Authors would like to thank Nancy Downer and all members from the laboratory of Analytical Chemistry, University of Rome "Tor Vergata" for their collaboration and logistic support. This work was supported by the EU project "BioCop".

1) Blocking coated Magnetic Beads:
Proceed as follow to block Coated Magnetic beads:
<ol>
 <li>Prepare a set of 2 ml Eppendorf tubes;</li>
 <li>Homogenize (shaking but not vortexing) coated magnetic beads (see the appendix for preparation of coated magnetic beads) using the sample rotating mixer;</li>
 <li>Immediately after homogenization, pipette 10 &mu;l of coated magnetic beads into each separate Eppendorf tubes;</li>
 <li>Add 1ml of skimmed milk blocking solution into each Eppendorf tu...

<ol>
	<li>Koch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+of+trichothecenes+analysis.">State of the art of trichothecenes analysis.</a> Toxicol. Letters. 153, 109-112 (2004).</li><li>Krska, R., Baumgartner, S., Josephs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+in+the+analysis+of+type-A+and-B+trichothecene+mycotoxins+in+cereals.">State of the art in the analysis of type-A and-B trichothecene mycotoxins in cereals.</a> Fresenius J. Anal. Chem. 371, 285-299 (2001).</li><li>Morozova, T. Y., Morozov, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+differentiation+in+recognition+of+cross-reactive+antigens+by+magnetic+beads.">Force differentiation in recognition of cross-reactive antigens by magnetic beads.</a> Anal. Biochem. , 263-271 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAO+Food+and+Nutrition+Paper+74.">FAO Food and Nutrition Paper 74.</a> JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES Fifty-sixth meeting. , 115 (2001).</li><li>Piermarini, S., Volpe, G., Ricci, F., Micheli, L., Moscone, D., Palleschi, G., Fuhrer, M., Krska, R., Baumgartner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Screening+Electrochemical+Methods+for+Aflatoxin+B1+and+Type-A+Trichothecenes:+a+preliminary+study.">Rapid Screening Electrochemical Methods for Aflatoxin B1 and Type-A Trichothecenes: a preliminary study.</a> Anal. Lett. 40, 1333-1346 (2007).</li><li>Findlay, J. W. A., Dillard, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appropriate+calibration+curve+fitting+in+ligand+binding+assays.">Appropriate calibration curve fitting in ligand binding assays.</a> AAPS J. 9 (2), E260-E267 (2007).</li><li>Ricci, F., Volpe, G., Micheli, l., Palleschi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+novel+developments+and+applications+of+immunosensors+in+food+analysis.">A review on novel developments and applications of immunosensors in food analysis.</a> Anal. Chim. Acta. 605 (2), 111-129 (2007).</li></ol>

The use of antibodies as biomolecular recognition probe has seen a widespread use for sensing technologies; immunochemical detection methodologies, such as ELISAs and MEIAs, are, nowadays, among the most used and applied platforms in many laboratories7.
While these approaches achieve exceptional sensitivity and specificity, a primary objective of many research groups in the last years has been the improvement and optimization of their performances.
In t...

Reagents
<table border="1" cellspacing="0" cellpadding="0">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Sigma</td>
 <td>P9333</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium dihydrogen phosphate</td>
 <td>Sigma</td>
 <td>P9791</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disodium hydrogen phosphate</td>
 <td>Sigma</td>
 <td>S3264</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma</td>
 <td>S3014</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MgCl2 anhydrous</td>
 <td>Sigma</td>
 <td>M8266</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DEA (99.5%)</td>
 <td>Sigma</td>
 <td>31589</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HCl 37%</td>
 <td>Sigma</td>
 <td>320331</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>H3BO3</td>
 <td>Sigma</td>
 <td>B6768</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris[hydroxymethyl]-aminomethane</td>
 <td>Sigma</td>
 <td>252859</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BSA (albumin bovine serum)</td>
 <td>Sigma</td>
 <td>A4503</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>Sigma</td>
 <td>S5881</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TWEEN 20</td>
 <td>Sigma</td>
 <td>P9416</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaN3</td>
 <td>Aldrich</td>
 <td>71290</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1-Naphthyl phosphate disodium salt</td>
 <td>Fluka</td>
 <td>N7255</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Skimmed milk blocking solution - non fat dry milk</td>
 <td>Bio-Rad</td>
 <td>170-6404</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HT-2 conjugated with KLH (Keyhole Limpet Hemocyanin), stock solution (1 mg/ml in PBS):</td>
 <td>Biopure</td>
 <td>004050</td>
 <td>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.</td>
 </tr>
 <tr>
 <td>Secondary labeled antibody: Anti-mouse IgG (H+L) from horse, conjugated with Alkaline Phosphatase, concentration 1 mg/ml.</td>
 <td>Vector Laboratories</td>
 <td>AP-2000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HT-2 toxin</td>
 <td>Biopure</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnetic beads: Dynabeads&reg;</td>
 <td>Dynal</td>
 <td>M-280 Tosylactivated</td>
 <td>Concentration 2 x 109 beads/ml. </td>
 </tr>
</table>
Solutions
<table border="1" cellspacing="0" cellpadding="0">
 <tr>
 <td>Name of the reagent </td>
 <td>Preparation</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS), pH 7.4</td>
 <td>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</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Diethanolamine buffer, DEA, 0.97 M + 1 mM MgCl2 + 0.15 M KCl, pH 9.8</td>
 <td>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.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Borate buffer, 0.1 M, pH 9.5</td>
 <td>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.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TRIS buffer, 0.2 M, pH 8.5</td>
 <td>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.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TRIS buffer + BSA solution (0.1%), pH 8.4</td>
 <td>Dissolve 0.050 g of BSA in 50 ml of TRIS buffer pH 8.4.</td>
 <td>This solution must be freshly prepared on the day of use.</td>
 </tr>
 <tr>
 <td>PBS buffer + TWEEN&reg; 0.05%</td>
 <td>Dissolve 0.250 g of TWEEN 20 in 500 ml of the previously prepared PBS buffer, pH 7.4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS buffer + BSA solution 0.1%</td>
 <td>Dissolve 0.050 g of BSA in 50 ml of PBS buffer, pH 7.3.</td>
 <td>This solution must be freshly prepared on the day of use.</td>
 </tr>
 <tr>
 <td>PBS buffer + BSA 0.1% + NaN3 0.02%</td>
 <td>Dissolve 0.050 g of BSA and 0.010 of NaN3 in 50 ml of PBS buffer, pH 7.3.</td>
 <td>Storage solution</td>
 </tr>
 <tr>
 <td>Enzymatic substrate</td>
 <td>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.</td>
 <td>This solution must be freshly prepared on the day of use.</td>
 </tr>
 <tr>
 <td>Skimmed milk blocking solution</td>
 <td>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.</td>
 <td>This solution must be freshly prepared on the day of use.</td>
 </tr>
 <tr>
 <td>HT-2 stock solution</td>
 <td>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.</td>
 <td>Split up this solution into single-use aliquots of 30 ml and store them at less than -30 &deg;C.</td>
 </tr>
 <tr>
 <td>HT-2 Working standard solution</td>
 <td>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 &mu;g /ml of trichothecene toxin.</td>
 <td>This solution should be freshly prepared on the day of use.</td>
 </tr>
 <tr>
 <td>HT-2 Working calibrant solutions</td>
 <td>Dilute the HT-2 working standard solution (10 &mu;g/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.</td>
 <td>These solutions must be prepared fresh on the day of use.</td>
 </tr>
 <tr>
 <td>HT-2 conjugated with KLH</td>
 <td>Split up HT-2 conjugated with KLH (Keyhole Limpet Hemocyanin) stock solution (1 mg/ml in PBS) into single-use aliquots of 350 &mu;l.</td>
 <td>Store aliquots at -30 &deg;C.</td>
 </tr>
 <tr>
 <td>Specific Monoclonal Antibody</td>
 <td>Dilute 1:350 (v:v) the stock concentration (1.383 mg/ml) of monoclonal antibody in PBS to use in competition step</td>
 <td>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.</td>
 </tr>
 <tr>
 <td>Secondary Labeled Antibody</td>
 <td>Anti-mouse IgG (H+L) conjugated with Alkaline Phosphatase is diluted 1:100 (v:v) in PBS to use in competition step.</td>
 <td>100 &mu;l 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.</td>
 </tr>
</table>
Apparatus
<table border="1" cellspacing="0" cellpadding="0">
 <tr>
 <td>Name of the reagent </td>
 <td>Company</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Magnetic Particle Concentrator: MPC&reg;-S</td>
 <td>Dynal</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PalmSens instrument</td>
 <td>PalmSens</td>
 <td>Provided with PalmSens Lite, Serial cable connecting PC laptop and Mux options software</td>
 </tr>
 <tr>
 <td>CH8 PalmSens Multiplexer</td>
 <td>PalmSens</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Eight channel Mux electric contact</td>
 <td>hand made</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Strip with eight screen printed electrodes (SPEs)</td>
 <td>hand made</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Specially designed support for electrodes strip</td>
 <td>hand made</td>
 <td>Includes 8 neodymium magnets each of which is placed below each working electrode surface of the SPE</td>
 </tr>
 <tr>
 <td>Calibrated microliter pipettes</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnetic stirrer and stir bars.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass beakers (100, 50, 20 ml).</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Volumetric flasks (2ml).</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.2 and 2ml Eppendorf tubes.</td>
 <td>Eppendorf</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Falcon&trade; tubes of 5ml and 15ml.</td>
 <td>Falcon</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laboratory Vortex Mixer</td>
 <td>&nbsp;</td>
 <td>Do not use vortex mixer to resuspend magnetic beads coated with HT2-KLH or linked with immunological chain to avoid denaturing of proteic parts</td>
 </tr>
 <tr>
 <td>Laboratory oven or thermostated room</td>
 <td>&nbsp;</td>
 <td>Choose a oven able to keep a temperature of 37&plusmn;3 &deg;C.</td>
 </tr>
</table>

elime (enzyme linked immuno magnetic electrochemical) method for mycotoxin detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

Watch this Scientific Journal Video about ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection at JoVE.com

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

elime-enzyme-linked-immuno-magnetic-electrochemical-method-for

Research

JoVE Journal

Biology

14.1K vues.  University of Rome, Tor Vergata. Un protocole pour détecter les trichothécènes (mycotoxines de préoccupation pour la santé humaine) en utilisant une méthode de dépistage nouvellement développé basé sur une méthode immunochimique compétitifs et une détection électrochimique dernière est démontrée.