BT Labs Horse Cortisol ELISA Kit

Assay Principle 
This kit is a Enzyme-Linked Immunosorbent Assay (ELISA). Add samples to the pre-coated plate. Then add biotinylated antigen. The antigens in the samples compete with the biotinylated antigen to bind to the capture antibody and incubate. Unbound antigen is washed away during a washing step. An avidin-HRP is then added and then incubate. Unbound avidin-HRP is washed away during a washing step. TMB Substrate is then added and color develops. The reaction is stopped by addition of acidic stop solution and color changes into yellow that can be measured at 450 nm. The intensity of the color developed is inversely proportional to the concentration of COR in the sample. The concentration of COR in the sample is then determined by comparing the O.D. of the samples to the standard curve.

Summary 
1. Prepare all reagents, samples and standards.
2. Add samples, standards and biotinylated antigen. Incubate for 60 minutes at 37°C.°
3. Aspirate and wash 5 times
4. Add avidin-HRP and incubate for 60 minutes at 37°C.
5. Aspirate and wash 5 times.
6. Add substrate solution A and substrate solution B and incubate in the dark for 10 minutes at 37°C.
7. Add stop solution.
8. Read the OD value within 10 minutes at 450nm.

Calculation of Results
Average the duplicate readings for each standard, control, and sample. Create a standard curve by plotting the mean absorbance for each standard on the Y-axis against the target antigen concentration on the X-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the target antigen concentration on the X axis versus the O.D. of the standards on the Y axis and the best fit line can be determined by regression analysis. The linear equation ( X = Y + Calibration Value ) can be used to calculate the standard curve where X is the log of the concentration of the standard and Y is the OD value of the standard. If samples have been diluted ( 2-5 times is recommended ), the concentration read from the standard curve must be multiplied by the dilution factor.