The ReaderFit.com Video Tutorial Series‘ fourth video goes over the Replicates Tab. Learn how to create ELISA replicate groups within ReaderFit.com using our easy draganddrop feature. Renaming your replicate groups couldn’t be easier!
Tag Archives: 5PL
ReaderFit.com Video Tutorial Series: ELISA Curve Fit Tab
Our third ReaderFit.com Video Tutorial demonstrates every feature in the Curves Tab within ReaderFit.com. The previous ReaderFit.com Video Tutorial explained how to generate a curve using nonlinear regression models, such as the 4PL and 5PL. Today, we will go over how to view your ELISA curve points and unknown points, where to find the Coefficient of Determination (Rsquared) and the Root Mean Square Error (RMSE) statistics. We will also go through changing the views of both x and yvalue axes on your nonlinear regression curve and recapitulate the Curve Point Data table.
View your nonlinear regression curves for ELISA analysis now in ReaderFit.com!
Check out our other ReaderFit.com Tutorial Videos:
ReaderFit.com Video Tutorial Series: Save and Fit Nonlinear Regression ELISA Curves
The ReaderFit.com Video Tutorial Series continues with a demonstration of how to manually fit curves or use the Best Fit feature. If you have predetermined curvefit parameters, like the 4 Parameter Logistic (4PL) nonlinear regression model, you will be able to manually select your parameters within the Save & Fit Curve menu. Our second video of this series will also go over the Best Fit feature, which iteratively goes through all combinations of model equations and weighting algorithms and selects the best one for your data.
Play with the ELISA curvefitting features using your own data in ReaderFit.com!
Check out ReaderFit.com Video Tutorial Series: Importing Your ELISA Data
MasterPlex ReaderFit Video Series: Manually Fit Your ELISA Curve
Our 10th video of the MasterPlex ReaderFit video series demonstrates how to perform ELISA curvefitting by manually selecting the curvefit parameters. Select your nonlinear or linear regression model and optional weighting algorithms in the “Fit Curves” tab. This method can be used if you have predetermined curvefit parameters that you want to use with your ELISA data.
Importing data into MasterPlex ReaderFit
With the large number of plate readers and/or file formats out there it would be very difficult for us to have native support for each one in MasterPlex ReaderFit. That being said we wanted to make data import as easy as possible so ReaderFit allows you to import your data in multiple ways:
1. Copying and Pasting data into ReaderFit
 Simply copy your raw data. It can be in plate shape or list form.
 Left click to select well A1 then Rightclick on it, select Paste Data and then Paste Response Values:
2. Using the File Import Wizard
There are 2 supported formats for the File Import Wizard:
 Plate  This format is in the 96well plate format where all the response or raw values are in a table that corresponds to all 96 wells.
Here is the procedure for handling Plate format:
1. Deliminator Type  Select the deliminator used to separate all the values in your file
2. Data Format  Select the Plate option
3. Select Data  Click and drag to select the area in the plate view window on the left where all the values are located and click Next
4. For each analyte set that you have (not common), highlight and select the corresponding wells and click Assign
5. Click Finish
 List  This format is a 2column format that indicates well location and the response or raw value. (Note: in later versions support for a 3rd column containing sample name was added.)
Here is the procedure for handling List format:
1. Deliminator Type  Select the deliminator used to separate all the values in your file
2. Data Format  Select the List option
3. Well Address Selection  Highlight and select all the address wells in your list file in the preview window on the left and click Done for Well Address. Please note that the only accepted well address formats accepted are the conventional addresses for 96well plates such as “H3” or just plain numbers.
4. Data Selection  Highlight and select all the data wells in your list file in the preview window on the left and click Done for Intensity.
5.Optional: If you have Sample Names you can follow the same procedure as in steps 3 and 4 to import sample names.
6. Click Next
7. For each analyte set that you have, highlight and select the corresponding wells and click Assign
8. Click Finish
Copying your raw data from an Excel sheet or text document and pasting it directly into ReaderFit is highly recommended as it is the fastest and easiest method for importing data!
Dose Response Curves with EC or IC(Anything) calculation feature such as IC90 in MasterPlex ReaderFit
Calculating the EC10 or IC90 values for a dose response curve is not as simple as the EC50 or IC50 values when using the 4PL or 5PL model equations.
The equation for calculating any EC value given a 4PL curve fit is the following:
EC_{F} = (F/(100F))^(1/H) * EC_{50}
0 < F < 100 (This is the EC value you are interested in. For example, F=10 for EC_{10}.)
H = Hill Slope for the 4PL curve fit
Luckily, our latest release of MasterPlex ReaderFit 2010 makes this easy. Yes, even with the 5PL curvefit!
Here’s how:
 Copy & paste your data into ReaderFit
Copy your response values from the source and paste it into ReaderFit via CTRL+V > Paste Response Values.
 Mark data as Sample Curve type (part 1)
Select all of the data points that will be used to fit one entire dose response curve including all replicates. In the example given below, there are 7 groups each done in duplicate.
 Mark data as Sample Curve type (part 2)
Click on the blue Mark Sample Curve button. (The Auto Fill dialog may pop up. Click the Close button if you see it.)
 Indicate known dose concentration values (part 1)
Select Independent Values(Xaxis) from the data pulldown menu. This will show the corresponding data in the plate view. By default, all of the Independent Values(Xaxis) will be 0.
 Indicate known dose concentration values (part 2)
Select the sample of interest and doubleclick the lowercell to enter edit mode. Once in edit mode, type in the concentration value. Make sure all values have the same unit. We will edit that later.
 Fitting the curve (part 1)
 Fitting the curve (part 2)
Select either the Four Parameter Logistic (4PL) or Five Parameter Logistic (5PL) from the Curve Fitting section and press the Calculate button.
 Edit dose response curve axes
Rightclick on the dose response curve chart and edit the x and y axes. You also have the option to add a chart title.
 Calculate EC or IC(Anything) value (part 1)
 Calculate EC or IC(Anything) value (part 2)
By default the EC50 and the log EC50 values are calculated for you. If you have an inhibition curve, press the ICxx radio button to switch to IC50 and log IC50 values.
 Calculate EC or IC(Anything) value (part 3)
If you have an inhibition curve and you are interested in the IC90 calculation, simply select the ICxx radio button and enter “90” into the Percentages section and press the Add button.
If you have not done so yet, I would like to invite you to try the free 14day trial of MasterPlex ReaderFit.
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The 5PL or 5 Parameter Logistic is a nonlinear regression model used for prediction of the probability of occurrence of an event by fitting data to a logistic curve. It differs from the 4PL or 4 Parameter Logistic model in that it is an asymmetric function which is a better fit for immunoassay or bioassay data. As the name suggests, there are 5 parameters in the 5PL model equation: