Product number: EIA05619
Read the instructions thoroughly before use. The enzyme-linked immunosorbent kit is based on the principle of classical enzyme-linked sandwich technology and can measure rat melatonin (MT). It can only be used for research purposes and should not be used for medical diagnosis.
Working principle Melatonin is a neuroendocrine hormone secreted by the pineal gland. Recent studies have found that melatonin has a wide range of biological effects, such as sedation, hypnosis, immune excitability, anti-aging, anti-tumor, etc. The Elisa Kit provided is a typical sandwich enzyme-linked immunosorbent assay kit (ELISA). The pre-coated antibody is a polyclonal antibody. The detection phase antibody is also a polyclonal antibody and is labeled with biotin. The sample and the biotin-labeled antibody were sequentially added to the wells of the plate, and washed with PBS or TBS. The peroxidase-labeled avidin reaction was then added; after thorough washing with PBS or TBS, the substrate was developed with TMB. TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color of zui under the action of an acid. The depth of the color is positively correlated with the factor to be tested in the sample.
Content in each kit (96T)
Material quantity standard 96T (2vial)
96-well plate 96T (8×12) pre-coated with antibody
Biotinylated antibody 96T (1:100)
ABC (Avidin-Peroxidase Complex) 96T (1:100)
Sample dilution 96T×2
Antibody dilution 96T×1
ABC dilution 96T×1
TMB coloring solution A 96T×1
TMB color developing solution B 96T×1
TMB stop solution 96T×1
ELISA special washing liquid 96T×1 (1:25)
Reagents and equipment that are needed but not provided
1. 37 ° C incubator.
2. Standard specification microplate reader.
3. Automatic washing machine.
4. Single or multi-channel adjustable pipettes and their tips.
5. Distilled water, volumetric flask, etc.
6. Clean test tubes and Eppendof tubes.
Precautions
1. Allow room temperature to equilibrate for at least 30 minutes before opening the kit.
2. When preparing various reagents, remember to mix!
3. When the user first uses the kit, the various reagent tubes should be centrifuged for a few minutes to allow the reagents to collect at the bottom of the tube.
4. It is recommended that all standards and samples be tested in duplicate.
5. It is necessary to strictly avoid drying of the microplate during operation. Drying will quickly deactivate the biological components on the microplate!
6. To avoid cross-contamination, avoid reusing the tips and tubes in your hand.
Washing method: Manually washing the plate: aspirate (not touch the wall) or pry off the liquid in the plate; pour a few layers of absorbent paper on the bench, and take a few times with the plate; use the recommended PBS or TBS wash buffer was injected into the well at least 0.35 ml and soaked for 1-2 minutes. Repeat this process several times as needed.
Automatic washing: If there is an automatic washing machine, it should be used in the formal experiment after skilled use.
Preparation and storage of samples If not immediately analyzed, they should be stored separately and stored frozen, and avoid repeated freezing and thawing.
Serum - Blood was collected in a clean tube, coagulated at room temperature for 2 hours or overnight at 4 ° C, centrifuged at 1000 x g for 10 minutes, and serum was collected. Immediately analyze or dispense and store at -20 °C.
Cell culture, tissue homogenate, body fluid - centrifugation to remove the precipitate, immediately analyzed or dispensed and stored frozen at -20 °C.
General Principles of Sample Dilution The user must consult the literature to understand the amount of the test factor in the sample and determine the appropriate dilution factor so that the concentration of the test factor in the diluted sample is within the range of the ELISA kit. The dilution of the sample should be recorded in detail.
Reagent preparation and storage
A. Dilution and use of standards: Prepare within 2 hours before use. Add 1 ml of the sample dilution to the lyophilized tube, dissolve it thoroughly, and dilute it.
The diluted standard was used within 2 hours.
B. Biotinylated antibody working solution: Prepared within 2 hours before use.
1. According to the need of 0.1ml per hole to calculate the total amount (in the actual preparation should be prepared 0.1-0.2ml).
2. The working solution was prepared in a ratio of 10 ul of biotin-labeled antibody plus antibody dilution 990 ul. Mix gently.
C. Preparation of avidin-peroxidase complex (ABC) working solution: Prepared within 1 hour before use.
1. According to the need of 0.1ml per hole to calculate the total amount (0.1-0.2ml should be prepared when the actual preparation).
2. The working solution was prepared in a ratio of 10 ul of avidin-peroxidase complex (ABC) plus ABC dilution 990 ul. Mix gently.
D. Preparation of TMB coloring working solution: TMB coloring working solution is prepared according to the ratio of TMB coloring solution A 9 parts and TMB coloring liquid B 1 part 30 minutes before use.
Operating procedure
1. Determine the number of well-coated antibody plate wells required for this assay.
2. 0.1 ml of each of the diluted standards was sequentially added to a row of 7 wells, and only 1 sample of the dilution was added as a control. The treated specimens were added in 100 ul per well.
3. The plate was capped and reacted at 37 ° C for 90 minutes.
4. After the reaction, the liquid in the microplate is sucked by an automatic washing machine; or the liquid in the microplate is removed, and then the absorbent paper is photographed a few times. Wash twice.
5. The prepared biotin antibody working solution was added in order of 0.1 ml per well. The reaction was carried out at 37 ° C for 60 minutes.
6. Wash 3 times with 0.01 M TBS or 0.01 M PBS, soak for about 1 minute each time.
7. The prepared ABC working solution was added in order of 0.1 ml per well. The reaction was carried out at 37 ° C for 30 minutes.
8. Wash with 0.01 M TBS or 0.01 M PBS 5 times, soak for 1-2 minutes each time.
9. TMB coloring solution which had been equilibrated at 37 ° C for 30 minutes was added in order, and the reaction was kept at 37 ° C in the dark at 37 ° C. During the reaction, it was necessary to observe frequently. When the standard 3-4 wells of the standard were visible, there was a clear gradient. Blue, when the difference between the 3-4 holes is not obvious, you can add TMB stop solution 0.1ml/well. (The color reaction is not longer than 30 minutes).
10. The OD value was measured at 450 nm using a microplate reader.
11. Find the corresponding concentration on the coordinates based on the absorbance of the sample. Users can also use a variety of application software to calculate. It should be remembered that since the sample is diluted N times, its actual concentration should be ×N.
Summary of operating procedures:
Prepare reagents, samples and standards
Add prepared samples and standards and react at 37 ° C for 90 minutes.
Wash the plate twice, add biotinylated antibody working solution, and react at 37 ° C for 60 minutes.
Wash the plate 3 times, add ABC working solution, and react at 37 ° C for 30 minutes.
Wash the plate 5 times, add TMB coloring solution, 37 ° C reaction
Add TMB Stop Solution
Read OD within 30 minutes
Calculate the test factor content of the specimen
Detection range: 1000pg/ml → 15.6pg/ml.
Sensitivity: <1.5pg/ml
Specificity: There is no cross-reactivity between the system and other cytokines.
Storage: -20°C [4°C in short term (eg two weeks)]
Validity: 12 months (-20 ° C).
Uses: For the in vitro quantitative analysis of liquid specimens (universal type).
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