Kit Code:

Lot Number:

Date: Name:
Experiment: Method:

Lot numbers: Conclusion:

Plate Rinsing buffer
Standard Substrate
Conjugate Dilution buffer
Antibody Stop solution

conc.(µg/ml) Absorbance 1 Absorbance 2 conc.1 conc.2 factor Final, Mean

(multiply) Concentration Stand. %Bmax. conc. (µg/ml)
max. 1.992 2.078 0.025 79 0.021
0.025 1.609 1.643 0.023 0.020 0.021 0.05 67 0.052
0.05 1.408 1.353 0.048 0.057 0.052 0.1 54 0.115
0.1 1.109 1.118 0.117 0.114 0.115 0.2 42 0.219
0.2 0.903 0.874 0.210 0.228 0.219 0.5 30 0.461
0.5 0.654 0.637 0.448 0.475 0.461 2 13 1.923
2 0.314 0.317 1.940 1.905 1.923 ED 75% 0.030
blank 0.046 0.054 ED 50% 0.141

R2= 0.9938
 Chart Title
10
O.D.

0.1
0.01 0.1 1 10

Concentration
 Chart Title
1.0
logit(B/B0)

0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
0.01 0.1 1 10

Concentration
Explanation of calculations

The dose-reponse relation of immunoassays most often can be approximated

by transforming the response variable (absorbance) into its logit function, and the dose into log dose.
An approximately linear relation results, of which slope and intercept can be calculated by least-squares approach.
To obtain the logit function, first the ratio B/B0 =(absorbance-blank absorbance)/(max absorbance- blank absorbance)
is calculated. Then, Logit B/B0= log((B/B0)/(1-B/B0)).
Once the relationship is known, any representation of the data can be constructed. The graphs on pages
"OD vs Conc" and "Logit vs Conc"are just examples of the many alternative representations, of which the latter is a linearising one.

The field "factor (multiply)" may be used to introduce a multiplication factor (either <1 or >1), depending on sample volume or dilution.
If the field is left empty, the factor is 1 by default.

EXCEPTIONS

Absorbance of standard point in italics:

The distance of the point from the fitted line exceeds what is expected on the basis of the scatter of the other points
about the regression curve, and the fitted concentration differs by more than 10% from its nominal value.
Additionally, the point is indicated in the graph by a closed symbol.

If the point is also printed in bold, it has been omitted by Simplefit III from the curve-fit procedure. It is not shown in graphs!

Note: First check for typing error!!

It is advised to delete outliers (to remove both duplicates), though a maximum of 2 standard points (both replicates) or 5 separate values, inclu
If more than 2 standard points (both replicates) or more than 5 separate values are deleted, no sample results are printed!
(Please note that the removed data should not be in a sequence, that is removal of a value and then another one immediately ab
Sample result in italics:
The difference between duplicates is larger than expected on the basis of the variation of standard points about
the fitted line, and the difference between duplicates exceeds 15%.

Exclamation mark (!)

A "!" is printed when the absorbance is either higher than the zero standard or lower than the blank

"<" and ">" symbols

These are printed if the absorbance of a sample exceeds 1.1 times the absorbance of the lowest, (non-zero) standard or
the absorbance is less than 0.9 times the absorbance of the highest standard point, respectively.

R2 values
The squared correlation coefficient ("coefficient of determination") of the linearized dose-response relation is given.
Note: R2 may improve by removing outliers.

Error messages
The message:"Curve-fit not possible, check your data!" is printed when a non-conform sequence of absorption values is encountered
(i.e. higher concentration gives higher absorption value than lower concentration or the reverse)
The message:"Too few valid standard points, check your data!" is printed when there are less than 7 valid standard points.