Bioassay Human Nesfatin 1 ELISA

Assay Principle 
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with human Nes-1 antibody. Nes-1 present in the sample is added and binds to antibodies coated on the wells. And then biotinylated human Nes-1 Antibody is added and binds to Nes-1 in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated Nes-1 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 Nes-1. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.

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.