Human Melatonin,MT ELISA Kit

Instructions
Code CSB-E08132h
Size 96T,5×96T,10×96T
Trial Size 24T ELISA kits trial application
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Product Details

Description

The Human Melatonin (MT) ELISA Kit enables the quantification of human (MT) in solution, including serum, plasma, and tissue homogenates. The kit uses the double antibody Sandwich-ELISA technique. Samples, standards, biotin-labeled MT detection antibody, and HRP-avidin are successively added to the microwell plate wells pre-coated with anti-human MT antibody. After incubation and thorough washing, the substrate TMB is pipetted into the wells. The HRP catalyzes TMB, rendering the solution turns blue. The solution finally changes from blue to yellow under the action of the stop solution. The intensity of the color is positively correlated with human melatonin in the sample. Read the absorbance (OD value) with a microplate reader at the wavelength of 450nm, and the MT concentration in the sample can be calculated. This kit has been verified to have high sensitivity, strong specificity, good linearity, recovery of 90%-110%, precision less than 10%, and consistency between batches. Refer to the product manual for details.

Melatonin is an important physiological sleep regulator in diurnal species including humans. It regulates circadian rhythms and the sleep-wake cycle. In humans and other diurnal species, melatonin acts at the suprachiasmatic nuclei (SCN) to abate the wake‐promoting signal of the circadian clock, thus promoting sleep. Its sleeping‐promoting effects become significant about 2 h after intake. Additionally, melatonin acts at the default mode network (DMN) regions in the brain to promote fatigue and sleep‐like changes in activation of the precuneus.

Target Name Melatonin,MT
Alternative Names N/A
Abbreviation MT
Species Homo sapiens (Human)
Sample Types serum, plasma, tissue homogenates
Detection Range 6.25 pg/mL-400 pg/mL
Sensitivity 1.56 pg/mL
Assay Time 1-5h
Sample Volume 50-100ul
Detection Wavelength 450 nm
Research Area Epigenetics and Nuclear Signaling
Assay Principle quantitative
Measurement Sandwich
Precision
Intra-assay Precision (Precision within an assay): CV%<8%      
Three samples of known concentration were tested twenty times on one plate to assess.  
Inter-assay Precision (Precision between assays): CV%<10%      
Three samples of known concentration were tested in twenty assays to assess.    
             
Linearity
To assess the linearity of the assay, samples were spiked with high concentrations of human MT in various matrices and diluted with the Sample Diluent to produce samples with values within the dynamic range of the assay.
  Sample Serum(n=4)  
1:1 Average % 84  
Range % 80-88  
1:2 Average % 99  
Range % 92-106  
1:4 Average % 88  
Range % 82-93  
1:8 Average % 90  
Range % 84-94  
Recovery
The recovery of human MT spiked to levels throughout the range of the assay in various matrices was evaluated. Samples were diluted prior to assay as directed in the Sample Preparation section.
Sample Type Average % Recovery Range  
Serum (n=5) 95 90-99  
EDTA plasma (n=4) 103 95-110  
             
             
Typical Data
These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.
pg/ml OD1 OD2 Average Corrected  
400 2.451 2.357 2.404 2.323  
200 1.627 1.616 1.622 1.541  
100 1.046 1.003 1.025 0.944  
50 0.437 0.473 0.455 0.374  
25 0.274 0.285 0.280 0.199  
12.5 0.189 0.196 0.193 0.112  
6.25 0.143 0.141 0.142 0.061  
0 0.082 0.079 0.081    
Materials provided
  • A micro ELISA plate --- The 96-well plate has been pre-coated with an anti-human MT antibody. This dismountable microplate can be divided into 12 x 8 strip plates.
  • Two vials lyophilized standard --- Dilute a bottle of the standard at dilution series, read the OD values, and then draw a standard curve.
  • One vial Biotin-labeled MT antibody (100 x concentrate) (120 μl/bottle) ---Act as the detection antibody.
  • One vial HRP-avidin (100 x concentrate) (120 μl/bottle) --- Bind to the detection antibody and react with the TMB substrate to make the solution chromogenic.
  • One vial Biotin-antibody Diluent (15 ml/bottle) ---Dilute the Biotin-antibody.
  • One vial HRP-avidin Diluent (15 ml/bottle) ---Dilute the HRP-avidin solution.
  • One vial Sample Diluent (50 ml/bottle)---Dilute the sample to an appropriate concentration.
  • One vial Wash Buffer (25 x concentrate) (20 ml/bottle) --- Wash away unbound or free substances.
  • One vial TMB Substrate (10 ml/bottle) --- Act as the chromogenic agent. TMB interacts with HRP, eliciting the solution turns blue.
  • One vial Stop Solution (10 ml/bottle) --- Stop the color reaction. The solution color immediately turns from blue to yellow.
  • Four Adhesive Strips (For 96 wells) --- Cover the microplate when incubation.
  • An instruction manual
Materials not provided
  • A microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
  • An incubator can provide stable incubation conditions up to 37°C±5°C.
  • Centrifuge
  • Vortex
  • Squirt bottle, manifold dispenser, or automated microplate washer
  • Absorbent paper for blotting the microtiter plate
  • 50-300ul multi-channel micropipette
  • Pipette tips
  • Single-channel micropipette with different ranges
  • 100ml and 500ml graduated cylinders
  • Deionized or distilled water
  • Timer
  • Test tubes for dilution
Troubleshooting
and FAQs
ELISA kit FAQs
Storage Store at 2-8°C. Please refer to protocol.
Lead Time 3-5 working days

Citations

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