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ELIME霉菌毒素的检测方法(酶联免疫磁电化学)

Biology

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Summary

检测trichothecenes(霉菌毒素对人类健康的关注),使用一种新开发的筛查方法的基础上有竞争力的免疫组织化学方法和最后的电化学检测的一个协议证明。

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Romanazzo, D., Ricci, F., Vesco, S., Piermarini, S., Volpe, G., Moscone, D., Palleschi, G. ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection. J. Vis. Exp. (32), e1588, doi:10.3791/1588 (2009).

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Abstract

免疫分析是一种有效的替代更昂贵和时间耗费定量高效液相色谱法或气相色谱1,2为食品类商品中的有害霉菌毒素的筛选检测方法。在这个协议中,我们将展示如何编造并询问电化学竞争性酶联酶联免疫检测,基于磁珠免疫组织化学链 3和丝网印刷电极感应平台的坚实支持。

我们的方法旨在确定总额的HT - 2和T - 2毒素,霉菌毒素属于trichothecenes家庭 4对人类健康的极大关注。使用了对HT - 2和T - 2的100%的交叉反应的抗体克隆允许同时检测两种毒素具有相似的灵敏度5。

我们检测的第一步是涂层的步骤,我们固定在磁珠表面HT2 - KLH偶联物毒素。后阻塞的一步,要避免非特异性吸收,另外一种单克隆抗体,使固定HT - 2和自由的HT - 2或T - 2样品中或溶解在一个标准的解决方案之间的竞争。

在年底的竞争一步,单克隆抗体的数量挂钩的固定HT - 2将样品溶液中的毒素量成反比。

碱性磷酸酶(AP)标记二抗是用来揭示之间的特异性抗体和固定的HT - 2的结合。最终的测量步骤是下降磁珠悬液等分的,对应一个特定的样品/标准的解决方案,一个丝网印刷工作电极表面上;磁珠固定和集中划归正是磁铁丝网印刷电极。经过两分钟的孵化磁珠和一个AP的基板之间,酶产品检测微分脉冲伏安法(DPV)使用便携式仪器(PalmSens)也能够启动几秒钟的时间间隔内自动八个测量。

Protocol

1)阻塞包被的磁珠:

继续如下块包被的磁珠:

  1. 准备一个2毫升Eppendorf管;
  2. 均质(摇晃,但没有涡旋)包被的磁珠(见附录准备包被的磁珠)使用样品旋转的搅拌机;
  3. 紧随之后,同质化,移液器10μL到每个单独的Eppendorf管包被的磁珠;
  4. 添加脱脂阻止到Eppendorf管中设置的每个解决方案的牛奶1毫升;
  5. 让我们为在室温下30分钟孵育旋转样品混合器磁珠;
  6. 取出阻塞的解决方案:这样做2分钟(见管侧壁上坚持磁珠)的磁性部分磁粉浓缩管;仔细移液器关闭上清,留下珠不受干扰;
  7. 加入1 ml PBS缓冲液+ NaN的3 + BSA作为存储解决方案,为每个管中;
  8. 商店涂层和封锁的磁珠在4 ° C(请注意,包被的磁珠至少2个月稳定在4 ° C);

2)免疫磁珠链建设的校准曲线和样品分析

继续为有准备的测量磁珠:

  1. 以涂层和阻止你要分析每个样品中提取的解决方案之一的Eppendorf管(2毫升);
  2. 以一个额外的Eppendorf管中,每一个工作的校准解决方案;
  3. 均质磁珠,使用前的旋转样品混合器2分钟;
  4. 删除存储磁粉浓缩液使用;
  5. 与PBS / BSA洗涤液洗磁珠三次。删除洗涤液;
  6. 比赛步 :添加每个包含管洗涤磁珠,200μL样品提取准备;每个样品使用一个磁珠管。每个标准溶液,重复同样的程序。单克隆抗体溶液中加入200μL,每个磁珠管。让磁珠孵育半小时旋转样品在室温下搅拌机;
  7. Eppendorf管中取出的竞争步使用的解决方案;
  8. 贴标步:每个磁珠Eppendorf管中添加标记二抗400μl。让磁珠孵育半小时旋转样品在室温下搅拌机;
  9. Eppendorf管中删除标签解决方案;
  10. 3次,PBS /吐温® 20洗涤液洗磁珠。删除的液体;
  11. 悬浮与DEA的缓冲区100μL每个等份磁珠。现在磁珠准备电化学测量步骤;

3)大会PalmSens多路复用器(MUX)选项

请注意:底物水解酶产品犯规电极表面,这样做的原因为每个测量都需要一个新的电极。

  1. 将PC连接笔记本电脑通过串行电缆PalmSens;
  2. CH8与PalmSens复用器连接9 - DIN插头。
  3. 八通道MUX CH8的多路复用器的电接触连接的串行电缆;
  4. 广场上的8个磁铁块电极地带。注意每个磁铁放置在每一个工作电极(注意,需要为每个电极的单磁铁是势在必行,否则磁粉不会在工作电极面积集中);
  5. 插入电极地带到8通道MUX电接点;

在相应的框中使用DPV测量以下参数:
个人开始:0(V),E端:0.6(V); E步骤:0.016(V); E脉冲:0.0339(V),E调理:0(V),E沉积:0(V),扫描速度:0.1(V / S),T脉冲:0.06(S),T空调:0(S),T沉积:0(S),T平衡:8(S)。

4)酶反应和电化学测量

  1. 均质通过轻轻晃动每个Eppendorf管中包含为了准备电化学测量步骤磁珠,获得了分散良好的悬浮磁珠;
  2. 移液器从每Eppendorf公司后,立即同质化,分装20μL到相应的工作电极表面的磁性珠分散。对于每一个地带使用的电极20μL磁珠取自不同的Eppendorf管(例如8个电极使用八个不同的Eppendorf);
  3. 第一电极上加入80μL的酶底物溶液。开始2分钟倒计时。 14秒后,加入80μl酶底物溶液的第二电极。另外14秒后,添加第三个电极上的酶底物溶液80μL。继续在同一时间INTerval(14秒),直到最后电极(这意味着:掉落每间隔14秒的基板等分)。 14秒的间隔时间的计算公式为电位进行测量所需的时间;注意,80μL每个传感器的解决方案应包括工作,参考电极。此外,每个传感器的解决方案不应接触到相邻电极的解决方案。
  4. 2分钟后计数的酶底物溶液此外,启动电化学测量;
  5. 使用PalmSens Lite软件获得的峰值电流(μA),为每个解决方案。

5)计算

使用后勤4个参数方程建立校准曲线,并计算在总HT-2/T-2毒素量浓度微克每毫升含有未知样品每个Eppendorf公司管。

实验数据,对校准物的浓度(ng / ml的)的峰高(μA),必须安装使用一种非线性的后勤(4 - PL)四参数方程图(1)(使用适马剧情8.0或Kaleidagraph)。通常采用4 - PL的非线性模型来形容的配体结合试验(的LBA)6

公式1 (1)
A,B,X0,Y0后勤参数Sigma的绘制曲线拟合程序。
使用阐释的公式来计算的稀释后的样品提取液HT-2/T-2毒素总量的内容
公式(2) (2)
x是在稀释后的提取液,后勤4个参数方程每milliltre纳克(ng / ml的)计算,总金额HT-2/T-2毒素的质量浓度
y是μA电流值未知样品获得

附录

6)包被的磁珠

继续作为包被的磁珠:

洗涤程序

  1. 均质tosylactivated磁珠® M - 280(2 × 109珠/毫升原液)摇晃和震荡(最大速度为1分钟(避免起泡)(请注意磁珠的浓度需求将始终是相同的,以确保可重复性在)测量);
  2. 立即到2 mL Eppendorf管吸管1毫升以上匀浆珠;
  3. 2分钟(见管侧壁上坚持磁珠)的磁性部分放置在磁粉浓缩管;
  4. 仔细移液器关闭上清,留下珠不受干扰;
  5. 从磁粉集中删除Eppendorf管中,重悬在1毫升0.1M的硼酸盐缓冲液pH值9.5的珠子。轻轻混匀,在2分钟旋转样品混合器2分钟;
  6. 移液器关闭上清;
  7. 吸取1毫升的硼酸缓冲液的Eppendorf管。磁珠现在准备涂层步骤;

涂层过程

  1. KLH(匙孔戚血蓝蛋白)原液1毫升磁珠)(HT - 2 - KLH最终浓度为375毫克/毫升)加入600毫升的HT - 2共轭;孵化磁珠和HT - 2 - KLH解决方案20H在37 ° C *缓慢倾斜旋转旋转样品混合器;
  2. 经过孵化的地方磁粉浓缩和移液器关闭上清管;
    用1毫升PBS / BSA缓冲液洗涂珠两次。彼此之间洗涤除去PBS / BSA溶液。设置为5分钟的旋转搅拌机磁珠和PBS / BSA的所有洗涤步骤进行;
  3. 使用1毫升的TRIS / BSA缓冲液洗一次磁珠。删除的TRIS / BSA溶液。开展这一步,通过磁珠的TRIS / BSA缓冲设置为4小时旋转搅拌机在37 ° C *;
  4. 磁珠洗一次使用1毫升刷新(在冰箱保存在4 ° C)的PBS / BSA缓冲液。删除PBS / BSA溶液。开展这一步骤设置磁珠和PBS / BSA缓冲液在室温5分钟旋转的搅拌机;
  5. 经过洗涤步骤删除所有的洗涤液,并添加1 ml PBS缓冲液中+为NaN 3 + BSA作为贮存液体;
  6. 商店包被的磁珠在4 ° C(请注意,包被的磁珠至少2个月稳定在4 ° C);

*为了这个目的,混频器是放置在烤箱配备了一个洞,允许插入电源电缆。或者混频器可放置于37℃在恒温室

7)样品制备和提取

25克磨细的样品(婴儿食品或早餐谷物),我们ighed成一个搅拌杯,用100毫升的乙腈水溶液(一十四分之八十六)/ 3分钟在高速搅拌器提取。在离心5分钟4000转(3000克),8毫升上清和Mycosep柱纯化后,4毫升清洗提取干下氮气流。干燥样品,可以储存在-30 ° C到几个月不等。

8)样品重组

早餐谷物

重组干早餐谷物提取物(谷物为基础的食品,对成人消费注定)与40毫升的PBS pH值7.4。这样等于所谓的法律限制(200纳克/克)的毒素浓度的样本将属于工作范围的中间一个信号,测量步骤。

婴儿食品

重组干的婴儿食品提取物(谷物为基础的食品,对婴幼儿消费注定)4毫升的PBS。这样等于所谓的法律限制(20-25毫微克/克)的毒素浓度的样本将属于工作范围的中间一个信号,测量步骤。

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Discussion

利用抗体作为生物分子识别探头已经出现了遥感技术的广泛使用;免疫组织化学检测方法,如酶联免疫吸附和MEIAs,时下许多实验室 7中最常用和应用平台之间, 。

虽然这些方法在过去几年中实现特殊的敏感性和特异性,许多研究小组的主要目标,已被其表演的改进和优化。

在这个协议中,我们已经展示了如何编造并询问霉菌毒素的检测的电化学免疫。我们相信,使用电化学的发展基于免疫传感器将被证明是在未来的地位日益重要。这是由于电化学检测的固有优势,在光学。电化学其实是不容易干扰,并已被证明对非特异性吸附一枝独秀,表现良好,即使在复杂的样品基质,如早餐麦片和婴儿食品的挑战。此外,电化学是更适合于小型化和多阵列适应。

耦合与磁性纳米粒子的这种做法也得到了改善分析的表演,并已被证明是特别适合在食物样本的污染物检测。

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Acknowledgements

作者想感谢他们的合作和后勤支持,罗马大学“的Tor Vergata”分析化学实验室南希唐纳和所有成员。这项工作是支持由欧盟项目“BioCop”。

Materials

Name Company Catalog Number Comments
Potassium chloride Sigma-Aldrich P9333
Potassium dihydrogen phosphate Sigma-Aldrich P9791
Disodium hydrogen phosphate Sigma-Aldrich S3264
Sodium chloride Sigma-Aldrich S3014
MgCl2 anhydrous Sigma-Aldrich M8266
DEA (99.5%) Sigma-Aldrich 31589
HCl 37% Sigma-Aldrich 320331
H3BO3 Sigma-Aldrich B6768
Tris[hydroxymethyl]-aminomethane Sigma-Aldrich 252859
BSA (albumin bovine serum) Sigma-Aldrich A4503
NaOH Sigma-Aldrich S5881
TWEEN 20 Sigma-Aldrich P9416
NaN3 Aldrich 71290
1-Naphthyl phosphate disodium salt Fluka N7255
Skimmed milk blocking solution - non fat dry milk Bio-Rad 170-6404
HT-2 conjugated with KLH (Keyhole Limpet Hemocyanin), stock solution (1 mg/ml in PBS): Biopure 004050 The HT-2-KLH conjugate was obtained by CDI-method where the free OH-groups on position 3 and 4 at the HT-2 toxin were activated by N,N'-carbonyldiimidazole (CDI) and the activated HT-2 toxin was let to react with aminogroups of the protein (KLH) to generate a stable carbammate linkage.
Secondary labeled antibody: Anti-mouse IgG (H+L) from horse, conjugated with Alkaline Phosphatase, concentration 1 mg/ml. Vector Laboratories AP-2000
HT-2 toxin Biopure
Magnetic beads: Dynabeads® Invitrogen M-280 Tosylactivated Concentration 2 x 109 beads/ml.
Phosphate buffered saline (PBS), pH 7.4 Dissolve 0.20 g of potassium chloride, 0.20 g of potassium dihydrogen phosphate, 1.16 g of disodium hydrogen phosphate and 8.00 g of sodium chloride in 900 ml of water
Diethanolamine buffer, DEA, 0.97 M + 1 mM MgCl2 + 0.15 M KCl, pH 9.8 Dissolve 0.0476g of MgCl2 anhydrous and 7.3.59 g of KCl in ~ 300 ml of water. After dissolution add 51 ml of DEA (99.5%). Adjust pH to 9.8 with HCl (6 M). Dilute to 500 ml with water.
Borate buffer, 0.1 M, pH 9.5 Dissolve 3.09 g of H3BO3 in ~ 300 ml of distilled water; adjust pH to 9.5 with NaOH and/or HCl (6 M or lower concentrations). Dilute to 500 ml with distilled water.
TRIS buffer, 0.2 M, pH 8.5 Dissolve 3.85 g of Tris[hydroxymethyl]-aminomethane in 100 ml of water. Adjust to pH 8.5 with NaOH and/or HCl (6 M or lower concentrations). Dilute to 200 ml with water.
TRIS buffer + BSA solution (0.1%), pH 8.4 Dissolve 0.050 g of BSA in 50 ml of TRIS buffer pH 8.4. This solution must be freshly prepared on the day of use.
PBS buffer + TWEEN® 0.05% Dissolve 0.250 g of TWEEN 20 in 500 ml of the previously prepared PBS buffer, pH 7.4
PBS buffer + BSA solution 0.1% Dissolve 0.050 g of BSA in 50 ml of PBS buffer, pH 7.3. This solution must be freshly prepared on the day of use.
PBS buffer + BSA 0.1% + NaN3 0.02% Dissolve 0.050 g of BSA and 0.010 of NaN3 in 50 ml of PBS buffer, pH 7.3. Storage solution
Enzymatic substrate Dissolve 0.010 g of 1-Naphthyl phosphate sodium salt in 100 ml of DEA buffer pH 9.8. Wrap the flask tightly in aluminium foil. This solution must be freshly prepared on the day of use.
Skimmed milk blocking solution Add 0.20 g of Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad, Hercules, CA, USA) to 200 ml of PBS buffer pH 7.3. This solution must be freshly prepared on the day of use.
HT-2 stock solution Dissolve 10 mg of trichothecene HT-2 toxin vial, in 10 ml of acetonitrile to give a solution with a concentration of 1 mg/ml. Split up this solution into single-use aliquots of 30 ml and store them at less than -30 °C.
HT-2 Working standard solution Pipette 20 ml of HT-2 toxin stock solution into a 2 ml calibrated volumetric flask and dilute with acetonitrile to obtain a HT-2 working solution containing 10 μg /ml of trichothecene toxin. This solution should be freshly prepared on the day of use.
HT-2 Working calibrant solutions Dilute the HT-2 working standard solution (10 μg/ml) to prepare working calibrant solution 100 ng/ml. Dilute HT-2 working calibrant solution 100 ng/ml to prepare working calibrant solutions of the following concentrations 0 (blank), 0.5, 1, 2, 4, 10 ng/ml. These solutions must be prepared fresh on the day of use.
HT-2 conjugated with KLH Split up HT-2 conjugated with KLH (Keyhole Limpet Hemocyanin) stock solution (1 mg/ml in PBS) into single-use aliquots of 350 μl. Store aliquots at -30 °C.
Specific Monoclonal Antibody Dilute 1:350 (v:v) the stock concentration (1.383 mg/ml) of monoclonal antibody in PBS to use in competition step. This solution must be prepared fresh at the moment of use. 10 ml antibody stock solution in PBS for a final volume of 3.5 ml, are enough to analyze 17 standards and /or samples.
Secondary Labeled Antibody Anti-mouse IgG (H+L) conjugated with Alkaline Phosphatase is diluted 1:100 (v:v) in PBS to use in competition step. 100 μl antibody stock solution in PBS for a final volume of 10 ml, are enough to analyze 25 standards and /or samples. This solution must be prepared fresh at the moment of use.
Magnetic Particle Concentrator: MPC®-S Invitrogen
PalmSens instrument PalmSens Provided with PalmSens Lite, Serial cable connecting PC laptop and Mux options software
CH8 PalmSens Multiplexer PalmSens
Eight channel Mux electric contact hand made
Strip with eight screen printed electrodes (SPEs) hand made
Specially designed support for electrodes strip hand made. Includes 8 neodymium magnets each of which is placed below each working electrode surface of the SPE
Calibrated microliter pipettes Gilson, Inc.
Magnetic stirrer and stir bars.
Glass beakers (100, 50, 20 ml).
Volumetric flasks (2ml).
0.2 and 2ml Eppendorf tubes. Eppendorf
Falcon tubes of 5ml and 15ml. Falcon BD
Laboratory Vortex Mixer Do not use vortex mixer to resuspend magnetic beads coated with HT2-KLH or linked with immunological chain to avoid denaturing of proteic parts
Laboratory oven or thermostated room Choose a oven able to keep a temperature of 37±3 °C.

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References

  1. Koch, P. State of the art of trichothecenes analysis. Toxicol. Letters. 153, 109-112 (2004).
  2. Krska, R., Baumgartner, S., Josephs, R. State of the art in the analysis of type-A and-B trichothecene mycotoxins in cereals. Fresenius J. Anal. Chem. 371, 285-299 (2001).
  3. Morozova, T. Y., Morozov, V. N. Force differentiation in recognition of cross-reactive antigens by magnetic beads. Anal. Biochem. 263-271 (2008).
  4. FAO Food and Nutrition Paper 74. JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES Fifty-sixth meeting. 2001 Feb 6-15, Geneva, Food & Agricultural Organization of the United Nations. 115 (2001).
  5. Piermarini, S., Volpe, G., Ricci, F., Micheli, L., Moscone, D., Palleschi, G., Fuhrer, M., Krska, R., Baumgartner, S. Rapid Screening Electrochemical Methods for Aflatoxin B1 and Type-A Trichothecenes: a preliminary study. Anal. Lett. 40, 1333-1346 (2007).
  6. Findlay, J. W. A., Dillard, R. F. Appropriate calibration curve fitting in ligand binding assays. AAPS J. 9, (2), E260-E267 (2007).
  7. Ricci, F., Volpe, G., Micheli, l, Palleschi, G. A review on novel developments and applications of immunosensors in food analysis. Anal. Chim. Acta. 605, (2), 111-129 (2007).

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