应用抗黄曲霉毒素单链抗体检测酱油中黄曲霉毒素B1

利用本实验室制备的抗黄曲霉毒素B1的单链抗体(ScFv),通过棋盘实验确定了抗原抗体的最适工作浓度,在此之上根据间接竞争酶联免疫法(ELISA)绘制标准曲线,检测酱油中AFB1的含量;通过改变样品的盐浓度及pH来确定其对ELISA检测结果的影响。研究结果表明,利用ScFv检测黄曲霉毒素的最小检测值为0.10ng/mL,平均加标回收率在84%～109%之间,本文建立的ELISA方法在pH5～8,盐浓度小于10%时较稳定。本文建立的利用抗AFB1的ScFv检测黄曲霉毒素含量的方法方便快捷,稳定性较好,并且成本较低,适合于食品中黄曲霉毒素的检测。     

黄曲霉毒素B1金标检测体系建立过程中的影响因素

详细研究了纳米金标记免疫检测体系建立过程中的影响因素，包括检测带的信号强度、封闭试剂，纳米金探针的浓度、包被试剂A和B的浓度、纳米金溶胶的颗粒尺寸，及检测过程中的化学体系等。通过参数优化，确立了建立快速、简易的金标试剂条检测方法的最佳条件。探针溶液中加入保护试剂K后，试剂条的稳定性有明显的提高，常温下试剂条保存期可达120d（信噪比保留70％以上）。     

抗黄曲霉毒素B1独特型纳米抗体的表达及复性

目的 快速制备大量具有生物活性的独特型纳米抗体F7。方法 构建pET22b-F7原核表达载体, 转化至大肠杆菌E. coli BL21(DE3)中进行诱导表达, 对诱导温度、诱导剂浓度和诱导时间进行优化。结果 独特型纳米抗体F7表达量有所增加但主要以包涵体形式存在。经过包涵体的溶解、复性获得了具有生物活性的抗AFB1独特型纳米抗体, ELISA证实复性后的蛋白依然存在对鼠源AFB1抗体的结合特性。结论 为后续抗体的性质研究和改造奠定了基础。     

磁分离结合CdTe发光量子点标记黄曲霉毒素B1免疫检测新方法

将发光量子点标记技术与磁分离富集技术相结合,基于竞争免疫分析,成功构建了黄曲霉毒素B1(AFB1)免疫检测新方法。首先合成了巯基丙酸包覆的CdTe发光量子点,同时采用水热法合成了氨基化磁性纳米粒子,通过TEM成像、荧光光谱、XRD、红外光谱等分别对其进行了表征。随后以AFB1人工抗原功能化磁性纳米粒子作为捕获探针,以发光量子点标记免疫球蛋白G(二抗)作为信号探针,基于磁性纳米粒子表面AFB1人工抗原和样品中AFB1与AFB1单克隆抗体之间的竞争免疫结合,建立了AFB1新型检测方法。实验优化条件下,荧光强度与黄曲霉毒素B1质量浓度在0.1~100 ng/mL范围内呈良好的线性关系,检测限为0.03 ng/mL。实际样品中加标回收实验结果表明,新方法准确性良好。     

Aflatoxin residues in eggs of laying Japanese quail after long-term administration of rations containing low levels of aflatoxin B1.

The aim was to evaluate the excretion of residues of aflatoxin B(1) (AFB(1)), aflatoxin M(1) (AFM(1)), aflatoxin B(2a) (AFB(2a)) and aflatoxicol (AFL) in eggs of laying Japanese quail fed rations with low levels of aflatoxin B(1) for 90 days. The quail were randomly assigned into four experimental groups and given prepared rations containing either 0 (controls), 25, 50 or 100 microg AFB(1) kg(-1) feed. Thirty-two eggs per treatment were collected on days 1-7, 10, 20, 30, 60 and 90 of the aflatoxin treatment period, and submitted to aflatoxin analysis by high-performance liquid chromatography. Average egg production and feed consumption were not affected ( p > 0.05) by AFB(1). Egg weight was significantly lower ( p<0.05) only for groups exposed to 100 microg AFB(1) kg(-1). Residues of aflatoxins were detected in eggs at levels that ranged from 0.01 to 0.08 microg kg(-1) (AFB(1)), 0.03-0.37 microg kg(-1) (AFM(1)), 0.01-1.03 microg kg(-1)(AFB(2a)) and 0.01-0.03 microg kg(-1) (AFL). Results indicate that the excretion of aflatoxin residues in quail eggs might occur at relatively low concentrations under conditions of long-term exposure of quail to low levels of AFB(1).     

LIF-HPCE法检测食品中的黄曲霉毒素B1

采用自行设计搭建的激光诱导荧光(LIF)- 高效毛细管电泳(HPCE)检测平台,建立LIF-HPCE 法对食品中的黄曲霉毒素B1(AFB1)进行高灵敏度检测。AFB1 在胶束电动毛细管电泳(MECC)模式下分离,LIF 检测,375nm 激光进行激发,检测波长440nm,操作电压15kV,电流104μA。AFB1 在0.5～50μg/kg 浓度范围内线性关系良好,r ＝ 0.9994;最低检出质量1.7 × 10-13g(S/N ＝ 3),最低定量限5.6 × 10-13g(S/N ＝ 10),方法精密度和重复性的相对标准偏差为5% 左右。测定食品中粮油等食品样品,加标回收率为84.1%～96.1%。所建立的方法无需进行衍生反应和荧光标记,快速灵敏,绿色环保,10min 左右完成分析,适用于食品中黄曲霉毒素的高灵敏度检测。     

HPLC determination of the total content of aflatoxins in naturally contaminated eggs in free and conjugate forms

A study was undertaken to evaluate the total aflatoxin content in naturally contaminated eggs. Two pools of eggs from laying hens were collected after 2 and 7 days of treatment with aflatoxin B1 (AFB1). An HPLC method has been developed for the determination of AFB1, aflatoxin M1 (AFM1), aflatoxin B2a (AFB2a) and aflatoxicol (Ro) both in free form and after release from water-soluble conjugates. The bound form was cleaned up after acid hydrolysis of the aqueous phase. Eggs collected after 2 days of treatment revealed residues of AFB1, AFB2a and Ro in the organic phase, but none in the aqueous portion. After 7 days of treatment both AFB1 and its hydroxy derivative were found in the organic phase but the aqueous portion showed only hydroxylated metabolites accounting for 35% of the total aflatoxin content.     

Long-term administration of low doses of mycotoxins to poultry. 1. Residues of aflatoxin B1 and its metabolites in broilers and laying hens

A study was performed to determine aflatoxin residues in tissues and organs of male broilers and hens that had been fed a diet contaminated with 50 micrograms/kg aflatoxin B1 (AFB1). Residue levels of AFB1, aflatoxicol (Ro), aflatoxin M1 (AFM1) and aflatoxin B2a (AFB2a) were determined by an HPLC method and, with the exception of AFB2a, were detected in the liver, kidney and thigh of both male broilers and hens. The highest levels found were for Ro in liver (1.10 and 0.60 micrograms/kg for male broilers and hens, respectively). On the other hand no detectable amounts of aflatoxins were found in any tissue after withdrawal periods of 14 and 33 days for male broilers and laying hens respectively.     

PRODUCTION AND CHARACTERIZATION OF ANTIBODY AGAINST AFLATOXIN G1

Antibody against aflatoxin G1 (AFG1) was obtained from rabbits after immunizing the animals with AFG1 hemiacetal (AFG2a) conjugated to bovine serum albumin. A direct heterogenous ELISA in which AFG2a was conjugated to horseradish peroxidase was used for monitoring the antibody titers and for toxin detection. Competitive ELISA assay revealed that the antibody was most specific for AFG2a and least for AFB2. The relative cross-reactivity of this antiserum with aflatoxins G2a, G2, G1, M1, E1 and B2 was found to be 1, 7, 13, 47, 48, and 63, respectively. The lower detection limits for detection of AFG1 after derivatizing to AFG2a was around 15–25 pg per assay.     

量子点标记荧光免疫法检测花生中黄曲霉毒素B_1

建立了基于量子点标记二抗的间接竞争荧光免疫吸附测定方法（indirect competitive fluorescence-linked immunosorbent assay,cFLISA）,并研究检测花生中黄曲霉毒素B1的可行性。采用谷胱甘肽为稳定剂,在水相中直接合成碲化镉（CdTe）量子点,并利用1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐（EDC）与兔抗鼠二抗进行共价偶联,以黄曲霉毒素B1的单克隆抗体建立cFLISA方法。结果表明,该方法的灵敏度和最低检测限值分别为0.023ng/mL和0.001ng/mL,与传统的有机染料FITC-二抗法比较,灵敏度提高了30倍,花生样品加标0.1、0.05和0.025ng/g,回收率范围在88%～116%之间,变异系数均小于10%。建立的cFLISA方法可以较好的检测花生中黄曲霉毒素B1,并为其它真菌毒素的检测提供参考。