2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 45
Technical Appendix
ELISA Data Analysis
Abstract
Enzyme-linked immunosorbent assays (ELISA) are specific and highly Standard curves were prepared using Mouse IFN Beta ELISA kit (PBL
sensitive procedures for identifying and quantifying analytes in product #42400) and Human Interferon Alpha ELISA kit (PBL product
samples. #41100). For each data set, two standard curves were created. One
This assay is based on the binding of a target molecule standard curve was plotted using a 4-Parameter fit algorithm and the
(analyte/antigen) to antibodies which recognize the compound. The second one generated using linear fit analysis. Mean OD @ 450 nm for
presence of an antigen-antibody complex is labelled using a secondary all data points vs. the actual concentration in pg/ml corresponding to
enzyme-conjugated antibody. Alternatively, if an analyte specific that data point were plotted.
antibody is biotinylated, enzyme-conjugated streptavidin may be used.
Detection is obtained by addition of a substrate which yields a
Figure 1A: Extended Range (ER) standard curve
measurable product. Enzyme-linked immunosorbent assays are
for Mouse IFN-β with 4-Parameter fit
routinely used in many areas of biological research.
The determination of the analyte concentration relies upon
construction of a calibration curve. The standard curve is prepared by
performing a dilution series of a known concentration of the analyte
across a range of concentrations near the expected unknown 0nm
concentration. The calibration curves are then used to calculate the
4
5
concentration of an unknown sample.
@
For many analyses, a plot of response versus concentration will
O
D
create a linear relationship, at least within a certain range of
concentrations, and can be analyzed with linear regression. However,
for those calibration plots which are sigmoid that is, a curve having an
[IFN-β] pg/ml (log scale, linear values)
“S” shape performing a linear fit leads to errors in estimating sample
values. These inaccuracies are most significant at the extremes of the
standard curve, most often in the low end but sometimes in the high
Figure 1B: Extended Range (ER) standard curve
end as well.
for Mouse IFN-β with Linear fit
In this study, we compare the results of using linear fit and 4-
Parameter analysis on ELISA data and report our findings.
Methods
Standard curves were prepared using kits from two different ELISA
4
5
0nm
products, VeriKine
TM
Mouse Interferon Beta ELISA (PBL product
@
#42400) and VeriKine
TM
Human Interferon Alpha ELISA (PBL product
O
D
#41100). The assays were performed following protocols for
each product.
After completion of each assay, plates were read at OD @ 450 nm
using Vmax Kinetic Plate Reader (Molecular Devices Corporation, CA,
[IFN-β] pg/ml (linear scale)
USA). Triplicate measurements were performed for each data set.
Data was analyzed using SoftMax Pro software (Molecular Devices
Corporation, CA, USA).
Analysis
The Mean of the ODs @ 450nm, % CV of the ODs @ 450nm, and the Standard
Deviation between the ODs @ 450 nm for each data point of the standard
curve was calculated. The concentration was plotted on the X-axis. The
Mean OD @ 450 nm was plotted on the Y-axis.
www.interferonsource.com Tel: +1 732-777-9123
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Fax: +1 732-777-9141
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