Human platelet activating factor,PAF ELISA Kit

Instructions
Code CSB-E07929h
Size 96T,5×96T,10×96T
Trial Size 24T ELISA kits trial application
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Product Details

Description

The product CSB-E07929h is a ready-to-use ELISA kit specifically designed and validated for the quantitative detection of human platelet-activating factor (PAF) in multiple biological solutions, including serum, urine, cell culture supernates, tissue homogenates, or cell lysates. It is not intended for diagnostic use. This assay kit was designed and optimized for biochemical research use in humans. The kit has undergone rigorous quality control in multiple parameters, including sensitivity, specificity, precision, linearity, recovery, and inter-batch difference. Refer to the product instructions for more details.

This assay employs the quantitative sandwich enzyme immunoassay technique, in which PAF in the samples or standards are sandwiched between pre-coated PAF antibody and Biotin-conjugated PAF antibody. HRP-avidin is then added to the wells. Following a wash to remove any unbound reagent, the TMB substrate solution is added to the wells and color develops in proportion to the amount of PAF bound in the initial step. The color development is stopped upon adding the stop solution, and the intensity of the color is measured at 450 nm via a microplate reader. The levels of PAF in the samples can be determined by referring to the O.D. (optical density) of the samples to the standard curve.

PAF is a potent phospholipid mediator originally described by its ability to cause platelet aggregation and dilation of blood vessels. It also potently mediates inflammation, allergic responses, and shock. It causes acute inflammation of the airway leading to asthmalike symptoms. PAF is involved in the development of cancer and other inflammatory conditions such as cardiovascular disease. It exerts a predominant role in angiogenesis, thrombosis, carcinogenesis, and metastasis.

Target Name platelet activating factor,PAF
Alternative Names N/A
Abbreviation PAF
Species Homo sapiens (Human)
Sample Types serum, urine, cell culture supernates, tissue homogenates, cell lysates
Detection Range 4.69 ng/mL-300 ng/mL
Sensitivity 1.17 ng/mL
Assay Time 1-5h
Sample Volume 50-100ul
Detection Wavelength 450 nm
Research Area Biochemicals
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 PAF 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 % 85  
Range % 81-92  
1:2 Average % 95  
Range % 90-105  
1:4 Average % 94  
Range % 91-102  
1:8 Average % 90  
Range % 80-98  
Recovery
The recovery of human PAF 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) 91 88-100  
             
             
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 Corrected  
300 2.097 2.093 2.095 1.994  
150 1.823 1.793 1.808 1.707  
75 1.476 1.374 1.425 1.324  
37.5 1.029 0.986 1.008 0.907  
18.75 0.782 0.772 0.777 0.676  
9.38 0.421 0.401 0.411 0.310  
4.69 0.218 0.202 0.210 0.109  
0 0.105 0.097 0.101    
Materials provided
  • A micro ELISA plate --- The 96-well plate has been pre-coated with an anti-human PAF 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 PAF 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-conjugateDiluent (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

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