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How to Analyze Data from ELISA

How to Analyze Data from ELISA
Prepare Data for Prism
a. First Subtract 540 Absorbance value from 450 absorbance
value (this “corrects” for optical imperfections in the plate)
b. Then arrange values like on the 96 well plate
c. Subtract the blank
i. Average the blank and subtract this absorbance
value from all measured values in excel
ii. Then create a column that looks like this in excel 
1. The second and third columns are the
duplicate values for the standards and
2. Copy First three columns (concentration of
standard and absorbance of duplicate
Open Prism
a. Select desired graph (choose ‘xy’)
i. Under select/import data, select ‘pick “2” replicate values
ii. Leave nothing highlighted for “use sample data”
iii. Click Create
b. To paste data, right click blank box under “x”, click paste, click paste data
c. Go to Graphs  Data 1 on left side of page
i. Click Data1
ii. Choose graph type: w/out connecting lines
1. Click Okay
iii. Under “Analysis” menu, click “interpolate
standard curves” button
1. Choose second order polynomial
2. Under “outliers/robust,” choose “no special handling of outliers”
3. Under option, check off boxes for:
a. Report each interpolated value with its NONE
b. Plot curve with “95% confidence band”
4. Click Okay
d. Go back to graph
i. Note:
1. The dashes = confidence band
2. The solid line = best fit
3. Make sure plotted dots are on best fit line
ii. To zoom in:
1. First, duplicate the graph by right clicking on graph tab “Data1” and
selecting duplicate current sheet
2. Double click on the y-axis of new graph
a. Uncheck “automatically determine the range and interval”
i. Change min = 0
ii. Change max to 1/4th the current value
b. Do the same to the x-axis by clicking on the x-axis tab
3. Make sure the plotted values are on the best fit line
e. Now, you can go to interpolated values tab to get concentration of samples based on
best fit line
i. Use first sheet under this tab – “interpolated x mean values”