Malachite Green Test Kit Instructions

Read the instructions before use (Chinese name is for reference only, please refer to the English version)
This kit is suitable for the quantitative detection of malachite green in aquatic products.
Principle The kit principle is a competitive enzyme-linked immunosorbent assay, which extracts malachite green from the sample using an extractant. Add standard and treated samples to the solid-phase carrier microplate, then add malachite green antibody and react for 30 minutes. Wash the wells, then add HRP-labeled malachite green for 30 minutes, wash the plate, add the coloring agent after washing, and combine the enzyme to convert the colorless developer into a blue product; add the reaction stop solution to make the color Blue turns yellow; at a wavelength of 450 nm, the concentration of malachite green in the sample is inversely proportional to the intensity of the absorbed light.
Cross-reactivity with specificity of malachite green analogues
Compound %
CR
Malachite green Malachite green
100%
Leucomalachite leuco malachite green
1%
Crystal Violet Crystal Violet
120%
Leucocrystal Violet leuco crystal violet
2%
Detecting water-restricted products: 0.5 ppb
Kit composition
96-well microtiter plate: 1 block (8 × 12 strips). Standard stock solution (Standard): 1 bottle (0.4ml / bottle), 100ppb.
Enzyme label: 1 bottle (25 times, 0.8 ml) HRP-labeled malachite green conjugate malachite green antibody: 1 bottle (9 ml)
Substrate coloring solution: 1 bottle (16ml)
Stop solution: 1 bottle (12 ml, 1 N HCl).
Conjugate dilution: 1 bottle (15 ml).
Sample Dilution Buffer: 2 bottles (25ml)
Concentrated washing solution: 1 bottle (5 times, 100ml / bottle), washing of the enzyme plate.
Instruction Manual Be careful not to use the Malachite Green Test Kit with the valid date. The contents of the reagents in different batches and different kits cannot be mixed. Do not use contaminated and diluted reagents. Allow all reagents to reach room temperature (20 ° C - 28 ° C) before use, but do not exceed 24 hours. Malachite green is an antibiotic should be treated with care. Stop the solution with 1M hydrochloric acid to avoid contact with the skin. If it is accidentally dripped onto the skin, wash it immediately with water. The lotion is 5 times and is diluted when used.

Also need equipment 
Deionized water or re-distilled water 
100ml or larger cylinder 
Glassware for sample extraction and collection 
Methanol 
25, 50, 100 and 250 μL single or multichannel pipettes 
Timer 
Vortex mixer 
Washing bottles 
Absorbent paper 
Centrifuge 
Sample preparation for the microplate reader of 450nm filter (meat 1:20 dilution)
1 Fish and shrimp are peeled and meat is taken. The homogenizer is used to homogenize the sample. Weigh 5g of homogenous sample into the centrifuge tube. 2 Add 10 mL of acetonitrile, and shake the oscillator for 30 minutes.
3 4000g centrifugation for 10 minutes
4 Pour off the supernatant and transfer to another centrifuge tube.
5 Activate the alumina column with 3 ml of acetonitrile (1-2 drops/sec).
6 Take 3ml of sample through the column.
7 Collect the sample in a clean tube.
8 Add 3 ml of acetonitrile to wash the column and collect the tube in step 7 above.
9 Dilute the eluate (50 ul + 450 ul) using a 1:10 dilution of the sample dilution and set aside.
The second pretreatment method: (1:10 dilution)
1. After the sample is homogenized, weigh 4g into the centrifuge tube;
2. Add 5ml 0.2MHCL, mix well for 5min; centrifuge at room temperature 2000g for 5min
3. Pipette 2.5 ml of the supernatant, add 5 ml of dichloromethane, and shake well for 2 minutes;
4. Centrifuge at room temperature 2000g for 5min to remove the upper layer of liquid;
5. Transfer 2.5 ml of the lower liquid to another glass test tube and evaporate at 50-60 ° C;
6. The residue is dissolved in 2 ml of acetonitrile and shaken;
7. Dilute the mixed eluate 10 times with the sample diluent (50 ul acetonitrile extract + 450 ul sample dilution);
8. Pipette 75 ul for analysis.
The enzyme cross-linking kit provided by the enzyme cross-linking preparation kit is 25 times and is diluted before each use.
Preparation of standard dilutions Mixing acetonitrile and sample dilutions at a ratio of 1:9 using standard preparation standard concentrations (ppb)
Standard dilution (ml)
Standard
1
4
40 uL
0.5
1
1.0ml 1ppb standard solution
0.1
1.8
0.2ml 1ppb standard solution
0.05
1
1ml 1ppb standard solution
0.025
0.5
0.5ml 0.05ppb standard solution
0.01
0.8
0.2ml 0.05ppb standard solution
0
1.000
0
Steps
1. Remove the kit and sample preparation from the refrigerator before use to reach room temperature. Wash the bottle with the lotion.
2. Remove the required number of microporous strips from the foil pouch, place the desiccant and reseal the pouch to protect the microporous strip from moisture.
3. Pipette 75 μl of the sample preparation into the corresponding test well and add 75 μl of antibody solution to each well.
4. Gently shake for 30 seconds and mix for 30 minutes.
5. After the incubation is complete, pour the solution from the microwell into the sink and rinse the wells with the appropriate wash buffer. Add at least 250 ul per well at a time, pour off after micro-shock washing, repeat three times, and wash the plate four times in total. Clapper, remove the residual lotion.
6. Add 150 μL of enzyme-labeled conjugate to each well.
7. Gently shake for 30 seconds to mix and incubate for 30 minutes.
8. After the incubation is complete, pour the solution from the microwell into the sink and rinse the wells with the appropriate wash buffer. Add at least 250 ul per well at a time, pour off after micro-shock washing, repeat three times, and wash the plate four times in total. Clapper, remove the residual lotion.
9. Add 150 μL of substrate to each well. Incubate for 30 minutes.
10. Add 100 μL of Stop Solution to each well.
11. Read the absorbance value (OD) of each well at 450 nm
Result analysis
1.
The determination of the semi-quantitative result can be determined according to the absorbance value of the sample and the standard absorbance value. The absorbance value of the sample is lower than the absorbance value of a certain standard, indicating that the malachite green content of the sample is greater than the standard concentration, and the absorbance of the sample. A value above the absorbance of a standard indicates that the malachite green content of the sample is less than this standard.
2.
For the quantitative result determination, the standard absorbance value is required to be the X-axis, the logarithm of the standard concentration is plotted on the Y-axis, and the point of the standard concentration absorbance value is connected by a straight line, and the absorbance value of the sample is also located on the straight line, according to the sample. The absorbance value of the sample can be found on the Y-axis. If the absorbance value of the sample is greater than the minimum standard absorbance value or less than the maximum standard absorbance value, the content of malachite green in the sample is less than 0.01 ppb or greater. 1ppb.
3. When the concentration of the sample is higher than 1 ppb, adjust the dilution factor to obtain the final concentration.

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