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Clenbuterol hydrochloride (clenbuterol) detection kit
This kit uses competitive enzyme-linked immunosorbent assay to quantitatively determine clenbuterol and other beta agonists in urine, muscle, liver, a
Product details
This kit uses competitive enzyme-linked immunosorbent assay to quantitatively determine clenbuterol and other beta agonists in urine, muscle, liver, and feed. The reagent kit includes 96 test wells, including standard tests and all reagents used for detection. If quantitative analysis is to be conducted, a microplate enzyme-linked immunosorbent assay (ELISA) reader is necessary.
summary
Clenbuterol (CLE) is an adrenal receptor agonist, also known as a nerve stimulant, used clinically to treat diseases such as bronchial asthma, chronic bronchitis, and emphysema. As a result, the drug can increase lean meat percentage, reduce fat deposition, and promote animal growth. It has been illegally used as a breeding promoter by some livestock farming companies. Its residual amount can cause symptoms such as muscle tremors, palpitations, nerve allergies, headaches, dizziness, nausea, vomiting, fever, shivering, etc., which are extremely harmful to consumers' health. It has been explicitly prohibited from being used in aquaculture. HPLC or GC-MS have been used as methods for detecting beta agonists, but their pre-treatment steps are expensive. The enzyme-linked immunosorbent assay kit can economically and rapidly detect CLE in urine, muscle, liver, and feed.
Measurement principle
The basis of measurement is antigen antibody reaction. The microporous plate is coated with rabbit antibodies targeting CLE. After the washing step, add CLE enzyme markers, standard or sample solutions. CLE competes with CLE enzyme markers for antibodies, and unconnected CLE enzyme markers are removed in the washing step. Add the enzyme reaction substrate (urea peroxide) and color reagent (tetramethylbenzidine) to the well and incubate. The combined enzyme marker converts colorless color reagent into a blue product. Add the reaction stop solution to change the color from blue to yellow. Measured at 450nm, the absorbed light intensity is inversely proportional to the concentration of CLE in the sample.
The provided reagents (stored at 2-8 ℃) should not be frozen
The reagents in each box are sufficient for 96 measurements (including standard analytical wells), and the materials in the box are as follows:
1 x 96 well plate (12 x 8 wells) coated with rabbit IgG antibody
6 x standard solution, (2ml) containing 0, 0.1, 0.3, 0.9, 2.7, 8.1ppb CLE aqueous solution
1 × CLE peroxidase marker concentrated solution
1 x enzyme reaction substrate (6ml); Containing urea peroxide
1 x color reagent (6ml); tetramethylbenzidine
1 x reaction stop solution (7ml); 1M sulfuric acid
1×PBS (10ml), Used for diluting concentrated solution of CLE peroxidase markers
1 x PBST concentrated solution (40ml), diluted with 800ml distilled water, used for plate washing
Sample processing
Sample processing method 1
Processing methods for tissue, meat samples (pure lean meat), and visceral samples:
1. Weigh 2 grams of homogenized sample and add 2 mL of solution 1 (colorless and odorless), stir or shake the sample to mix evenly.
2. Add 6 ml of solution 2 (light yellow, with a pungent odor), and it is recommended to stir the sample with clean sanitary chopsticks or bamboo sticks for 5 minutes; Or oscillate for 15 minutes to evenly distribute it in the solution.
Centrifuge at 3.3000g for 10 minutes.
4. Take 3ml of the supernatant and place it in a clean petri dish. Steam it dry at 60 ℃ in a constant temperature incubator for about 30 minutes; It can also be heated and evaporated directly in a water bath in a test tube.
5. Rinse and dissolve the residue with 1ml of solution 2 (colorless and odorless).
Centrifuge at 6300g for 10 minutes.
7. Take 50ul of the clear middle part and add the sample directly, Perform ELISA analysis.
Note: If the solution accidentally gets on the skin, please immediately wipe it with 75% medical alcohol cotton and rinse with water. Do not drink or splash into the eyes.
Sample Processing Method 2 (Faster)
1. Weigh 2 grams of homogenized sample and add 2 mL of solution 1 (colorless and odorless), stir or shake the sample to mix evenly.
2. Add 4mL of solution 2 (light yellow, with a pungent odor), vigorously shake, meat sample for about 2 minutes, tissue for about 6 minutes.
3. Centrifuge 3000g for 5 minutes.
4. Take 1 ml of the supernatant and place it in a clean petri dish. Steam it dry at a constant temperature of 70 ℃~80 ℃ (approximately 5 minutes). Or use a hair dryer, it only takes 3 minutes.
5. Rinse the residue with 0.5 ml of solution 3 (colorless and odorless), and wash all the white residue at the bottom into the solution.
6. Take 50ul of eluent for testing purposes.
Enzyme linked immunosorbent assay program
1. Precautions before measurement
a) Raise all reagents to room temperature of 20-25 ℃ before use
b) Immediately put all reagents back to 2-8 ℃ after use
c) Do not let the micropores dry during use
d) The repeatability in ELA analysis largely depends on the consistency of the washing process, and carefully following the recommended washing sequence is a key point in the ELA measurement program
e) During all constant temperature incubation processes, avoid exposure to light and cover the microplate with a lid
2. Enzyme markers and microporous Flat noodles
a) The CLE enzyme marker provided is a concentrated solution. Due to the poor stability of the diluted enzyme label, only the actual required amount of enzyme label was diluted 1:14 (1 μ l enzyme label solution plus 14 μ l PBS). Before extracting the concentrated solution, shake carefully and store the remaining at 2-8 ℃.
b) Take out the required number of microporous plates and frames from the microporous Flat noodles, seal the unused microporous plates with adhesive tape, put them into the original bag for resealing, and store them at 4 ℃ or -20 ℃ to avoid moisture.
3. The concentration of CLE standard solution provided by the standard solution is 0, 0.1, 0.3, 0.9, 2.7, 8.1ppb(ng/ml)
4. Measurement procedure (operated at room temperature of 25 ℃)
a)
Cut open the aluminum foil bag and insert enough hole strips for the standard and sample into the microporous holder. Conduct two parallel experiments on the standard and sample, and record their positions. Add 20 μ l of standard solution or processed sample into their respective micropores. If the microporous Flat noodles cannot be used up in one experiment, put it back into the original aluminum foil bag and seal it tightly. It is better to put another plastic self sealing bag and clamp it, and then store it at 4 ℃ or -20 ℃. Avoid moisture.
b) Add 100 μ l of diluted enzyme label to the bottom of the micropore and incubate at 25 ℃ for 30 minutes (recommended to use a constant temperature incubator).
c) Pour out the liquid from the hole, invert the microporous holder and pat it on absorbent paper (3 times per row) to ensure complete removal of the liquid from the hole. Use 250 μ l
Fill PBST into the hole, pour out the liquid from the micropores again, and repeat the operation twice. After washing the board, add the washing solution and wait for 2 minutes before pouring it out. It is recommended to use bottle washing and plate washing, which can speed up the speed and avoid the difference of OD value caused by the inconsistent incubation time between Flat noodles.
d) Mix the reaction substrate and colorimetric reagent in a 1:1 ratio, add 100 μ l to the micropores, mix thoroughly, and incubate in the dark at 25 ℃ for 15 minutes.
e) Add 50 μ l of reaction stop solution to the micropores, mix well, and measure the absorbance value at 450nm. The absorbance value must be read within 10 minutes after adding the stop solution.
result
The average absorbance value of the obtained standard and sample is divided by the absorbance value of the first standard (0 standard) and multiplied by 100. Therefore, 0 standard equals 100% and the absorbance value is given in percentage form.
Standard absorbance value (or sample)
-----------------------100=% absorbance value
0 standard absorbance value
The calculated standard value is plotted as a semi logarithmic coordinate system curve corresponding to CLE concentration (ng/g, or ng/ml), and the calibration curve should be linear within the range of 0.2-2ng/g (ppb). The concentration (ng/g, or ng/ml) corresponding to each sample can be read from the calibration curve.
sensitivity
The average detection limit of the clenbuterol assay kit is about 0.1ng/g (0.1ppb).
specificity
Cross reactivity with clenbuterol analogues:
Clenbuterol 100%
Brombuterol 110%
Cimbuterol 40%
Mapenterol 17%
Mabutrol 16%
Salbutamol 9%
Tulobuterol
2%
Terbutalin 2%
Lackadine<1%
Isoproterenol<1%
Adrenaline<1%
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