5 -hydroxytryptamine ELISA kit instructions
(Germany IBL: RE59121)
1, the scope of application
This kit can be used for in vitro quantitative detection of serotonin in human serum, plasma, platelets and urine. Further research indicates that this kit can also be used for tissue homogenate and cell culture supernatant studies.
2 , the preface  Â
Serotonin is an intermediate in the metabolism of tryptophan, which is found mainly in intestinal chromaffin cells, serotonin-activated neurons, and platelets, and has been identified as a neurotransmitter in the central nervous system. Serotonin in circulating blood is almost exclusively concentrated in platelets. Changes in circulating serotonin concentrations can reflect some of the pathological changes in the human body. These pathological changes include: chronic myogenic headache, schizophrenia, hypertension, chorea, Duchenne muscular atrophy, and early acute appendicitis. The detection of serum serotonin has important clinical significance for the detection of carcinoid syndrome. It is expected that in the near future, research interest in serotonin in platelets (including studies on the absorption and release kinetics of pentahydroxyamine) should continue to grow.
3 , the principle of experiment
Sample preparation (conversion of serotonin to N-acetyl serotonin) is part of the dilution of the sample, and each sample is incubated with an acylating reagent to obtain an acylated serotonin. This experiment uses the principle of a competitive ELISA in which biotinylated and non-biotinylated antigens compete for binding to a number of antibody binding sites. Finally, the amount of biotinylated antigen bound to the antibody is inversely proportional to the concentration of the analyte in the sample. When the reaction system reaches equilibrium, the plate is washed to remove unbound biotin-labeled antigen, labeled with avidin alkaline phosphatase, and p-nitrophenyl phosphate is used as a substrate to detect the amount of biotin-labeled antigen bound to the antibody. . The concentration of serotonin in an unknown sample can be obtained by standardizing a known standard with a standard curve and calibrating the enzyme activity of the unknown sample on a standard curve.
4 , storage and stability
The kit and transport environment and storage environment temperature is 2-8 ° C, avoiding heat or direct sunlight. Sample storage and stability and reagent preparation will be described in the relevant sections. The opened kit is stable for the period of validity, provided that the kit is sealed with a bag and stored at 2-8 °C.
5 , sample collection and storage
note | Some foods contain a certain amount of serotonin. In addition, some drugs also stimulate the release of serotonin, which leads to an increase in the concentration of serotonin. Patients should fast foods rich in serotonin such as aspirin, adrenocorticotropic hormone, MAO inhibitors, phenazetin, catecholamine, reserpine and nicotine. |
serum
Pay attention to the routine precautions for collecting whole blood by venipuncture. The chemical integrity of the blood sample should be maintained from the moment the blood sample is collected to the time the test is performed. Do not use obvious hemolysis, jaundice, and lipemia samples. If the sample is turbid, it should be centrifuged to remove all particulate matter before the experiment. | ||||
Storage | 18-25 ° C | 2-8 ° C | ≤-20°C | Avoid heat or direct sunlight, avoid repeated freezing and thawing, and transport samples in a frozen state. |
stability | 2 hours | 6 hours | 3 months | |
Urine
It can be natural urine or 24h urine. All urine within 24 hours of the patient was collected and then added to 10-15 ml of 6N HCl preservative. Determine the total volume used for settlement of results. Mix all samples and centrifuge before use. | ||
Storage | ≤-20°C | Avoid heat or direct sunlight and avoid repeated freezing and thawing. |
stability | 6 months | |
Plasma, platelet
More than 98% of circulating serotonin is located in platelets and can be released when blood is agglutinated. Whole blood was collected by venipuncture into plastic tubes containing EDTA or citric acid (e.g., 1 ml of citric acid solution was added to a 10 ml Monovette NC tube). Whole blood samples were centrifuged at 200 x g for 10 minutes at room temperature to obtain platelet rich plasma (PRP). Transfer the PRP-supernatant to another tube. In order to obtain platelet chylomicrons, we added 200 ul of PRP (350,000 to 500,000 platelets/ul) to 800 ul of physiological saline, followed by centrifugation at 4500 x g for 10 minutes at 4 ° C (or 10,000 x g at 4 ° C) 2 minute). Discard the supernatant after centrifugation. Add 200 ul of double distilled water to the platelet chylomicron, and then the sample can be frozen for several weeks under -20 °C. The freeze-thawed sample was centrifuged at 1000 x g for 2 minutes at room temperature. A total of 20 ul of supernatant was used throughout the experiment (see acylation). If you want to measure the concentration of serotonin in platelet-free plasma (PFP), we can obtain PDP without centrifugation at 4 °C for 10 minutes at 4500 x (or 2 min at 10,000 x g at 4 °C). Plasma (PFP). An ELISA experiment for free serotonin used 50 ul of supernatant (see acylation). Note: Direct detection of serotonin in PRP has shown that approximately 10% of PRP samples can detect unpredictable high concentrations of serotonin (results obtained by high performance liquid chromatography and flow cytometry). To avoid this difference, we recommend that you detect serotonin in platelets and serotonin in platelet-free plasma. | ||||
Platelet-free plasma | Platelet microparticles (isolated from plasma) | Avoid heat or direct sunlight, avoid repeated freezing and thawing, and transport samples in a frozen state. | ||
store | ≤-20°C | ≤-20°C | ≤-80°C | |
stability | 2 weeks | 4 weeks | 1 year | |
Tissue homogenate and cell culture supernatant
Use tissue homogenate and cell culture supernatant as samples, generally no special precautions Note: Cell culture media may contain a certain amount of serotonin! | ||
store | ≤-20°C | Avoid heat or direct sunlight and avoid repeated freezing and thawing. |
stability | 6 months | |
6 , kit components
note | The kit has enough reagent components for 96 single well assays or 48 double well assays. |
Quantity | mark | ingredient |
1×12×8 | MTP | The coated plate was detachable and the micro-pore was coated with goat anti-rabbit antiserum. |
1×5ml | ANTISERUM | Histamine antiserum, blue, ready to use, contains: rabbit antiserum, Tris buffer and 0.01% NaN 3 . |
1×5ml | BIOTIN | Serotonin biotin, yellow, ready to use, contains 0.1% NaN 3 . |
1 x 0.2 ml | ENZCONJ CONC | The enzyme conjugate was concentrated 10 times and contained alkaline phosphatase-labeled avidin antibody, Tris buffer, HCl and 0.01% NaN 3 . |
1×7×0.5ml | CAL AG | Standards AG, 0; 0.08; 0.24; 0.73; 2.2; 6.6; 19.8 ng/ml, 0; 0.45; 1.4; 4.1; 12.5; 37.4; 112.3 nmol/l, ready-to-use, containing acylated serotonin, phosphate Buffer and 0.1% NaN 3 . |
1×2×0.5ml | CONTROL 1+2 LYO | Control 1+2, lyophilized, containing human serum and 0.02% NaN 3 . See the reagent bottle label for specific concentrations and allowable ranges. |
1×3ml | ACYLREAG | Acylation reagent Containing acetic anhydride and acetone, ready to use |
1×50ml | ASSAYBUF CONC | The assay buffer was concentrated 10 times and contained phosphate buffer, BSA and 1% NaN 3 . |
1×50ml | WASHBUF CONC | The washing solution was concentrated 20 times and contained phosphate buffer, Tween and 0.1% thimerosal. |
1×9× | PNPP SUBS | The PNPP substrate is sealed in an aluminum-platinum metal bag containing p-nitrophenol phosphate. |
1×27ml | PNPP BUF | PNPP substrate buffer, i.e. with, diethanolamine containing, water and 0.05% NaN 3. |
1×15ml | PNPP STOP | The PNPP stop solution, ready to use, contains 1 M NaOH and 0.25 M EDTA. |
3× | FOIL | Sticky metal plate |
7. Equipment required for the experiment but the kit does not provide
1) Pipette, volume: 20; 25; 50; 100; 1000ul
2) Glass test tube (12×75mm)
3) Test tube rack
4) Oscillator (500rpm)
5) Vortex mixer
6) Water bath, 37 ° C
7) 8-channel pipette with reservoir
8) Washing bottle, automatic or semi-automatic washing machine
9) Centrifuge: ≥1500×g
10) Microplate reader that can read at 405nm (reference wavelength: 600-650nm)
11) Double distilled or deionized water
12) Absorbent paper, sampling tip and timer
8. Preparation instructions before the experiment
For manual and automatic operation reference
note | The reagent components used for the 96-well assay can be divided into 3 uses. The volume described below is the volume required when using 4 strips (32 holes). If the customer wants to reduce the standard from 7 to 6, we can save the standard G. The detection range was correspondingly reduced to 155 ug/l (plasma) or 706 ug/l (serum, urine, tissue homogenate and cell culture supernatant). Serum, urine, tissue homogenate and cell culture supernatant samples can be selected in a short procedure, only 3.5 hours of incubation (but plasma samples cannot be performed in a short procedure), and the above samples can also be selected for optional procedures. Detection (the sample is incubated overnight) and the plasma sample must be tested overnight as it is incubated. |
8.1 Preparation of lyophilized or concentrated ingredients
Dilution/dissolution | ingredient | Join | Thinner | proportion | Remarks | store | stability |
15ml | Detection buffer | 150ml | Double distilled water | 1:10 | Yellowish brown does not affect the results of the experiment | 2-8 ° C | 2 weeks |
15ml | detergent | 300ml | Double distilled water | 1:20 | 2-8 ° C | 4 weeks | |
Quality control | 0.5ml | Double distilled water | Allow to stand for 15 min, no foam when mixing | ≤-20°C | Stable during the validity period | ||
60ul | Enzyme complex | 6ml | Diluted assay buffer | 1:101 | Temporary configuration, can only be used once | 18-25 ° C | 2 hours |
3 | PNPP substrate | 8ml | PNPP bottom Buffer | Temporary configuration, can only be used once | 18-25 ° C | 5 hours |
8.2 dilution of the sample
Samples with serotonin concentrations higher than the highest standards must be further diluted with assay buffer.
8.3 Acylation of samples and controls
Sample A: serum, urine, platelet extract, tissue homogenate and control
Sample B: Platelet-free plasma
note | Please do not acylate the standard, the standard has been acylated. Sample preparation allowed the serum, urine, platelet chylomicrons, tissue homogenate, and cell culture supernatant to be diluted 107-fold, while plasma samples were diluted 23.5-fold. This dilution factor must be taken into account in the calculation of the results. |
8.3.1 Sample A: serum, urine, platelet extract, tissue homogenate and control
1 | 20 ul of the control and sample A, respectively, were added to the glass test tube. |
2 | Add 100 ul of diluted assay buffer to each tube and vortex to mix. |
3 | 25 ul of acylating reagent 1 (3%) was added to each tube, and immediately vortexed after the addition. |
4 | The tube was capped and the tube was incubated for 15 minutes in a 37 ° C water bath. |
5 | Add 2 ml of diluted detection buffer to each tube, vortex mixing |
6 | Centrifuge at 1500 × g for 10 minutes |
note | The prepared sample must be tested immediately. The supernatant can be stored stably at 18-25 ° C for 1 hour. |
8.3.2 Sample B: Platelet-free plasma
1 | 50 ul of Sample B was added to a glass test tube. |
2 | Add 100 ul of diluted assay buffer to each tube and vortex to mix. |
3 | 25 ul of acylating reagent was added to each tube and vortexed immediately after the addition. |
4 | The tube was capped and the tube was incubated for 15 minutes in a 37 ° C water bath. |
5 | Add 1 ml of diluted detection buffer to each tube, vortex mixing |
6 | Centrifuge at 1500 × g for 10 minutes |
note | The prepared sample must be tested immediately. The supernatant can be stored stably at 18-25 ° C for 1 hour. |
9 , experimental steps
9.1 Short procedure (for sample A only, not for plasma)
1 | 50 ul of each of the standard, the acylated control, and the acylated sample was added to the corresponding microwell. |
2 | 50 ul of serotonin biotin was added to each well. |
3 | 50 ul of serotonin antiserum was added to each well. |
4 | The cover was incubated for 90 min at room temperature (18-25 ° C) with shaking (500 rpm). |
5 | The adhesive metal plate was removed, the reaction solution in the well was discarded, and the plate was washed 3 times with 250 ul of diluted washing solution per well, and the absorbent paper was patted dry to remove residual liquid. |
6 | 150 ul of temporarily prepared enzyme conjugate was added to each well. |
7 | The cover was incubated for 60 min at room temperature (18-25 ° C) with shaking (500 rpm). |
8 | The adhesive metal plate was removed, the reaction solution in the well was discarded, and the plate was washed 3 times with 250 ul of diluted washing solution per well, and the absorbent paper was patted dry to remove residual liquid. |
9 | If available, an 8-channel pipette can be used to add substrate and stop solution. When adding the substrate solution and the stop solution, the time interval between each well should be the same. Use an active displacement pipette and avoid air bubbles. |
10 | 200 ul of temporarily configured PNPP substrate solution was added to each well. |
11 | Incubate for 60 min at room temperature (18-25 ° C) with shaking (500 rpm). |
12 | 50 ul of PNPP stop solution was added to each well to terminate the substrate reaction and the plate was gently shaken to homogenize the solution. |
13 | The OD value was read at 405 nm within 60 min after the addition of the stop solution. |
9.2 Optional procedure (all nights, sample A and sample B are applicable)
9.2.1 first day
1 | 50 ul of the standard, the acylated control and the sample were respectively added to the corresponding micropores. |
2 | 50 ul of serotonin biotin was added to each well. |
3 | 50 ul of serotonin antiserum was added to each well. |
4 | Cover plate, gently shake the plate, incubate at 20-8 °C for 16-20 hours (all night). |
9.2.2 the next day
1 | Remove the viscous metal plate, discard the reaction solution in the well, wash the plate 3 times with 250 ul of diluted washing solution per well, and pat dry on the blotting paper to remove the participating liquid. |
2 | 150 ul of temporarily configured enzyme conjugate was added to each well. |
3 | The cover was incubated for 60 minutes on a room temperature shaker (500 rpm). |
4 | The adhesive metal plate was removed, the reaction solution in the well was discarded, and the plate was washed 3 times with 250 ul of diluted washing solution per well, and the absorbent paper was patted dry to remove residual liquid. |
5 | If available, an 8-channel pipette can be used to add substrate and stop solution. When adding the substrate solution and the stop solution, the time interval between each well should be the same. Use an active displacement pipette and avoid air bubbles. |
6 | 200 ul of temporarily configured PNPP substrate solution was added to each well. |
7 | Incubate for 30 minutes on a room temperature shaker (500 rpm). |
8 | Add 50 ul of PNPP stop solution to each well and shake the plate to mix the contents of the well. |
9 | 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
The OD value of all standards, controls, and samples was subtracted from the OD value of the blank wells. On a semi-logarithmic coordinate paper, a standard curve is drawn on the concentration (X-axis, logarithm) of the standard OD value (Y-axis, linear), or a standard curve is drawn by an automatic calculation method. A better plot can be obtained with Cubic spline, 4 parameters or double logarithm. In the calculation of the standard curve, it should be applied to each standard value (in the two values, the apparent escape value should be ignored, using a more reasonable value). The concentration of the sample can be quantified from the standard curve.
Since the sample is diluted, the final sample result must be multiplied by the corresponding dilution factor in ng/ml.
Serum, urine, platelets, tissue homogenate, cell culture supernatant, quality control: ×107
Platelet-free plasma: ×23.5
Samples that have been further diluted should be multiplied by the corresponding dilution factor.
The experiment must meet the following criteria to meet the requirements:
50% OD/Odmax (ED50): 0.60-1.00 ng/ml (average 0.8 ng/ml)
△OD standard A-standard product G≥0.80OD
Conversion factor:
Serotonin (ng/ml) × 5.67 = nmol/l
If the serotonin concentration in the sample is higher than the highest standard, the sample must be diluted and retested according to the pre-test preparation instructions.
11 , expected value
The results of the experiment cannot be considered as the sole cause of any treatment outcome, and the judgment of the disease should be combined with other clinical observations and diagnostic tests. The following results are normal values ​​for the normal population (97.5%):
sample | Quantity | unit | average value | range |
serum | 99 | Ng/ml | 88.6 | 30-200 |
Platelet-free plasma | 35 | Ng/ml | 3.7 | 1.8-7.5 |
Platelet | 35 | Ng/10 9 platelets | 490 | 217-861 |
24-hour urine | 49 | Gg/d | 83.1 | ≤200 |
(It is recommended that each lab read to determine its normal range of values)
This translation is for reference only, please refer to the original for details.
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