96 Well Plate Human High Precision and Sensitivity 5-Nucleotidase ELISA Assay Kit For Research

*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.

Intended Use
This sandwich kit is for the accurate quantitative detection of human 5-Nucleotidase (also known as 5-NT) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

Assay Principle
This ELISA Assay Kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with human 5-NT antibody. 5-NT present in the sample is added and binds to antibodies coated on the wells. And then biotinylated human 5-NT Antibody is added and binds to 5-NT in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated 5-NT antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of human 5-NT. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.

Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (160ng/ml) with 120μl of standard diluent to generate a 80ng/ml standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (80ng/ml) 1:2 with standard diluent to produce 40ng/ml, 20ng/ml, 10ng/ml and 5ng/ml solutions. Standard diluent serves as the zero standard(0 ng/ml). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:

Wash Buffer Dilute 20ml of Wash Buffer Concentrate 25x into deionized or distilled water to yield 500 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.

Cat.No E0831Hu
Standard Curve Range: 0.5ng/ml - 150ng/ml
Sensitivity: 0.34ng/ml

Reagent Provided

Material Required But Not Supplied

• 37°C±0.5°C incubator
• Absorbent paper
• Precision pipettes and disposable pipette tips
• Clean tubes
• Deionized or distilled water
• Microplate reader with 450 ± 10nm wavelength filter

Summary
1. Prepare all reagents, samples and standards.
2. Add sample and ELISA reagent into each well. Incubate for 1 hour at 37°C.
3. Wash the plate 5 times.
4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.
5. Add stop solution and color develops.
6. Read the OD value within 10 minutes.

Calculation of Result
Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.