Rat 5-Hydroxytryptamine,5HT ELISA Kit

Instructions
Code CSB-E08364r
Size 96T,5×96T,10×96T
Trial Size 24T ELISA kits trial application
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Product Details

Description

The Rat 5-Hydroxytryptamine (5HT) ELISA kit is used to detect and quantify the concentrations of INS in serum, plasma, cell lysates, and tissue homogenates. This kit exclusively recognizes rat 5HT proteins. This ELISA kit has been confirmed to have high sensitivity, strong specificity, premium precision, high recovery, good linearity, and lot-to-lot consistency. See the instructions for more details.

Standards or samples are added to the appropriate microtiter plate wells pre-coated with goat-anti-rabbit 5HT antibody. HRP-conjugated 5HT and anti-rat 5HT antibody are pipped into the wells, where 5HT in the sample or standard competes with the HRP labeled 5HT for limited binding sites on the anti-rat 5HT antibody. These Ag-Ab complexes are captured by the antibody immobilized on the plate. The solution color turns blue after the addition of substrate A and B. The color reaction is immediately terminated after the addition of the stop solution. And the color changes from blue to yellow. The color develops in opposition to the amount of 5HT in the sample, and the intensity of the color is measured by a Microplate reader at 450 nm.

5-HT, also called serotonin, is a monoamine neurotransmitter that plays a critical role in the central nervous system (CNS). 5-HT effects are mediated through its interaction with 5-HT receptors (5-HTRs). 5-HT regulates mood stability, pain tolerance, and sleep patterns. It is also involved in the modulation of individual behaviors that organize social group dynamics.

Target Name 5-Hydroxytryptamine,5HT
Abbreviation 5HT
Species Rattus norvegicus (Rat)
Sample Types serum, plasma, cell lysates, tissue homogenates
Detection Range 0.8 ng/mL-160 ng/mL
Sensitivity 0.4 ng/mL
Assay Time 1-5h
Sample Volume 50-100ul
Detection Wavelength 450 nm
Research Area Neuroscience
Assay Principle quantitative
Measurement Competitive
Precision
Intra-assay Precision (Precision within an assay): CV%<15%      
Three samples of known concentration were tested twenty times on one plate to assess.  
Inter-assay Precision (Precision between assays): CV%<15%      
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 rat 5-HT 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 % 94  
Range % 89-98  
1:2 Average % 95  
Range % 92-99  
1:4 Average % 97  
Range % 89-103  
1:8 Average % 95  
Range % 90-100  
Recovery
The recovery of rat 5-HT 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) 96 91-102  
EDTA plasma (n=4) 96 92-103  
             
             
Typical Data
These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.
ng/ml OD1 OD2 Average    
160 0.069 0.074 0.072    
40 0.181 0.195 0.188    
12.8 0.426 0.439 0.433    
3.2 0.802 0.855 0.829    
0.8 1.155 1.231 1.193    
0 2.158 2.227 2.193    
Materials provided
  • A 96-well Assay plate -- The 96-well plate has been pre-coated with goat-anti-rabbit 5HT antibody.
  • Standard 6 x 0.5 ml -- Dilute the standard at dilution series, read the OD values, and then draw a standard curve.
  • Anti-rat 5HT Antibody 1 x 6 ml -- Act as the detection antibody.
  • HRP-conjugated 5HT 1 x 6 ml -- Bind to the 5HT antibody, and HRP catalyzes the substrate A and B to elicit a chromogenic reaction.
  • Wash Buffer (20 x concentrate) 1 x 15 ml -- Wash away unbound or free substances.
  • Substrate A 1 x 7 ml -- Mix with substrate B, and the TMB is catalyzed by HRP to develop color.
  • Substrate B 1 x 7 ml -- Mix with substrate A, and the TMB is catalyzed by HRP to develop color.
  • Stop Solution 1 x 7 ml -- Stop the color reaction. The solution color immediately turns from blue to yellow.
  • Four Adhesive Strips (For 96 wells)
  • An Instruction manual
Materials not provided
  • A microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 600 nm - 630 nm.
  • An incubator that 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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