Intended Use
The REAGEN™Malachite green plate kit is a competitive elisa for the quantitative analysis of Malachite green in aquatic products.
Assay Principles
The REAGEN™Malachite green plate kit is a competitive enzyme-labeled immunoassay. Malachite green is extracted from a sample by blending or shaking with extraction solvent. The diluted Malachite green sample extract and calibrators are pipetted into the test wells followed by malachite green antibody into the test wells to initiate the reaction. After this 30 minute incubation the wells are washed with laboratory grade water. Add Malachite green HRP conjugate for 30 minutes. Following a second wash after the incubation, a clear substrate is added to the wells and any bound enzyme conjugate causes the conversion to a blue color. After the 15 minute incubation, the reaction is stopped and the amount of color in each well is read . The color of the unknown samples is compared is compared to the color of the calibrators and the Malachite green concentration of the samples is derived.
Detection Linmit
Aquatic products: 0.5ppb
Materials Required But Not Provided
1. Laboratory quality distilled or deionized water.
2. Graduated cylinder,100ml or larger .
3. Glassware for sample extraction and extract collection.
4. Acetonitrile
5. MgSO4
6. Pipet with disposable tips capable of dispensing 50μl
7. Multi-channel pipet; 8 channel capable of dispensing 50 and 100 μL
8. paper towels or equivalent absorbent material
9. Microwell plate or strip reader with 450nm filter
10. Timer.
11. Vortex mixer.
Warnings and Precautions
1. Each reagent is optimized for use in Reagen Malachite green Plate Kit. Do not substitute reagents from any other manufacturer into the test kit. Do not combine reagents from other Reagen Malachite green Plate Kites with different Lot numbers.
2. . Dilution or adulteration of reagents or samples not called for in the procedure may result in inaccurate results.
3. Do not use reagents after expiration date.
4. Reagents should be brought to room temperature , 20-28℃(62-82℉)prior to use . Avoid prolonged (>24hours) storage at room temperature.
5. Malachite green is an antibiotic and should be treated with care.
6. The Stop Solution is 1N hydrochloric acid,Avoid contact with skin and mucous membranes. Immediately clean up any spills and wash area with copious amounts of water. If contact should occur. Immediately flush with copious amounts of water.
Preparation Reagents:
Reagent II: Dilute Reagent II 1:9 with 100% Acetonitrile(for example, 1 mL of Reagent II, diluted with 9 mL of Acetonitrile). Prepare fresh before each assay.
Sample Dilution Buffer: Mix 1 volume of 10X Sample Dilution Buffer with 9 volume of distilled water.
Wash Buffer: Mix 1 volume of 20X Wash Buffer with 19 volume of distilled water.
HRP-conjugate solution: The conjugate provided in the kit is 100X concentrate. Before each assay, the conjugate should be diluted into HRP conjugate diluent. For example mix 100 uL of 100X concentrate conjugate with 9.9 mL of HRP conjugate diluent to obtain the working conjugate.
Standard Preparation:
1. Take out six clean tube, mark of distinction“1.5, 0.5, 0.15, 0.05, 0.015, 0”
2. 1.5ppb Standard preparation: Remove 30 ul 100ppb to 1.97ml standard diluent in the tube, mixing.
3. 0.5ppb Standard preparation: Remove the 300 ul has Prepared of 1.5 ppb standard to 600 ul standard diluent in the tube,mixing
4. 0.15ppb Standard preparation: Remove the 300 ul has Prepared of 0.5 ppb standard to 700 ul standard diluent in the tube,mixing
5. 0.05ppb Standard preparation: Remove the 300 ul has Prepared of 0.15 ppb standard to 600 ul standard diluent in the tube,mixing
6. 0.015ppb Standard preparation: Remove the 300 ul has Prepared of 0.05 ppb
standard to 700 ul standard diluent in the tube,mixing
7. 0 ppb Standard preparation:Just use the standard diluent for negative.
Sample Preparation (Meat 1:10 Dilution)
1. Homogenize the sample with a suitable mixer.
2. Take 2.0 g of the homogenized sample and place into 15 mL tube.
3. Add 6.0 mL of acetonitrile, and add 1.0 g of anhydrous MgSO4, vortex (at max speed) for 1 minute.
4. Place the vials in shaker or rotator for 10 minutes.
5. Centrifuge the sample for 10 minutes at 4,000 rpm at room temperature.
6. Take 1.0 mL of supernatant to a new 2 mL tube containing 250 mg of Reagent A
7. Vortex vigorously for 1 minutes. Centrifuge the sample for 10 minutes at 4,000 rpm at room temperature.
8. Take 600 μL of supernatant to a 2 mL tube. Use an evaporator to dry the sample at 50°C under reduced pressure. Alternatively, the sample can be dried by blowing nitrogen gas in a 50-60°C water bath.
9. Add 150 μL of freshly prepared Reagent B, vortex for 1 minutes.
10. Centrifuge the sample for 30 seconds at 4,000 rpm, and leave the tube at room temperature for 15 minutes.
11. Add 1.35 mL of freshly prepared Sample Dilution Buffer, vortex for 10 seconds.
12. Use 75 μL of the extraction solution per well in the ELISA test.
Note: Dilution factor: 10 [2 g meat x 0.6/(6 ml of Acetonitrile + 2g of the sample)=0.15 g in 1.5 mL of the solution]