Melatonin ELISA Kit Instructions
(Germany IBL: RE54021)
1 , the scope of application
This kit can be used for in vitro quantitative detection of melatonin in human serum and plasma.
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 reached 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. Biotin and non-biotinylated antigens competitively bind to a limited antibody binding site on the plate. The amount of biotin antigen bound to the antibody is inversely proportional to the amount of antigen in the sample. After the system reaches equilibrium, the plate is washed to remove free biotin antigen, and the biotin antigen bound to the antibody can be detected with an avidin alkaline phosphatase marker and a p-nitrophenyl phosphate substrate. A standard curve is made using known standard data, and the amount of the unknown sample can be quantified by comparing the enzyme activity of the unknown sample with a 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 section. If the coated board is sealed and stored in an environment of 2-8 ° C, the reagent will be stable during the validity period. The extraction column eluted with methanol can be used for extraction or storage of other samples at 2-8 °C. The extraction column can be reused 4 times.
5 , sample collection and storage
Serum, plasma (EDTA, heparin)
Samples are collected according to routine precautions for venipuncture, and blood samples must be chemically intact from collection to testing. Do not use significant hemolysis, jaundice, and lipemia samples. The turbid sample should be centrifuged before the start of the experiment to remove all particulate matter. |
store | 2-8 ° C | ≤-20°C | ≤-70°C | Avoid direct sunlight and avoid repeated freezing and thawing. |
stability | 24 hours | 3 months | 1 year |
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). |
3×2ml | BIOTIN LYO | Melatonin biotin, lyophilized, contains stabilizers. |
3×2ml | ANTISERUM LYO | Melatonin sulfate antiserum, lyophilized powder containing antiserum (rabbit, polyclonal) and stabilizer. |
1×250ul | ENZCONJ CONC | Enzyme conjugate, 80-fold concentrated, containing alkaline phosphatase-labeled avidin antibody (sheep), Tris buffer and stabilizer. |
1×6×2ml | CAL AF LYO | Standard AF, lyophilized powder, containing stabilizer, please refer to the reagent bottle label or QC control product for extraction concentration. |
1×2×2ml | CONTROL 1+2 LYO | Quality control 1+2, lyophilized powder, containing stabilizer, concentration/acceptable range, please refer to the label of the reagent bottle. |
1×50ml | ASSAYBUF CONC | The assay buffer was concentrated 10 times and contained phosphate buffer, Tween and stabilizer. |
1×9× | PNPP SUBS | The PNPP substrate is sealed in an aluminum-platinum metal bag containing p-nitrophenyl sulfate. |
1×27ml | PNPP BUF | PNPP substrate buffer, ready to use, contains diethanolamine and water. |
1×15ml | PNPP STOP | The PNPP stop solution, ready to use, contains 1 M NaOH and 0.25 M EDTA. |
2×10 | EXTRCOL | Extraction column, ready to use, C18 RP, 1 m 3 /100 mg. Another column to be extracted is available from IBL (number: KEME761) |
3× | FOIL | Sticky metal plate |
7. Equipment required for the experiment but the kit does not provide
1) Pipette, volume: 50; 500ul
2) Test tube (12 × 75mm)
3) Orbital oscillator (400-600rpm)
4) Vortex mixer
5) 8-channel pipette with reservoir
6) Washing bottle, automatic or semi-automatic washing machine
7) Centrifuge (referred centrifuge is more suitable): 200-500 × g, or multi-head vacuuming device (for example: Mallinekrodt-Baker or Waters).
8) Methanol (HPLC grade)
9) Evaporation centrifuge
10) Microplate reader that can read at 405nm (reference wavelength 600-650nm)
11) distilled or deionized water
12) Absorbent paper, sampling tip and timer
8. Preparation instructions before the experiment
note | The kit for the 96-color detection kit can be divided into 3 times. The volume described below is the amount required to detect 32 servings. |
8.1 Preparation of lyophilized or concentrated ingredients
Dilution/dissolution | ingredient | | Thinner | proportion | Remarks | store | stability |
15ml | Detection buffer | 150ml | Double distilled water | 1:10 | | 2-8 ° C | 4 weeks |
| Standards and controls | 2.0ml | Double distilled water | | Allow to stand for 15 min, avoid bubbles when mixing | ≤-20°C | Until the validity period |
| Melatonin biotin | 2.0ml | Distilled water | | Allow to stand for 15 min, avoid bubbles when mixing | Temporary configuration, can only be used once |
| Melatonin antiserum | 2.0ml | Distilled water | | Allow to stand for 15 min, avoid bubbles when mixing |
70ul | Enzyme complex | 5.6ml | Diluted assay buffer | 1:81 | |
3 | PNPP substrate | 8ml | PNPP substrate buffer | | |
10ml | Undiluted methanol | 100ml | Distilled water | 10% (v/v) | |
If the amount of solution required is large, the solution in the tube should be mixed to avoid repeated freezing and thawing.
8.2 dilution of the sample
If the melatonin concentration in the sample is suspected to be higher than the highest standard, the sample must be further diluted with the assay buffer prior to the extraction step.
8.3 Extraction of samples, standards and controls (extraction column)
According to this step, the extraction amount is about 90-100%.
To avoid clogging of the column, the sample is filtered or centrifuged before the sample is taken.
note | Each sample, standard, and control must be extracted and the extraction must be done in advance. The dried extract (after evaporation of methanol) can be stored at 2-8 ° C or -20 ° C for 24 hours. After elution with methanol, the extraction column can be used for the extraction of the next sample or storage at 2-8 °C. The extraction column can be used 4 times repeatedly. If it is repeated, start again from A.1 (modulation of the column). |
A , standard version: centrifugation and evaporation centrifugation steps
1. Modulation of the column
1 | The extraction column was placed in a polystyrene or glass test tube (12 x 75 mm). |
2 | 2 x 1 ml of methanol (undiluted) was added to the extraction column, and the mixture was centrifuged at 200 g for 1 min to pass the solvent through the extraction column, and the eluate was discarded. |
3 | 2 x 1 ml of distilled water was added to the extraction column, and the mixture was centrifuged at 200 g for 1 min to pass the solvent through the extraction column, and the eluate was discarded. |
4 | To avoid drying the column, take the sample immediately. |
2. Sample extraction
5 | Place the extraction column in the corresponding labeled polystyrene or glass test tube (12 x 75 mm). |
6 | 0.5 ml of the standard, the control and the sample were added to the extraction column, and the mixture was centrifuged at 200 g for 1 min to pass the solvent through the extraction column, and the eluate was discarded. |
3, washing
7 | 2 x 1 ml of 10% methanol (diluted with distilled water) was added to the extraction column, and the mixture was centrifuged at 500 g for 1 min to pass the solvent through the extraction column, and the eluate was discarded. |
4, the elution of the extract
8 | Place the extraction column in a new labeled polystyrene or glass test tube (12 x 75mm) |
9 | 1 ml of undiluted methanol was added to the extraction column, and the mixture was centrifuged at 200 g for 1 min to pass the solvent through the extraction column. |
10 | Remove the extraction column from the tube to prevent the droplets from remaining in the column. The column was used for the extraction or storage of the next sample at 2-8 °C. The extraction column can be reused four times. |
5. Evaporation and reconfiguration of extracts
11 | The methanol was evaporated using an evaporating centrifuge. |
12 | Re-diluted the configuration sample with 0.15ml of distilled water |
13 | The vortex was mixed for 1 minute and the sample was immediately tested. |
B , optional version: replace the centrifuge with a multi-head vacuum
The extraction step is still carried out in accordance with A1-5 and the column is unchanged. The solvent was passed through the column with a vacuum and a flow rate of < 5 ml/min. Samples and extracts were flowed at ≤ 2 ml/min. The solvent was dried by evaporation using an evaporating centrifuge or nitrogen. |
9 , experimental steps
1 | 50 ul of the extracted standard, control and sample were respectively added to the corresponding micropores. |
2 | 50 ul of melatonin biotin was added to each well. |
3 | 50 ul of melatonin antiserum was added to each well. |
4 | Cover plate, gently shake the plate, incubate for 14-20 hours at 2-8 °C. |
5 | Remove the viscous metal plate, discard the reaction solution in the well, wash the plate 3 times with 250 ul of diluted detection buffer per well, and pat dry on the blotting paper to remove the residual liquid. |
6 | 150 ul of temporarily configured enzyme conjugate was added to each well. |
7 | The cover was incubated for 120 min on a room temperature (18-25 ° C) orbital shaker (500 rpm). |
8 | Prepare the PNPP substrate solution approximately 10 minutes before the end of the incubation. |
9 | Remove the viscous metal plate, discard the reaction solution in the well, wash the plate 3 times with 250 ul of diluted detection buffer per well, and pat dry on the blotting paper to remove the residual liquid. |
10 | If there are 8 pipettes, use an 8-channel pipette to add the substrate and stop solution. The time interval between the addition of the substrate and the stop solution should be the same. Use an active displacement pipette and avoid air bubbles. |
11 | 200 ul of temporarily configured PNPP substrate solution was added to each well. |
12 | Incubate for 20-40 min on a room temperature (18-25 ° C) orbital shaker (500 rpm). |
13 | Add 50 ul of PNPP stop solution to each well and gently shake the plate to mix the solution evenly. |
14 | The OD value was read at 405 nm within 60 min after the addition of the stop solution. (Reference wavelength: 600-650nm) |
10 , the result of calculation
On a semi-logarithmic paper or an automatic calculation method, a standard curve is made on the concentration (X-axis, logarithm) of the standard OD value (Y-axis, linear). A better standard curve can be obtained using a cubic regression, four-parameter or double logarithmic curve equation. When extrapolating the standard curve, you should make full use of each standard value (the apparent escape value should be ignored, and replace it with a more reasonable value). The concentration of the sample can be read directly from the standard curve. The initial dilution factor should be taken into account when reading the results from the coordinate paper. Multiply the diluted sample result by the dilution factor to get the corresponding result. Samples with higher antibody concentrations than the highest standards in the sample should be diluted and retested as described in the pre-experiment preparation instructions.
Conversion factor: melatonin (pg/ml) × 4.30 = pmol / l
Typical standard curve
(Example: Cannot be used for quantification)
Standard | Melatonin (pg/ml) | Average OD value | OD/OD max (%) |
A | 0.0 | 1.517 | 100 |
B | 3.0 | 1.383 | 91.1 |
C | 10 | 1.214 | 80.1 |
D | 30 | 0.867 | 57.1 |
E | 100 | 0.434 | 28.6 |
F | 300 | 0.260 | 17.1 |
11 , 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. A study of a group of people who are clearly healthy found that the levels of melatonin in the human body have obvious physiological cycle changes. The level of melatonin during the day is very low, while the level at night is high, and the differences between individuals are also very high. Big. In addition, the level of melatonin is also related to age, and the highest concentration is found in infant (3 years old) samples. The melatonin physiological cycle of six apparently healthy blood donors was studied. Melatonin is about the lowest at 4 pm during the day, with an average of 4.6 pg/ml; the best at 4 am, with an average of 77.5 pg/ml. The highest values ​​of melatonin vary among healthy individuals, and it is recommended that each laboratory determine the normal range of values ​​in their laboratory.
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
Chu Hou Sauce
Lishida Chuhou Sauce is perfectly for brasing meats and vegetables.You could also use for marinate meat like pork,beef,chicken,duck and so on.Brewed from non-GMO soybean and other safe ingredients,please enjoy the healthy and yummy mealtime with your families and friends!
Chu Hou Sauce,Chinese Seasoning Chu Hou Sauce
KAIPING CITY LISHIDA FLAVOURING&FOOD CO.,LTD , https://www.lishidafood.com