2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 46
Technical Appendix
ELISA Data Analysis (continued)
Figure 2A: High Sensitivity Range (HS) standard Figure 2B: High Sensitivity Range (HS) standard
curve for Human IFN-α with 4-Parameter fit curve for Human IFN-α with Linear fit
0nm
4
5
0nm
4
5
@ @
O
D
O
D
[IFN-α] pg/ml (log scale, linear values) [IFN-α] pg/ml (linear scale)
4-Parameter fit Linear fit
Actual Concentration Mean OD Backfit % Difference: Backfit % Difference:
(pg/ml) @ 450 nm Concentration Backfit and Actual Concentration Backfit and Actual
(pg/ml) Concentration (pg/ml) Concentration
15.625 0.167 14.8 2.48 -27.1 279
31.25 0.268 35.9 14.8 6.59 78.9
62.5 0.374 58.7 6.05 41.9 3.0
125 0.645 121 3.66 132 5.73
250 1.19 259 3.50 312 24.9
500 1.94 495 1.10 565 12.9
1000 3.11 1002 0.15 954 4.60
Table 1:The backfit concentrations calculated with 4-Parameter fit and linear fit for points on the standard curve for Mouse IFN-β ELISA kit (PBL product #42400).
4-Parameter fit Linear fit
Actual Concentration Mean OD Backfit % Difference: Backfit % Difference:
(pg/ml) @ 450 nm Concentration Backfit and Actual Concentration Backfit and Actual
(pg/ml) Concentration (pg/ml) Concentration
12.5 0.167 12.3 1.50 0.20 98.4
25 0.268 25.1 0.44 16.8 33.1
50 0.374 50.4 0.78 49.0 2.08
100 0.645 99.5 0.49 109 9.21
200 1.19 200 0.10 222 11.2
500 1.94 500 0.01 490 1.99
Table 2: The backfit concentrations calculated with 4-Parameter fit and Linear fit for points on the High Sensitivity standard curve for Human IFN-α ELISA (PBL product #41100).
Concentrations were interpolated from the fitted curves using the Mean OD Conclusions
values. The percent difference between actual concentrations and the Different methodologies are applied to mathematically fit data generated
backfit estimated concentrations obtained from a 4-Parameter curve fit from ELISA experiments. The use of an inappropriate model to plot a
and linear fit equation are shown in Tables 1 and 2. standard curve can lead to inaccurate concentration estimates.
In this technical note, we have demonstrated that there are calculated
Results concentration differences obtained when using a linear fit and a 4-
Comparing the backfitted interferon concentration values from 4- Parameter curve fit. The backfit values derived from the 4-Parameter fit
Parameter fit versus Linear analysis revealed disparities between the two standard curves varied less from the actual concentrations than did the
methods. The largest discrepancies were noted at the lower backfit values from the linear fit.
concentrations. This trend was observed for both the Mouse IFN-β and Thus, we recommend using 4-Parameter fit algorithm for plotting
Human IFN-α products. Overall, the estimated interferon concentration standard curves prepared using ELISA kits manufactured by PBL
values more closely matched the actual concentration when applying a 4- InterferonSource.
Parameter fit. (Table 1 and Table 2).
Significant differences were seen for Mouse IFN-β at 15.125 pg/ml
(∼278%) and for Human IFN-α (∼98%) at 12.5 pg/ml when using a linear
fit equation.
PBLInterferonSource Toll Free: 1 877- PBL-8881
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