免疫亲和层析净化-高效液相色谱法同时测定牛奶和奶粉中黄曲霉毒素B1、B2、G1、G2、M1、M2

建立牛奶和奶粉中黄曲霉毒素B1、B2、G1、G2、M1、M2的免疫亲和层析净化和柱后衍生高效液相色谱测定方法。样品经溶解、离心、过滤后,通过免疫亲和柱,黄曲霉毒素特异性抗体选择性地与存在的黄曲霉毒素抗原键合,形成抗体-抗原复合体。甲醇-乙腈混合溶液(4:5,v:v)洗脱,带荧光检测器的高效液相色谱仪经柱后衍生测定,外标法定量。标准曲线线性良好,添加回收率在57.0%～88.7%,相对标准偏差在3.37%～16.9%,牛奶中各黄曲霉毒素检出限:B1为2ng/kg,B2为1ng/kg,G1、G2为3ng/kg,M1、M2为5ng/kg;奶粉中B1为20ng/kg,B2为10ng/kg,G1、G2为30ng/kg,M1、M2为50ng/kg,检测低限能够满足各国对牛奶和奶粉中黄曲霉毒素的限量要求。该方法准确、快速、灵敏度高,适用于牛奶和奶粉中黄曲霉毒素的测定。     

总黄曲霉毒素ELISA定量检测方法的研制

为敏感、特异和快速地检测食品中总黄曲霉毒素的污染水平,在抗总黄曲霉毒素单克隆抗体的基础上,研制了间接竞争ELISA检测方法,包被抗原为AFB1-BSA,包被量为0.0625μgml,抗体工作浓度为1:1.6×106。该方法对黄曲霉毒素B+G标准品的最低检出浓度为0.13ngml,对样品的最低检出量为1.50μgkg。校正曲线的线性范围为0.26～20.00ngml,线性方程y=-0.4463x+0.3532(R2=0.9915);对黄曲霉毒素B+G50%的抑制浓度为2.08ngml;对玉米2个加标水平的平均回收率分别为94.56%和112.05%,对花生2个加标水平的平均回收率分别为87.50%和85.60%。该方法所用抗总黄曲霉毒素单克隆抗体对黄曲霉毒素B1、B2、G1、G2的交叉反应率分别为100%、57.5%、104%和19%,与其它真菌毒素无交叉反应。     

以赭曲霉毒素A单克隆抗体建立竞争酶联免疫吸附分析方法的研究

本研究通过活性酯法合成赭曲霉毒素A人工抗原，免疫小鼠制备单克隆抗体，在此基础上建立了竞争ELISA检测方法，线性范围为200-6000pg／ml，检测下限为150pg／ml。单抗与赭曲霉毒素B的交叉反应率为35％，与桔霉素、黄曲霉毒素B1和展青霉素等毒素交叉反应低于0．01％。在小麦样品中的加标回收率为83％～116％，变异系数为9.4％-19．8％。测试23份样品，赭曲霉毒素A的含量在0．6～2．56μg／kg之间。     

粮油作物中伏马菌素B1免疫测定方法的建立

旨在制备伏马菌素B_1单克隆抗体,并建立伏马菌素B_1免疫学检测方法。以制备的免疫原FB_1-BSA免疫小鼠,利用细胞融合技术建立能分泌抗FB1抗体的杂交瘤细胞株,体内诱生腹水的方法制备FB_1单抗。基于制备的FB_1单抗,建立间接竞争ELISA检测分析方法,并对检测方法性能进行了初步鉴定。结果表明,通过动物免疫、细胞融合筛选到1株分泌抗FB_1抗体的杂交瘤细胞株3F6。建立的间接竞争ELISA检测分析方法检测范围40~600 ng/mL之间,检测下限达到40 ng/mL,半数抑制浓度IC_(50)为136.81 ng/mL,除与FB1特异性反应外,与同系物FB2及FB3交叉反应率分别为11.65%和7.85%,与黄曲霉毒素B_1、黄曲霉毒素M_1、β-玉米赤霉烯醇、呕吐毒素、T-2毒素、玉米赤酶烯酮、赭曲霉毒素A、玉米赤霉酮、α-玉米赤霉醇交叉反应率均低于0.2%,样品回收率在88.89%到115.45%之间,平均101.91%,变异系数为7.43%。本研究建立的ELISA方法与LC-MS-MS检测相同的样品时,二者的检测结果没有显著差异(P0.05)。本研究初步研发出灵敏度符合检测要求、特异较强、准确度较高、简便快捷的FB1免疫学检测方法 。     

重金属铅多克隆抗体的制备及鉴定

目的：合成铅离子完全抗原,并对其进行鉴定;用合成的完全抗原免疫Balb/c 小鼠制备多克隆抗体,鉴定效价和特异性。方法：用双功能螯合剂p-SCN-Bn-DTPA 将Pb2+ 与载体蛋白BSA 和OVA 偶联在一起,合成免疫抗原(Pb-DTPA-BSA)和检测抗原(Pb-DTPA-OVA),并通过SDS-PAGE、紫外扫描、电感耦合等离子体(ICP) 检测抗原中Pb2+ 含量,以及通过免疫Balb/c 小鼠,采集血清进行间接ELISA 和竞争ELISA 检测。结果：两种合成的抗原相对于各自的载体蛋白均出现了蛋白条带滞后,紫外吸收峰偏移等现象;免疫的4 号和5 号Balb/c 小鼠的抗血清效价达1:400000;与OVA 无交叉反应,ELISA 检测IC50 值为1.5ng/mL,与Fe3+ 有近5% 的交叉反应,与其他重金属离子的交叉反应均小于1%。结论：铅离子多克隆抗体制备成功。     

荧光免疫定量法即时检测谷物中黄曲霉毒素B1

目的 基于时间分辨荧光免疫层析技术,研制快速定量检测试纸条,用于粮谷物中黄曲霉毒素B1的检测。方法 以荧光微球为标记物,采用DNP独立质控体系和竞争法检测原理,构建了粮谷物中黄曲霉毒素B1荧光免疫定量即时检测（point-of-care testing ,POCT）方法。评价其准确度、重复性、特异性、与仪器确证方法符合度。结果 该方法最适条件为：pH7.8硼酸盐（BB）活化,pH7.5磷酸盐（PB）偶联,微球抗体质量比为5:4,抗原抗体质量比为50:1。检测限在0.299 ng/g -0.997 ng/g之间,定量限在0.544 ng/g -2.663 ng/g。代表性样本准确度为92.2%-111%,重复性为3.2%-7.5%。除与黄曲霉毒素B2有交叉反应（6.1%）,与其他类似物无（<0.1%）。该法检测结果与仪器确证法一致性好,在-12.5%—15.9%之间。结论 提供了一种准确、快速、定量、灵敏、便捷、适合现场检测粮谷物中黄曲霉毒素B1的时间分辨荧光免疫检测试纸条。     

光化学衍生检测玉米中4种黄曲霉毒素

建立了HPLC-免疫亲和柱净化,柱后光化学衍生检测玉米中4种黄曲霉毒素B1,B2,G1,G2的分析方法。玉米样品提取、浓缩、过滤后经免疫亲和柱净化,液相色谱分离后采用光化学衍生器对黄曲霉毒素B1,B2,G1,G2进行在线衍生,通过配制荧光检测器的液相色谱同时检测玉米中B1,B2,G1,G2这4种黄曲霉毒素。结果表明,4种黄曲霉毒素B1,B2,G1,G2的色谱分析时间在6~11 min内完成,检测定量限分别为:0.10,0.03,0.10,0.03μg/kg,完全符合并高于欧盟检测标准定量限,通过光化学衍生器在线衍生将B1、G1的分析定量限提高到2.7倍和3.6倍。玉米基质中B1,B2,G1,G2在添加水平为10,3,10,3μg/L时其回收率分别为:90.3%,85.6%,93.5%,92.8%。其线性范围分别为0~20μg/L,0~6μg/L,0~20μg/L,0~6μg/L,RSD分别为2.3%,1.5%,2.7%,3.2%。文章建立了分析玉米中4种黄曲霉毒素B1,B2,G1,G2的分析方法,经验证该方法定性准确,定量灵敏度高,可同时检测出玉米中4种黄曲霉毒素B1,B2,G1,G2含量。     

抗黄曲霉毒素B_1单抗独特型抗体的制备

目的研制黄曲霉毒素B1标准品的替代品并应用于酶联免疫吸附试验(ELISA)。方法在研制了抗黄曲霉毒素B1单抗的基础上,将抗体用无花果蛋白酶进行酶切,制备出抗黄曲霉毒素B1单抗F(ab’)2片段,并以F(ab’)2片段作为免疫原免疫日本大耳白兔,制备出抗黄曲霉毒素B1单抗独特型抗体,并对该独特型抗体进行鉴定。结果该独特型抗体是Ab2β型,与黄曲霉毒素B1存在内影像关系。结论该抗体可以替代黄曲霉毒素B1标准品应用于ELISA检测。     

玉米赤霉烯酮单克隆抗体制备及免疫分析

采用活泼酯法将玉米赤霉烯酮(Zaralenone,ZEN)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原ZEN-BSA和包被原ZEN-OVA,经紫外扫描和聚丙烯酰胺凝胶电泳鉴定后免疫BALB/c小鼠;挑选产生高效价和高敏感性抗体的小鼠进行抗原超强免疫,取脾细胞与SP2/0细胞融合获得1株稳定分泌抗玉米赤霉烯酮单克隆抗体的杂交瘤细胞(3D8),经鉴定,该抗体亚类为IgG1,轻链为k型。经体内诱生腹水并纯化获得效价为1∶2.048×106的单克隆抗体。建立了基于单克隆抗体的ZEN间接竞争ELISA法(ic-ELISA),该方法IC50和检出限分别为22.89pg/mL和10.07pg/mL。与α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、α-玉米赤霉醇和β-玉米赤霉醇的交叉反应率分别为95%、8%、12%、6%和5%,而与黄曲霉毒素B1、呕吐毒素、伏马毒素B1和赭曲霉毒素A未见有交叉反应。在玉米、大麦、小麦和燕麦中加标的回收率在86.4%~104.8%之间。该方法灵敏特异,可作为快速检测谷物中玉米赤霉烯酮的初筛方法。     

绿色无毒黄曲霉毒素B_1免疫检测方法

建立了一种基于抗独特型抗体的绿色无毒的ELISA方法,并用于检测面粉中黄曲霉毒素B1。通过酶解2种抗黄曲霉毒素小鼠单克隆抗体(11A9和1G3)制备F(ab’)2片段,并将其免疫新西兰大白兔制备抗独特型抗体。最终得到两种抗独特型抗体,并对抗独特型抗体和AFB1进行相关性分析。通过实际样品添加回收,其批内回收率为115.60%~121.88%,变异系数在5%以内;而批间回收率为111.89%~126.98%,变异系数低于8%。分析结果准确可靠,表明此无毒绿色ELISA是一种安全可靠的AFB1分析方法。     

Occurrence of aflatoxins B1, B2, G1, and G2 in peanuts and their products marketed in the region of Campinas, Brazil in 1995 and 1996.

Samples of peanuts and their products marketed in the region of Campinas, Brazil in 1995 and 1996 were analysed in terms of aflatoxin B1, B2, G1, and G2 by thin-layer chromatography, aiming at the protection of consumer health. Of the 80 samples analysed, 35 of peanuts and 45 of peanut products, 41 (51%) presented contamination with aflatoxin, 11 of them being peanuts (27%) and 30 being products (67%), in a range of 43 to 1099 micrograms/kg for B1 + B2 + G1 + G2 with a 90th percentile of 415 (B1 + G1) and of 615 (B1 + B2 + G1 + G2). The results also demonstrated that aflatoxin B1 (AFB1) reached the highest incidence and the highest upper limits compared with all the other aflatoxins.     

光化学衍生-高效液相色谱法测定 粮谷类样品中黄曲霉毒素

目的建立光化学衍生-高效液相色谱荧光法测定粮谷类样品中的黄曲霉毒素(AFT)含量。方法样品经Romer Labs免疫亲和柱净化,经SW-3光化学柱后衍生,经高效液相色谱分离和荧光检测器测定,分析其中黄曲霉毒素B_1、B_2、G_1、G_2的含量。同时对免疫亲和柱洗脱条件、流动相的洗脱程序进行了优化。结果在0.5~10 ng/mL(AFT B_1,G_1)和0.15~3.0 ng/mL(AFT B_2,G_2)线性范围内,所得回归方程的相关系数均大于0.999。黄曲霉毒素B_1、G_1方法检出限为0.15 ng/g,黄曲霉毒素B_2、G_2方法检出限为0.05 ng/g,加标回收率为89.5%~107%,精密度为1.4%~7.2%。采集粮谷类样品222件,其中有5件样品检出AFT,但均未超过国家限值标准。结论该方法灵敏度和准确度较高,可适用于粮谷类食品中黄曲霉毒素的检测。     

Occurrence of aflatoxins and aflatoxin-producing moulds in fresh and processed meat in Egypt.

A survey was carried out to detect aflatoxins and isolate aflatoxigenic moulds contaminating fresh and processed meat products. The fungal contamination was examined in 215 samples of fresh and processed meat products and 130 samples of spices used in the meat industry collected from different local companies in Cairo, Egypt. Processed meat products such as beefburger, hot-dog, kubeba, sausage, luncheon meat had the highest count of moulds as compared with fresh and canned meat. Out of 150 samples of meat products and 100 samples of spices, aflatoxin B1 was detected in five samples of beefburger, (8 micrograms/kg), four samples of black pepper (35 micrograms/kg), and four samples of white pepper (22 micrograms/kg). Aflatoxins B1 and B2 were detected in one sample of kubeba (150 micrograms B1/kg and 25 micrograms B2/kg); hot-dog (5 micrograms B1/kg and 2 micrograms B2/kg) sausage (7 micrograms B1/kg and 3 micrograms B2/kg) and luncheon meat (4 micrograms B1/kg and 2 micrograms B2/kg). Also, aflatoxins B1 and G1 were detected in two samples of turmeric (12 micrograms B1/kg and 8 micrograms G1/kg) and coriander (8 micrograms B1/kg and 2 micrograms G1/kg). Aspergillus flavus (24 isolates), and Aspergillus parasiticus (16 isolates) were the predominant aflatoxin-producing moulds isolated from both processed meat products and spices. Aflatoxins were absent in fresh meat, canned meat, salami, beefsteak and minced meat. The contamination of processed meat with aflatoxin was shown to correlate with the addition of spices to fresh meat.     

Occurrence of aflatoxins, ochratoxin A, and fumonisins in retail foods in Japan

We conducted a survey of aflatoxin B1, B2, G1, and G2, ochratoxin A, and fumonisin B1, B2, and B3 contamination in various foods on the retail market in Japan in 2004 and 2005. The mycotoxins were analyzed by high-performance liquid chromatography, liquid chromatography-mass spectrometry, or high-performance thin-layer chromatography. Aflatoxins were detected in 10 of 21 peanut butter samples; the highest concentration of aflatoxin B1 was 2.59 microg/kg. Aflatoxin contamination was not found in corn products, corn, peanuts, buckwheat flour, dried buckwheat noodles, rice, or sesame oil. Ochratoxin A was detected in oatmeal, wheat flour, rye, buckwheat flour, green coffee beans, roasted coffee beans, raisins, beer, and wine but not in rice or corn products. Ochratoxin A concentrations in contaminated samples were below 0.8 microg/kg. Fumonisins were detected in popcorn, frozen corn, corn flakes, and corn grits. The highest concentrations of fumonisins B1, B2, and B3 in these samples were 354.0, 94.0, and 64.0 microg/kg, respectively.     

免疫亲和柱净化-光化学柱后衍生高效液相色谱 荧光法测定粮食中黄曲霉毒素

建立了粮食中黄曲霉毒素B1、B2、G1、G2的免疫亲和柱净化-光化学柱后衍生高效液相色谱荧光检测法。样品经甲醇-水提取,免疫亲和柱净化,高效液相色谱分离,光化学柱后衍生,荧光检测器测定。结果表明,黄曲霉毒素B1、B2、G1、G2的检出限分别为0.50、0.25、0.50、0.25μg/kg,回收率为67.2%～91.7%,RSD小于10%。该方法快速、准确、灵敏度高、重现性好,能满足我国对粮食中黄曲霉毒素限量的检测要求。     

Distribution of aflatoxins in product and by-products during glucose production from contaminated corn

Aflatoxins are known to be hepatotoxic, carcinogenic, and teratogenic. A positive correlation has been established between the consumption of aflatoxin-contaminated foods and the increased incidence of liver cancer worldwide. A survey of Egyptian corn and corn-based products and by-products shows that the majority of the samples had higher limits of aflatoxin. We have conducted experiments to determine the fate and distribution of aflatoxin during wet-milling process fractions and investigate the aflatoxin destruction during starch conversion to glucose syrup. The present results showed that about half of the aflatoxin content (48.1%) in the infected corn grain was found to be lost in steep liquor, depending upon the aflatoxin type, arranged in the order G1 > G2 > B1 > B2. After wet-milling aflatoxins were distributed into starch, gluten, fiber, and germ. Gluten, fiber, and germ were the most highly contaminated fractions. The loss of aflatoxin during process of starches reached 54.4% in steep water and water process. Although the gluten fraction represents only 9.6% of corn, the higher percentage (25.3%) of aflatoxin was found in this fraction, the fiber and germ account for nearly 29% of the milled corn and contain 11.6% of the aflatoxin. On the other hand, 8.7% of the total aflatoxins in start corn was found in starch fraction which accounts 61% of the milled corn. Aflatoxins G1 and G2 were found lost in higher concentrations compared to the aflatoxin B1 and B2. A higher percentage of AfG1 (86.35%) and AfG2 (78.36%) and a lower percentage of AfB1 (16.3%) and AfB2 (14.7%) were found in starch fraction. The conversion percent of contaminated starch was 89.5% compared with control starch. It can be concluded that aflatoxins were destroyed during starch conversion. Consequently, glucose syrup produced from contaminated starch was found aflatoxin-free.     

固相萃取柱净化-超高效液相色谱-串联质谱法快速测定液态乳中14 种真菌毒素

建立使用固相萃取柱净化液态乳中14 种真菌毒素（黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1、赭曲霉毒素A、橘霉素、T-2毒素、杂色曲霉素、伏马毒素B1、伏马毒素B2、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、青霉酸）多残留的超高效液相色谱-串联质谱检测方法。样品经含0.1%甲酸-乙腈沉淀蛋白和提取,固相萃取柱Oasis PRiME HLB净化后,以0.1%甲酸溶液与乙腈为流动相,经ACQUITY UPLC HSS T3色谱柱（2.1?mm×100?mm,1.8?μm）分离,梯度洗脱,采用电喷雾-正离子多反应监测模式,外标法定量。结果表明：14?种真菌毒素的定量限（RSN≥10）为0.5～5?μg/kg,高、中、低3?个添加水平时,平均回收率为67.7%～112.7%,相对标准偏差为0.43%～7.28%。该方法的检测速度快,净化效果较好,基质干扰较少,灵敏度高,结果准确、可靠,可用于液态乳中真菌毒素的检测分析。     

高效液相色谱-串联质谱法同时测定花生制品中的黄曲霉毒素B_1、B_2、G_1、G_2

采用高效液相色谱-串联质谱建立了高效液相色谱-串联质谱法同时测定花生制品中4种黄曲霉毒素（B1、B2、G1、G2）的方法。结果表明,在ESI正离子模式下,高效液相色谱-串联质谱法的最低检出限为0.2μg/kg,定量限为0.6μg/kg;标准工作液在0.5~50.0μg/kg的范围内线性良好,相关系数达到0.9990。     

Co-occurrence of aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumonisin B 1 in Brazilian corn

Two hundred and fourteen unprocessed corn samples (1997-98 harvest), collected at wholesale markets in different regions in Brazil, were surveyed for the occurrence of mycotoxins. The samples were analysed for aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumoni1sin B 1 using in-house validated methods. The occurrence of aflatoxin B 1 , zearalenone and fumonisin B 1 was found in 38.3, 30.4 and 99.1% of the samples, respectively. Aflatoxin B 1 , zearalenone and fumonisin B 1 contamination levels varied from 0.2 to 129, 36.8 to 719, and 200 to 6100 μg/kg, respectively. The cooccurrence of the two carcinogenic mycotoxins aflatoxin B 1 and fumonisin B 1 was observed in 100% of the aflatoxin-contaminated samples (82 samples). Cooccurrences of aflatoxin B 1 : zearalenone: fumonisin B 1 and aflatoxin B 1 : aflatoxin B 2 : fumonisin B 1 were found in 18 and 43 samples, respectively.     

微波消解一电感耦合等离子体质谱法测定畜禽肉中的硼和铝

建立了畜禽肉中硼和铝元素的电感耦合等离子体质谱（ICP-MS）测定方法。样品用HNO3＋H2O2＋H2O经微波消解后,用ICP-MS进行分析。以Sc和Li作为内标物质,补偿了基体效应,优化仪器参数,考察了方法的检出限、线性范围、回收率和精密度等。结果表明：硼和铝在0-2．0μg／mL线性范围内,线性关系良好,相关系数均大于0．999,铝和硼的方法检出限分别为70．5ng／g和30．5ng／g,相对标准偏差（n=6）分别为1．35％和2．01％;在5种不同样品基质（牛肉、猪肝、猪肚、牛百叶、鸡肉）的添加回收试验中,添加水平为0．3-3．2μg／g时,两种元素的平均回收率为90．5％107．2％。利用该方法分析鸡肉的国家标准物质,结果与标准值一致。所建立的方法简单、快速和准确,适用于检验分析畜禽肉中硼和铝元素。