ELISA Development Guide - OriGene

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19. Add 100 l of Stop Solution to each well. Incubate for 1-2 minutes.
20. Read the absorbance at 450 nm.
Data Analysis
1. Use commercial data analysis software to analyze the data as analysis of ELISA data with
Excel can be quite difficult. Many plate readers are sold with a software package that
can be used to perform the analysis. Stand-alone analysis software is also available.
ReaderFit from MiraiBio (Hitachi), Prism from GraphPad, and SigmaPlot from Systat are
all suitable. A free (but limited) web-based analysis package is available at
www.readerfit.com.
2. Generate a standard curve by fitting the results of the protein standard data to a model
equation.
3. For microplate ELISA data, generally a 4-parameter logistic (4-PL) model gives a good fit.
For some assays, a 5-parameter logistic (5-PL) model may give a better fit than the 4-PL
model.
4. To account for greater variance at the low end of the curve, it is recommended to
consider 1/Y or 1/Y 2 weighting in conjunction with the 4-PL or 5-PL models.
5. Use the standard curve to calculate the concentration of the analytes in the samples
(unknowns). Be sure to adjust for any dilution of the samples relative to the standards.
6. The following on-line articles provide additional in-depth discussion and analysis of the
various factors and parameters to consider for ELISA data analysis.
Tips for ELISA Data Analysis
The 4 Parameter Logistic Regression Model
The 5 Parameter Logistic Regression Model
The Importance of Weighting with 4PL and 5PL Regression Models
OriGene Technologies, Inc. 9620 Medical Center Drive, Suite 200, Rockville, MD 20850
www.origene.com techsupport@origene.com 1-301-340-3188 (P) 1-301-340-9254 (F) Page 10