ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

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。

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^和 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： 准备一个2毫升Eppendorf管; 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; 紧随之后，同质化，移液器10μL到每个单独的Eppendorf管包被的磁珠; 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; 让我们为在室温下30分钟孵育旋转样品混合器磁珠; </l…

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?…

Materials

Reagents

Name of the reagent	Company	Catalogue number	Comments (optional)
Potassium chloride	Sigma	P9333
Potassium dihydrogen phosphate	Sigma	P9791
Disodium hydrogen phosphate	Sigma	S3264
Sodium chloride	Sigma	S3014
MgCl2 anhydrous	Sigma	M8266
DEA (99.5%)	Sigma	31589
HCl 37%	Sigma	320331
H3BO3	Sigma	B6768
Tris[hydroxymethyl]-aminomethane	Sigma	252859
BSA (albumin bovine serum)	Sigma	A4503
NaOH	Sigma	S5881
TWEEN 20	Sigma	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®	Dynal	M-280 Tosylactivated	Concentration 2 x 109 beads/ml.

Solutions

Name of the reagent	Preparation	Comments (optional)
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.

Apparatus

Name of the reagent	Company	Comments (optional)
Magnetic Particle Concentrator: MPC®-S	Dynal
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
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
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.