Furaltadone (AMOZ) ELISA Test Kit For Detection Honey Shrimp / Meat / Hepar

Furaltadone(AMOZ) ELISA Test Kit

Product Description

REAGEN^™Furaltadone(AMOZ) ELISA Test Kit provides a competitive enzyme immunoassay for the quantitative analysis of furaltadone in fish, shrimp, meat (chicken,beef,pork and hepar), eggs, hone.

• Rapid (4 hours), high recovery (80 - 105%), and cost-effective extraction methods for various samples.

• High sensitivity (0.05 ng/g or ppb) and low detection limit (0.1 ng/g or ppb) for various samples.

• High reproducibility.

• A quick ELISA assay (less than 1 hours regardless of number of samples).

Procedure Overview

The method is based on a competitive colorimetric ELISA assay. The AMOZ-BSA has been coated in the plate wells. During the analysis, sample and the AMOZ antibody are added along with secondary antibody, tagged with a peroxidase enzyme. If the AMOZ residue is present in the sample, it will compete for the AMOZ antibody, thereby preventing the antibody from binding to the AMOZ-BSA attached to the well. The resulting color intensity, after addition of the HRP substrate (TMB), has an inverse relationship with the AMOZ residue concentration in the sample.

Kit Contents, Storage and Shelf Life

REAGEN^™Furaltadone (AMOZ) ELISA Test Kit has the capacity for 96 determinations or testing of 42 samples in duplicate (assuming 12 wells for standards). Return any unused microwells to the foil bag and reseal them with the desiccant provided in the original package. Store the kit at 2-8°C*. The shelf life is 12 months when the kit is properly stored.

* If you are not planning to use the kit for over 1 month, store Antibody#1 and HRP-Conjugated Antibody #2 at -20°C or in a freezer.

Sensitivity (Detection Limit)

Specificity (Cross-Reactivity)

Required Materials Not Provided With the Kit

1.Microtiter plate reader (450 nm)

2.Incubator

3.Tissue Mixer (e.g. Omni TissueMaster Homogenizer)

4.Rotary evaporator or nitrogen gas

5.Vortex mixer (e.g. Gneie Vortex mixer from VWR)

6.10, 20, 100 and 1000 mL pipettes

7.Multi-channel pipette: 50-300 mL (Optional)

8.Ethyl acetate

9.0.1 M K2HPO4

10.n-Hexane (or n-Heptane)

11.1M NaOH

12.1M HCL

Warnings and Precautions

• The standards contain Furaltadone. Handle with particular care.

• Do not use the kit past the expiration date.

• Do not intermix reagents from different kits or lots except for components with the same part No’s within their expiration dates. ANTIBODIES AND PLATES ARE KIT- AND LOT-SPECIFIC.

• Try to maintain a laboratory temperature of 20°–25°C (68°–77°F). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Cold bench tops should be avoided by placing several layers of paper towel or some other insulation material under the assay plates during incubation.

• Make sure you are using only distilled or deionized water since water quality is very important.

• When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic.

• Incubations of assay plates should be timed as precisely as possible. Be consistent when adding standards to the assay plate. Add your standards first and then your samples.

• Add standards to plate only in the order from low concentration to high concentration as this will minimize the risk of compromising the standard curve.

• Always refrigerate plates in sealed bags with a desiccant to maintain stability. Prevent condensation from forming on plates by allowing them equilibrate to room temperature (20 – 25°C / 68 – 77°F) while in the packaging.

SAMPLE PREPARATION

Be sure samples are properly stored. In general, samples should be refrigerated at 2-4°C forno more than 1-2 days. Freeze samples to a minimum of -20°C if they need to be stored for a longer period. Frozen samples can be thawed at room temps (20 – 25°C / 68 – 77°F) or in a refrigerator before use.

Fish

• Mix 1 g of the homogenized sample with 0.5 mL of 1X Sample Extraction Buffer, 3.5 mL of distilled water, 0.5 mL of 1 M HCl and 20 mL of 50 mM 2-Nitrobenzaldehyde by vortexing for 30 seconds.

• Incubate at 50°C -55°C for 3 hours. Vortex the sample for 5 seconds every hour during the incubation.

• Add 5 mL of 0.1 M K2HPO4, 0.4 mL of 1 M NaOH and 6 mL of ethyl acetate, vortex for 2 minutes.

• Centrifuge at 4,000 x g for 5 minutes at room temperature (20 – 25°C).

• Transfer 3 mL of the ethyl acetate supernatant (corresponding to 0.5 g of the original sample) into a new vial (F Avoid the lower aqueous layer! If contaminated with lower layer, centrifuge the extracted ethyl acetate for 5 minutes at 4,000 x g again and get the upper organic layer).In case emulsion happened and the upper ethyl acetate layer was less than 3 mL,incubate the sample in water bath for 3 minutes at 85°C.Use a rotary evaporator to dry the sample in a 60-70°C water bath under reduced pressure. Alternatively,the sample can be dried by blowing nitrogen gas in a 60-70°C water bath.

• Dissolve the dried residue in 1 mL of n-hexane (or n-heptane).

• Add 1 mL of 1X Sample Extraction Buffer, vortex the sample for 2 minutes.

• Centrifuge the sample at 4,000 x g for 10 minutes at room temperature (20 – 25°C / 68 – 77°F).

• Use 50mL of the lower aqueous layer per well for the assay.

Note: Dilution factor: 2. To avoid high background, it is recommended that a solvent blank sample be prepared in parallel, starting with the reduction of 3 mL of ethyl acetate to dryness and continue with the rest extraction procedure. Subtract the result of the solvent control from the sample results.

Shrimp /Meat/Hepar

• Mix 1 g of the homogenized sample with 0.5 mL 1X Sample Extraction Buffer, 3.5 mL of distilled water, 0.5 mL of 1 M HCl and 20 mL of 50 mM 2-Nitrobenzaldehyde by vortexing for 30 seconds.

• Incubate at50°C -55°C for 3 hours. Vortex the sample for 5 seconds every hour during the incubation.

• Add 5 mL of 0.1 M K2HPO4, 0.4 mL of 1 M NaOH and 6 mL of ethyl acetate, vortex for 5 minutes.

• Centrifuge at 4,000 x g for 5 minutes at room temperature (20 – 25°C / 68 – 77°F).

• Transfer 3.0 mL of the ethyl acetate supernatant (corresponding to 0.5 g of the original egg sample) into a new vial (F Avoid the lower aqueous layer! If contaminated with lower layer, centrifuge the extracted ethyl acetate for 5 minutes at 4,000 x g again and get the upper organic layer). Use a rotary evaporator to dry the sample in a 60-70°C water bath under reduced pressure. Alternatively, the sample can be dried by blowing nitrogen gas in a 60-70°C water bath.

• Dissolve the dried residue in 1 mL of n-hexane (or n-heptane).

• Add 1 mL of 1X Sample Extraction Buffer and vortex the sample for 2 minutes.

• Centrifuge the sample at 4,000 x g for 10 minutes at room temperature (20 – 25°C / 68 – 77°F).

• Use 50 mL of the lower aqueous layer per well for the assay.

Note: Dilution factor: 2. To avoid high background, it is recommended that a solvent blank sample be prepared in parallel, starting with the reduction of 3 mL of ethyl acetate to dryness and continue with the rest procedure. Subtract the result of the solvent control from the sample results.

Egg

• Mix 1 g of the egg sample with 0.5 mL of 1X Sample Extraction Buffer, 3.5 mL of distilled water, 0.5 mL of 1 M HCl and 20 mL of 50 mM 2-Nitrobenzaldehyde by vortexing for 2 minutes.

• Incubate at 50°C -55°C for 3 hours. Vortex the sample for 5 seconds every hour during the incubation.

• Add 5mL of 0.1 M K2HPO4, 0.4 mL of 1 M NaOH and 6 mL of ethyl acetate, vortex 5 minutes at max speed.

• Centrifuge at 4,000 x g for 10 minutes at room temperature (20 – 25°C / 68 – 77°F).

• Transfer 3.0 mL of the ethyl acetate supernatant (corresponding to 0.5 g of the original honey sample) into a new vial (F Avoid the lower aqueous layer! If contaminated with lower layer, centrifuge the extracted ethyl acetate for 5 minutes at 4,000 x g again and get the upper organic layer). Use a rotary evaporator to dry the sample in a 60-70°C water bath under reduced pressure. Alternatively, the sample can be dried by blowing nitrogen gas in a 60-70°C water bath.

• Dissolve the dried residue in 1 mL of n-hexane (or n-heptane).

• Add 1 mL of 1X Sample Extraction Buffer and vortex for 2 minutes.

• Centrifuge the sample at 4,000 x g for 10 minutes at room temperature (20 – 25°C / 68 – 77°F).

• Use50 mL of the lower aqueous layer for the assay.

Note: Dilution factor: 2. (1) After evaporation of the ethyl acetate extract, the resulting residue is not completely dissolved. However, the recovery rate will not be compromised (>80%). (2) For this preparation, we recommend using standard 0.5 ng/g or ppb as the cut off value for positive samples since negative samples could show considerable background effects (in some cases between standards 0.05 ng/g and 0.15 ng/g).