标准样品的吸光值(A值)对标准样品的浓度绘制4-参数拟合曲线,根据标准曲线计算出待测样品中的CT浓度。结果显示,在浓度范围(0.6～16)ng/ml之间,CT标准浓度和检测浓度成线性关系,r2=0.9986。精确度在浓度范围(0.6～16)ng/ml,CT的平均回收率在96.24%～114.44%之间。精密度:批内变异CV%≤12.98%,批间变异CV%≤18.48%。特异性CT浓度在10ng/ml时,平均回收率为102.6%;CT浓度在5ng/ml时,平均回收率为111.17%;CT浓度在2.5ng/ml时,平均回收率为123.83%。实验表明该方法可检测霍乱疫苗原液中CT的含量。     

基于纳米磁珠和双标记抗体的玉米赤霉烯酮快速、高灵敏检测方法的建立及应用

玉米赤霉烯酮(zeralenone,ZEN)具有雌激素活性,主要污染谷物和饲料,大量聚积可导致流产和死胎,给动物和人类健康带来严重威胁。本研究通过将ZEN偶联抗原ZEN-BSA包被于纳米磁珠(magnetic nanoparticles,MNPs),制备纳米磁珠-偶联抗原复合物(MNPs-BSA-ZEN),同时使用金颗粒(Au nanoparticles,AuNPs)和辣根过氧化物酶(horseradish peroxidase,HRP)双标记的ZEN单克隆抗体,建立新型酶联免疫检测方法(MNPs-HRP-AuNPsIC-ELISA)。检测下限(IC10)达到0.03ng/mL,检测区间(IC20–IC80)为0.05–0.89ng/mL,半数抑制率(IC50)为0.22ng/mL,与ZEN类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol和β-zearalanol)的交叉反应性依次为19.2%、11.7%、8.3%、1.2%和4.3%,与黄曲霉毒素B1、赭曲霉毒素A、伏马毒素B1、桔青霉素和展青霉毒素几乎不存在交叉反应。在玉米、面粉和大豆样本中的加标回收率可达81.6%–113.5%,与LC-MS/MS同时对天然样本中ZEN含量的检测结果表明,两种方法相关性良好。本研究建立的MNPs-HRP-AuNPs IC-ELISA具备快速和高灵敏的双重优势,也可为其他霉菌毒素精准检测技术的开发提供参考。     

小麦中脱氧雪腐镰刀菌烯醇酶联免疫吸附测定方法的研究

用B淋巴细胞杂交瘤技术制备了能稳定分泌抗脱氧雪腐镰刀菌烯醇(DON)单克隆抗体的杂交瘤细胞株,命名为3D7.3D7的亚类为lgG_1.运用该抗体,建立了检测小麦中DON的间接竞争性酶联免疫吸附测定法.该法最低检出量为5ng/ml,敏感范围为5—1000ng/ml;平均回收率为96.6—107.3%;精密度为6.2—13.3%.运用该方法,检测了10份小麦样品.均为阳性,其含量为56.4—1002.0ppb.     

赭曲霉毒素 A 的高灵敏时间分辨荧光免疫分析

采用时间分辨荧光免疫分析 (TRFIA) 技术建立快速的高灵敏度的赭曲霉毒素 A (OTA) 全自动检测方法 . 将 OTA-BSA 作为免疫分子免疫 Balb/c 小鼠, OTA-KLH 为筛选用抗原包被板,用间接酶联免疫分析 (ELISA) 法筛选出专一针对 OTA 的高效价的阳性克隆 3G9 ,用杂交瘤细胞制备抗 OTA 单克隆抗体 . 用 OTA-BSA 包被 96 孔板为固相抗原,与游离 OTA 共同竞争有限的抗 OTA 单克隆抗体,以稀土离子 Eu3+ 标记的羊抗鼠抗体进行示踪,采用间接竞争免疫分析方法在解离增强荧光免疫分析体系中建立 OTA-TRFIA. 该方法的灵敏度为 0.03 μg /L ,测量范围为 0.03 ~1 000 μg /L ,批内和批间变异分别为 3.7% 和 5.3% ,平均回收率为 94.2% ,与赭曲霉毒素 B 的平均交叉反应为 3.7% ,与黄曲霉毒素 B1 、 牛血清白蛋白和苯基丙氨酸无交叉反应,说明抗体的特异性很好 . 8 条不同时间进行的间接竞争 OTA-TRFIA 的效应点均值 ED80 、 ED50 、 ED20 分别为 (0.33±0.02) μg /L、 (1.44 ±0.08) μg /L 和 (5.22 ± 0.12) μg /L ,说明方法的稳定性好,样品经 TRFIA 和 ELISA 试剂盒同时检测 OTA ,两者的相关系数为 0.925 ,结果相符 . 研究表明, OTA-TRFIA 是目前报道的 OTA 检测中最灵敏的方法,该分析方法稳定性好,可测范围宽,具有很好的应用前景 .     

土拉弗朗西斯菌胶体金免疫层析快速检测方法的建立

目的建立胶体金免疫层析技术快速定量检测土拉弗朗西斯菌。方法利用胶体金标记和双抗体夹心免疫层析技术,建立土拉弗朗西斯菌的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测。在面粉、饼干、果冻、梨汁等食品样品中添加土拉弗朗西斯菌的FopA蛋白模拟污染样品,评价该方法对固体、半固体、液体等食品样品的检测能力。结果该法可在10min内完成定性和定量检测,灵敏度为750ng／ml,线性范围750～24000ng/ml、回收率为56．7％-89．2％。结论所建立的检测土拉弗朗西斯菌的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的土拉弗朗西斯菌,适用于现场快速检测。     

A case report on a minor contamination of nivalenol in cereals harvested in Canada

An investigation for the occurrence of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) in cereals (ten wheat, one rye and one corn) harvested in Canada have been carried out using a procedure, which is rapid and sensitive for Fusarium mycotoxins. NIV, DON and ZEN were detected in 4, 9 and 9 out of ten wheat samples, and their average levels in the positives were 23 ng/g, 1257 ng/g and 9 ng/g, respectively. One rye and one corn were also contaminated with a minor amount of NIV. This is the first evidence for the natural occurrence of NIV in cereals grown in Canada, though its level was far less than DON.     

A case of sporotrichosis caused by two genetically differentSporothrix schenckii strains

A survey for the natural occurrence of Fusarium mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN), in Dutch cereals (totaling 29 samples) harvested in 1984/1985, showed that 90%, 79% and 62% of samples were contaminated with DON, NIV and ZEN, respectively. Average contents (ng/g) in the total of positive samples were 221 (DON), 123 (NIV) and 61 (ZEN). Among the cereals examined, the highest concentrations (ng/g) was 3198 (DON), 1875 (NIV) and 677 (ZEN) in a yellow corn sample for animal feed. The results of this survey show that Dutch cereals were relatively significantly contaminated with Fusarium mycotoxins.     

基于纳米颗粒的赭曲霉毒素A高灵敏酶联免疫检测方法的建立及应用

赭曲霉毒素(ochratoxins)主要是由青霉菌Penicillium和曲霉菌Aspergillus产生的有毒次级代谢产物,常见于发霉或发酵的农产品中,其中赭曲霉毒素A(ochratoxin A,OTA)毒性最强且最为普遍。OTA是粮食作物和饲料的重要污染物,在加工、储存或运输过程中均可产生,具有肾毒性和免疫毒性,可通过蓄积作用发挥毒性效应,对人类和动物健康造成严重威胁。本研究通过将OTA单克隆抗体包被于纳米磁珠(magnetic nanoparticles,MNPs)表面,获得具有免疫活性的磁珠抗体复合物(MNPs-Anti OTA),并制备生物素标记的偶联抗原OTA-BSA-Bio,后续采用链酶亲和素标记的纳米金颗粒(Strep-HRP-AuNPs)催化底物进行信号检测,最终建立了OTA高灵敏检测方法(MNPs-bs-AuNPs-ELISA)。在最优条件下,经计算该方法检测下限(IC10)为0.01ng/mL,检测区间(IC20-IC80)为0.02-0.73ng/mL,半数抑制率(IC50)为0.13ng/mL。与OTA类似物OTB、OTC交叉反应性为4.3%和8.1%,对其他常见真菌毒素AFB1、ZEN、FB1、DON、CIT和PAT均无交叉反应。玉米、面粉和大豆样本中的加标回收率可达85.6%-115.7%,对天然样本中OTA含量的检测结果表明,该方法与LC-MS/MS相关性良好。本研究建立的MNPs-bs-AuNPs-ELISA可满足谷物及饲料样本中OTA的快速、高灵敏度定量检测,成本较低,具有很好的应用前景。     

基于量子点标记的赭曲霉毒素A快速、高灵敏荧光免疫层析检测方法的建立及应用

赭曲霉毒素A（ochratoxin A,OTA）具有肾毒性、致畸性、致癌性和免疫毒性,广泛存在于各种粮食作物及其副产品中,是食品和饲料原料的重要污染物,可在人类及动物体内蓄积,在已知发现的真菌毒素中,重要性和危害性仅次于黄曲霉毒素。本研究通过采用量子点荧光微球（quantum dots,QDs）标记OTA单克隆抗体,并基于免疫层析原理,优化、建立了OTA高灵敏荧光免疫层析检测方法（FICGA）,15min即可实现对农产品中OTA污染的快速定量检测。该方法检测下限（IC₁₀）达到0.04ng/mL,检测区间（IC₂₀-IC₈₀）为0.05-0.59ng/mL,半数抑制率（IC₅₀）为0.18ng/mL。与OTA类似物OTB、OTC交叉反应性为7.3%和11.9%,对其他常见真菌毒素AFB₁、ZEN、FB₁和DON均无交叉反应。在玉米、面粉和大豆样本中的加标回收率可达83.2%-117.8%,与LC-MS/MS同时对天然样本中OTA含量的检测结果表明,两种方法相关性良好。本研究建立的FICGA快速、灵敏,可满足基层单位和现场的快速检测需求,具有很好的应用前景。     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination.

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

玉米赤霉烯酮单克隆抗体和免疫酶技术研究

采用蛋白质连接技术合成玉米赤霉烯酮抗原,免疫Balb/c鼠,通过淋巴细胞杂交瘤技术建立六株分泌抗玉米赤霉烯酮的单克隆抗体杂交瘤细胞株。间接酶联免疫吸附试验测定细胞上清抗体效价为1:2084(4H8)、1:256(6H9、4H3、2H5、2C8)、1:16(3F10);腹水抗体效价为10~9(4H3、4H8)、10~8(2H5)、10~7(6H9)、10~5(3H10)。竞争间接酶联免疫吸附试验测定六株单克隆抗体对玉米赤霉烯酮的敏感度为0.3—0.8ng/ml。六株抗体与玉米亦霉烯醇的交叉反应率为1.3—9.0％。六株单克隆抗体均属IgG类。细胞体外传代培养和冻存复苏后分泌抗体稳定。纯化抗体在37℃保存12天稳定,-30℃保存90天抗体滴度不变。用该抗体建立竞争间接酶联免疫吸附试验检测掺合玉米赤霉烯酮的玉米、小麦、饲料,平均回收率分别为105％、90％、103％,平均批间变异系数为5.8％、2.8％、6.8％,批内变异系数为3.8％、12.7％、15.7％。样品中玉米赤霉烯酮掺合量与竞争间接酶联免疫吸附试验检出量有良好相关性(r≥0.9996)。     

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of zearalenone,deoxynivalenol and their metabolites in pig serum

A sensitive and selective liquid chromatography tandem mass spectrometry method using negative electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, β-zearalanol and de-epoxy-deoxynivalenol in pig serum. For method development, different sample preparation columns were tested for their suitability for extraction and clean up. Finally, preparation of serum samples was carried out using Oasis? HLB solid-phase extraction (SPE) columns. The analyte concentrations were determined by the use of isotopically labelled internal standards (IS). The method was in-house validated for all analytes. Calibration graphs (0.3–480 ng/ml) were prepared and high degree of linearity was achieved (r?≥?0.99). Results for method precision ranged between 2.7 and 21.5 % for inter-day and between 1.1 and 11.1 % for intra-day. The recoveries were in the range of 82–131 %. Limits of detection and quantification ranged 0.03–0.71 and 0.08–2.37 ng/ml, respectively. The method has been successfully used for quantitative determination of ZEN, DON and their metabolites in pig serum from a feeding trial with practically relevant ZEN and DON concentrations. This method is precise and reproducible and can be used as a multi-biomarker method to assess animal exposure to these mycotoxins and for diagnosis of intoxications.