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

Summary

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.

Abstract

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC^{1, 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.

Protocol

1) Blocking coated Magnetic Beads:

Proceed as follow to block Coated Magnetic beads:

1. Prepare a set of 2 ml Eppendorf tubes;
2. Homogenize (shaking but not vortexing) coated magnetic beads (see the appendix for preparation of coated magnetic beads) using the sample rotating mixer;
3. Immediately after homogenization, pipette 10 μl of coated magnetic beads into each separate Eppendorf tubes;
4. Add 1ml of skimmed milk blocking solution into each Eppendorf tube of the set;
5. Let magnetic beads incubate for 30 min at room temperature on the rotating sample mixer;
6. Remove blocking solution: t....

Discussion

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.......

Materials

List of materials used in this article

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.