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 tube of the set;</li>
 <li>Let magnetic beads incubate for 30 min at room temperature on the rotating sample mixer;</li>
 <li>Remove blocking solution: to do so place the tube in magnetic particle concentrator with the magnetic part put on for 2 min (see magnetic beads sticking on the lateral wall of the tube); carefully pipette off the supernatant, leaving beads undisturbed; </li>
 <li>Add 1 ml of PBS buffer + NaN3 + BSA as storage solution for each tube; </li>
 <li>Store coated and blocked magnetic beads at 4&deg;C (please note that coated magnetic beads are stable for at least 2 months at 4&deg;C); </li>
</ol>
2) Immunological chain on magnetic beads for construction of the calibration curve and sample analysis
Proceed as follow to have magnetic beads ready for the measurement:
<ol>
 <li>Take one Eppendorf tube (2 ml) of coated and blocked solution for each sample extract you are going to analyze;</li>
 <li>Take an extra Eppendorf tube for each working calibrant solution;</li>
 <li>Homogenize magnetic beads for 2 min on the rotating sample mixer before use;</li>
 <li>Remove storage liquid using magnetic particle concentrator; </li>
 <li>Wash magnetic beads three times with PBS/BSA washing solution. Remove washing liquid; </li>
 <li>Competition step: Add for each tube containing the washed magnetic beads, 200&mu;l of sample extract prepared; use one magnetic bead tube for each sample. Repeat the same procedure for each standard solution. Add 200 &mu;l of monoclonal antibody solution to each magnetic bead tube. Let magnetic beads incubate for half an hour on the rotating sample mixer at room temperature; </li>
 <li>Remove from Eppendorf tubes the solution used for the competition-step;</li>
 <li>Labeling step: Add 400&mu;l of labeled secondary antibody to each magnetic bead Eppendorf tube. Let magnetic beads incubate for half an hour on the rotating sample mixer at room temperature;</li>
 <li>Remove labeling solution from Eppendorf tubes; </li>
 <li>Wash magnetic beads three times with PBS/TWEEN&reg; 20 washing solution. Remove the liquid; </li>
 <li>Resuspend with 100&mu;l of DEA buffer each aliquot of magnetic beads. Now the magnetic beads are ready for electrochemical measurement step;</li>
</ol>
3) Assembly of PalmSens with Multiplexer (Mux) options
Please note: enzymatic product of substrate hydrolysis fouls the electrode surface; for this reason a new electrode is needed for each measurement. 
<ol>
 <li>Connect a PC laptop to PalmSens through the serial cable; </li>
 <li>Connect the 9-DIN plug of CH8 Multiplexer with PalmSens.</li>
 <li>Connect serial cable of Eight channel Mux electric contact with CH8 Multiplexer;</li>
 <li>Place electrode strip on the eight magnet block. Pay attention to place each magnet under each working electrodes (of note, the need of a single magnet for each electrode is imperative otherwise the magnetic particle will not be concentrated on the working electrode area);</li>
 <li>Plug in electrodes strip into eight channel Mux electric contact;</li>
</ol>
For DPV measurement use the following parameters in the appropriate boxes: 
 E begin: 0 (V);E end: 0.6 (V); E step: 0.016 (V); E pulse: 0.0339 (V); E conditioning: 0 (V); E deposition: 0 (V); Scan rate: 0.1 (V/s); T pulse: 0.06 (s); T conditioning: 0 (s); T deposition: 0 (s); T equilibration: 8 (s).
4) Enzymatic reaction and Electrochemical Measurement
<ol>
 <li>Homogenize by gently shaking each Eppendorf tube containing magnetic beads ready for electrochemical measurement step in order to obtain a well dispersed suspension of magnetic beads;</li>
 <li>Pipette from each Eppendorf, immediately after homogenization, an aliquot of 20 &mu;l of the magnetic bead dispersion onto the corresponding working electrode surface. For each electrode of one strip use 20 &mu;l of magnetic beads taken from different Eppendorf tubes (e.g. for eight electrode use eight different Eppendorf); </li>
 <li>Add 80 &mu;l of enzymatic substrate solution on the first electrode. Start 2 min count down. After 14 sec, add 80 &mu;l of enzymatic substrate solution on the second electrode. After further 14 sec, add 80 &mu;l of enzymatic substrate solution on the third electrode. Proceed at the same time interval (14 seconds) till the last electrode (that means: drop each aliquot of substrate with intervals of 14 sec). The interval time of 14 sec is calculated as the time required by the potentiostat to perform the measurement; of note, the 80 &mu;l solution on each sensor should cover the working, reference and counter electrode. Moreover, the solutions for each sensor should not come into contact with solutions of neighboring electrodes. </li>
 <li>After the 2 min count down from the first addition of enzymatic substrate solution, start the electrochemical measurement;</li>
 <li>Use the PalmSens Lite software to obtain current peaks (&mu;A) for each solution. </li>
</ol>
5) Calculation
Establish a calibration curve using a logistic 4 parameters equation and calculate the concentration of total HT-2/T-2 toxin amount in nanograms per millilitre for each Eppendorf tube containing unknown sample.
The experimental data, peak height (&mu;A) against the concentration of the calibrants (ng/ml), have to be fitted using a non-linear four-parameter logistic (4-PL) equation plot (1) (using Sigma Plot 8.0 or Kaleidagraph). The nonlinear 4-PL model is usually adopted to describe ligand binding assays(LBAs) 6.
<img src="/files/ftp_upload/1588/1588eq1.jpg" alt="Equation 1" />(1) 
 a, b, x0, y0 are logistic parameters as given by the Sigma Plot curve fit programme. 
 Use the explicated formula to calculate the content of HT-2/T-2 toxin total amount in the diluted sample extract solution 
 <img src="/files/ftp_upload/1588/1588eq2.jpg" alt="Equation 2" />(2) 
 x is the mass concentration of the total amount of HT-2/T-2 toxins in the diluted extract solution, calculated from the logistic 4 parameters equation in nanogram per milliltre (ng/ml) 
 y is the current value in &mu;A obtained for the unknown sample
 Appendix
 6) Coated Magnetic Beads
 Proceed as follow to have Coated Magnetic beads:
 Washing procedure
<ol>
 <li>Homogenize tosylactivated Dynabeads&reg; M-280 (stock solution 2 x 109 beads/ml) by shaking and vortexing (max speed) for 1 min (avoid foaming) (please note the concentration of magnetic beads needs to be always the same to ensure reproducibility in the measurement);</li>
 <li>Immediately pipette 1 ml of the above homogenized beads into a 2 ml Eppendorf tube;</li>
 <li>Place the tube in magnetic particle concentrator with the magnetic part put on for 2 min (see magnetic beads sticking on the lateral wall of the tube);</li>
 <li>Carefully pipette off the supernatant, leaving beads undisturbed; </li>
 <li>Remove the Eppendorf tube from the magnetic particle concentrator and resuspend the beads in 1 ml of 0.1 M borate buffer pH 9.5. Mix gently for 2 min in the rotating sample mixer for 2 min; </li>
<li>Pipette off the supernatant;</li>
<li>Pipette 1 ml of borate buffer solution into the Eppendorf tube. Magnetic beads are now ready for coating step;</li>
</ol>
Coating procedure
<ol start="8">
<li>Add 600 ml of HT-2 conjugated with KLH (Keyhole Limpet Hemocyanin) stock solution to 1 ml of magnetic beads) (final HT-2-KLH concentration of 375 mg/ml); incubate magnetic beads and HT-2-KLH solution for 20h at 37&deg;C* with slow tilt rotation on rotating sample mixer;</li>
<li>After incubation place the tube in the magnetic particle concentrator and pipette off the supernatant; 
 Wash coated beads two times with 1 ml of PBS/BSA buffer solution. Between each washing remove PBS/BSA solution. All washing step were performed setting magnetic beads and PBS/BSA on rotating mixer for 5 min;</li>
<li>Wash magnetic beads once using 1 ml of TRIS/BSA buffer solution. Remove TRIS/BSA solution. Carry out this step by setting magnetic beads and TRIS/BSA buffer on rotating mixer for 4 h at 37&deg;C*;</li>
<li>Wash magnetic beads once using 1ml of refreshed (kept in the fridge at 4&deg;C) PBS/BSA buffer solution. Remove PBS/BSA solution. Carry out this step setting magnetic beads and PBS/BSA buffer on rotating mixer for 5min at room temperature;</li>
<li>After washing step remove all washing liquid and add 1 ml PBS buffer + NaN3 + BSA as storage liquid;</li>
<li>Store coated magnetic beads at 4&deg;C (please note that coated magnetic beads are stable for at least 2 months at 4&deg;C);</li>
</ol>
*For this purpose the mixer is placed in oven equipped with a hole which allows the insertion of the cable for the power supply. Alternatively the mixer could be placed in a thermostated room at 37&deg;C.
7) Sample preparation and extraction
25 g of finely ground sample (baby food or breakfast cereals) are weighed into a blender jar and extracted with 100 ml of acetonitrile/water solution (86/14) for 3 min in a high-speed blender. After centrifugation at 4000 rpm (3000 g) for 5 min, 8 ml of supernatant were taken and purified with Mycosep column, 4 ml of the cleaned extract were dried under nitrogen flow. Dried samples can be stored at -30 &deg;C up to several months.
8) Sample reconstitution
Breakfast cereals
Reconstitute the dried breakfast cereal extract (cereal based food destined for adult consumption) with 40 ml of PBS pH 7.4. In this way a sample with a concentration of toxin equal to the supposed legal limit (200 ng/g) will give in the measurement step a signal falling in the middle of the working range.
Baby food
Reconstitute the dried baby food extract (cereal based food destined for infant consumption) with 4 ml of PBS. In this way a sample with a concentration of toxins equal to the supposed legal limit (20-25 ng/g) will give in the measurement step a signal falling in the middle of the working range.

<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 Views.  University of Rome, Tor Vergata. 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. 

Video: ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.