Melatonin Sulfate ELISA Kit Instructions
2021-06-05 10:02:54
Melatonin Sulfate ELISA Kit Instructions
(Germany IBL: RE54031)
1 , the scope of application
This kit can be used for in vitro quantitative detection of melatonin sulfate (also known as 6-hydroxy melatonin sulfate and 6-melatonin) in human urine.
2 , the preface
The pineal gland has been considered as a neuroendocrine sensor because of its important role in photoperiod phenomena. The hormones secreted by the pineal gland are: N-acetyl-5-methoxytryptamine and melatonin. Melatonin is generally synthesized from tryptophan. The concentration of plasma melatonin can be best at night. The fluctuating changes characteristic of melatonin can reflect some information about time changes, such as the length of the night. The secretion of melatonin is regulated by the nervous system. The sympathetic nervous system plays an important role in regulating melatonin secretion, which is mainly regulated by the release of norepinephrine. According to reports, changes in the mode and level of melatonin secretion are consistent with some physiological states of humans, such as: insomnia, jet lag, depression, stress, schizophrenia, hypothalamic amenorrhea, pregnancy, nervous appetite deficiency, certain The effects of cancer, immune system disorders and adolescent sexual maturity. Most circulating melatonin can be metabolized in the liver to 6-hydroxy melatonin, which later becomes 6-melatonin and is ruled out with urine. The concentration of 6-hydroxymelatonin sulfate in the urine is well correlated with the total level of melatonin in the blood.
3 , the principle of experiment
This ELISA kit utilizes the principles of competition law. An unknown amount of antigen in the sample and a certain amount of the enzyme-labeled antigen compete for binding to the antibody binding site coated in the microwell. The plate was washed after incubation to terminate the competitive reaction. After the addition of the substrate solution, the color intensity exhibited by the solution is inversely proportional to the amount of antigen in the sample. The experimental results of the sample can be obtained directly from the standard curve.
4 , storage and stability
The kit must be transported and stored at 2-8 ° C to avoid direct sunlight. Sample storage and stability and reagent preparation will be described in the relevant sections. If the coated sheet is sealed and stored in an environment of 2-8 ° C, the reagent is stable for the period of validity.
5 , sample collection and storage
Urine
The experiment must use naturally occurring urine, or use urine generated within 24 hours. The urine produced by the patient within 24 hours was collected and added to a container containing 10-15 ml of 6N HCl preservative. To calculate the results, determine the total volume of urine. Centrifuge the sample before starting the experiment. | |||
store | 2-8 ° C | 2-8 ° C | Avoid direct sunlight and avoid repeated freezing and thawing |
stability | 4 days | 15 years | |
6 , kit components
Quantity | mark | ingredient |
1×12×8 | MTP | The coated plate was detachable and the micropores were coated with anti-rabbit IgG (sheep, polyclonal). |
1×5ml | ANTISERUM | Melatonin sulfate antiserum, ready to use, contains: rabbit antiserum, Tris buffer and 0.01% thimerosal |
1 x 0.2 ml | ENZCONJ CONC | Enzyme-linked enzyme, 40-fold concentrated, containing peroxidase-labeled melatonin sulfate, Tris buffer, and 1% thimerosal. |
1×7×0.1ml | CAL AG | Standards AG, 0; 1.7; 5.2; 15.6; 46.7; 140; 420 ng/mL 0; 5.2; 15.9; 47.6; 142; 427; 1281 nmol/L. Ready to use, containing melatonin sulfate, Tris buffer and 0.01% thimerosal. |
1×2×0.1ml | CONTROL 1+2 | Quality control 1+2, ready to use, containing 0.02%; thimerosal. See the reagent bottle label for specific concentrations and allowable ranges. |
1×60ml | ASSAYBUF CONC | Detection buffer, red, ready to use, containing Tris buffer, BSA and 0.01% thimerosal. |
1×50ml | WASHBUF CONC | The washing solution was concentrated 20 times and contained phosphate buffer, Tween and 0.1% thimerosal. |
1 x 0.9 ml | TMB SUBS CONC | TMB substrate solution, 31 times concentrated, containing TMB, buffer and stabilizer. |
1×27ml | TMB buffer | TMB substrate buffer, stored in direct sunlight, contains H 2 O 2 , citrate buffer and stabilizer. |
1×12ml | TMB STOP | TMB stop solution, ready to use, 1 M H 2 SO 4 . |
3× | FOIL | Sticky metal plate |
7. Equipment required for the experiment but the kit does not provide
1) Pipette, volume: 10; 50; 100; 1000ul
2) Test tube (12 × 75mm)
3) Test tube rack
4) Oscillator (500rpm)
5) Vortex mixer (500 rpm)
6) 8-channel pipette with reservoir
7) Washing bottles, automatic or semi-automatic washing machine
8) Microplate reader (reference wavelength: 600-650nm)
9) distilled or deionized water
10) Absorbent paper, sampling tips and timer
8. Preparation instructions before the experiment
8.1 Preparation of lyophilized or concentrated ingredients
Dilution/dissolution | ingredient | Thinner | proportion | Remarks | store | stability | |
15ml | detergent | 300ml | Double distilled water | 1:20 | Dissolve the crystal at 18-25 ° C | 2-8 ° C | 4 weeks |
50ul | Enzyme complex | 2ml | Slow detection Flush | 1:41 | Temporary configuration, can only be used once | 18-25 ° C | 30min |
300ul | TMB substrate liquid | 9ml | TMB substrate Buffer | 1:31 | Temporary configuration, can only be used once | 18-25 ° C | 10min |
8.2 dilution of standards, controls and patient urine samples
1 | Add 10 ul of standard, control, and patient urine samples to polystyrene or polypropylene or glass tubes to avoid direct sunlight. |
2 | Add 500 ul of detection buffer to each tube and vortex to mix. |
Samples with melatonin sulfate concentrations above the highest standard concentration must be further diluted with assay buffer.
9 , experimental steps
1 | 50 ul of the diluted standard, control, and patient samples were added to the corresponding microwells. |
2 | 50 ul of temporarily configured enzyme conjugate was added to each well. |
3 | 50 ul of melatonin sulfate antiserum was added to each well. |
4 | The cover was incubated for 2 hours at room temperature (18-25 ° C) coated plate shaker (500 rpm). |
5 | Prepare the TMB substrate solution 10 minutes before the end of the incubation period. |
6 | Remove the viscous metal plate, discard the reaction solution in the well, wash the plate 4 times with 250 ul of diluted washing solution per well, and pat dry on the absorbent paper to remove the residual liquid. |
7 | If possible, use an 8-channel pipette to add substrate and stop solution. The time interval between the addition of the substrate solution and the stop solution should be the same, using |
8 | 200 ul of TMB substrate solution was added to each well. |
9 | Incubate for 30 min at room temperature (18-25 ° C) coated plate shaker (500 rpm). |
10 | Add 100 ul of TMB stop solution to each well and gently shake the plate to mix the solution. |
11 | The OD value was read at 450 nm within 60 min after the addition of the stop solution. |
10 , expected value
The results of the experiment cannot be considered as the sole factor in determining the outcome of the treatment, and the judgment of the disease should be combined with other clinical observations and diagnostic tests. The following are the normal values ​​of melatonin sulfate in the apparent normal population:
age | number | Melatonin sulfate | |||
24-hour urine (ug) | Nocturnal urine (ug/h) | ||||
average value | 90% of the proportion | average value | 90% of the proportion | ||
20-35 | 26 | 36.8 | 15.6-58.1 | 2.8 | 0.9-5.6 |
36-50 | 17 | 29.6 | 9.9-52.9 | 2.1 | 0.6-3.6 |
51-65 | 16 | 20.4 | 12.3-32.8 | 1.5 | 0.9-2.5 |
> | 16 | 15.8 | 7.5-32.7 | 1.0 | 0.3-2.3 |
It is recommended that each laboratory read to determine its normal range of values.
This translation is for reference only, please refer to the original for details.
Exclusive distributor in China: Shenzhen Kerunda Bioengineering Co., Ltd.
Address: 6th Floor, No. 10, Yanshan Road, Shekou, Shenzhen
Telephone, 26680196 Postcode: 518067
Free call fax
With the improvement of living standards, snack foods have been deeply loved by the masses of the people. When you walk into the supermarket, you will see snacks such as preserved fruit, sour horns, potato chips, almonds, pistachios, fish fillets, dried meat, and spiced fried meat. Snack foods are gradually upgrading to become the daily necessities of the people. As the economy develops and the level of consumption increases, consumers' demand for the quantity and quality of snack foods continues to grow.
The whole body of the duck is not only high in nutritional value and delicious, but also the various parts of the duck are also very delicious. The nutritional value of the snack is very high, such as duck's paw, duck, duck neck, duck tongue, duck gizzard, etc., delicious coveted, aftertaste It is a casual food for all ages.
Duck Feet,Duck Wings,Chickens Leg,Chickens Wings Snacks
Hangzhou Aiyomi food co.,LTD , https://www.aiyomisnacks.com