This practical document outlines how to use Excel to create standard curves for ELISA experiments and calculate sample concentrations. After completing the ELISA experiment, we obtain OD values from both standards and samples using a microplate reader. The concentration of the standards is known, allowing us to plot a standard curve with OD values on the x-axis and corresponding concentrations on the y-axis. By substituting the sample's OD value into this equation, we can determine its concentration.
Here’s how to achieve this step by step:
- First, organize your data in Excel by inputting the OD values of standards along with their corresponding concentrations.
- Select the completed data range with your mouse, then click on Chart Wizard; choose 'XY Scatter Plot' as your chart type and select 'Scatter' (the first option without connecting lines).
- Click 'Next,' which will bring up an interface like shown below. If you have entered horizontal lists as in this example, no changes are needed; if vertical lists were used, select 'Columns.'
When creating an ELISA standard curve and calculating sample concentrations based on it, since we know the sample's OD value, we need to set it as X. As seen above, X represents OD values while Y represents standard concentrations—exactly what we want. 4. Sometimes manual selection of X values is necessary; click ‘Series’ to change them accordingly. 5. To swap x-axis and y-axis labels if needed, click on small icons next to text boxes for X or Y values. 6. Once adjusted correctly, proceed by clicking ‘Next’ until reaching chart options screen. 7. Continue clicking ‘Next,’ then finish in pop-up window completion stage—now you have a complete scatter plot showing standard values! 8. At this point when asked about where the curve is: simply click on one of those points representing standard values (make sure it's selected), right-click it again selecting ‘Add Trendline.’ 9. A dialog box appears offering choices such as linear or polynomial trends among others—observe visually whether points form a straight line before making selections! Generally speaking though most ELISAs won’t yield perfectly linear relationships so opting for “Polynomial” usually works best here—with degree options available (we typically choose 2nd degree yielding equations like y=ax²+bx+c). 10 After choosing appropriately trendlines appear indicating our desired results! Next display formulas required for calculating concentration based upon obtained sample’s specific od readings just previously established earlier through chosen equation format displayed via R-squared correlation coefficient method outlined beforehand confirming accuracy levels reached at .99+. In our case noted high level achieved being .9999 indicates strong reliability regarding derived conclusions drawn concerning resultant calculations made herein.