Dose response assays are generally characterized by a nonlinear relationship between the response and the amount of drug/agonist/antagonist given. When plotted, this response will usually exhibit a sigmoidal curve that is best described using the 4 Parameter Logistic (4-PL) or 5 Parameter Logistic (5-PL) nonlinear regression model equation.

The **C parameter** in the 4-PL model represents the concentration value for the inflection point of the curve. Depending on whether you have an inhibition or activation assay, the C parameter also represents the **half maximal inhibitory concentration (IC50)** or the **half maximal effective concentration (EC50)**respectively. Due to the symmetry around the inflection point, the 4PL makes it very convenient to determine the EC50/IC50 value.

The 5PL is a different story because of the asymmetry around the inflection point of the curve. The formula for calculating the EC50/IC50 from the 5PL still involves the **C parameter** but also a couple of others:

**E parameter**– This is fifth or the “asymmetry factor” for the 5PL model equation. A value of 1 for this parameter represents perfect symmetry around the inflection point making it equal to the 4PL model equation.**B parameter**– This is the**Hill Slope**and it a measure of the rapidity of the curve’s transition between the asymptotes.

The equation for calculating the EC50/IC50 from the 5PL is as follows:

**x = (C(2^(1/E) – 1))^(1/B)**

MasterPlex ReaderFit offers full support for the **4 Parameter Logistic (4-PL)** and **5 Parameter Logistic (5-PL)** model equation making it an ideal tool for automated fitting of dose response curves and calculating the **EC50** or **IC50** values.

Here is a **Quick Guide** for this procedure:

- To acquire MasterPlex ReaderFit, download and install the free 14-day trial.
- Importing the raw response values is a simple
**copy & paste**OR you can use the file import wizard to import Excel, .csv, or text files. - This is a sample dose response data set which contains 3 different sets of data from which we will generate 3 dose response curves (each outlined with a different color). Each curve set contains 8 data points with duplicates that are side-by-side.
- Select
**1 entire set of data points**(including replicates) and click the blue Sample Curve button. Please note that you can hold down the CTRL button and left click non-contiguous wells. - The
**Auto Fill**dialog will pop up. This feature is only helpful if you have a serial dilution for your drug concentrations. If not, you will have to enter them in manually.In this example, Drug ABC was serially diluted from top (20,000 pg/mL) to bottom (0.26 pg/mL) with a dilution factor of 5.

- Click
**Apply & View**to preview the values in plate tab to confirm. - Repeat steps 2-4 for any other data sets you may have. In this example, there are 2 other data sets.

For each cell, you will notice 2 numbers: the response value on the top and the drug dose on the bottom. - Click on the
**Fit Curves**tab.

- Make sure the
**Use Best Fit feature**checkbox is**unchecked**and select either the**4-PL**or**5-PL**model equation for the curve fit and then click**Calculate**. - MasterPlex ReaderFit will then draw the dose response curve(s) according to the parameters you have chosen.
- To view the
**EC50**or**IC50**calculations, click on the**View Results**tab and press the**Show Columns Selector**button.

- Drag and drop the
**EC50 / IC50**column to the table header row.

- (Optional) You can click on the
**Merge Cells By Group**button for a cleaner output. This function basically merges cells of replicate groups and just outputs a single result instead of repeating the same value for all replicates. - (Optional) If you have more than 1 dose response curve, you may want to group them together by dragging and dropping the
**Replicate Group Name**column header onto**Analyte Name**.

The end results will look something like this:

If you are looking for curve-fitting software with EC50 and IC50 determination we have a couple options:

ReaderFit.com – Free online curve-fitting application

**Sign Up for Free Account**

ReaderFit Desktop – Robust curve-fitting, quality control and reporting desktop software

**Download Free Trial**

You may also be interested in reading our blog post on:

Hi:

I need to make a rabbit serum titration (1/100, 1/200, 1/400, …………1/51200) by indirect ELISA and I don´t know how can I calculate it with your software. Could you help me?

Hi Carmen,

MasterPlex ReaderFit can certainly help you with this type of curve fitting and I will be more than happy to help you out.

Do you need to interpolate concentration values or do you simply want to plot raw fluorescence values vs. dilution factor?

Allen

Only Absorbance values vs dilution factor

In this case, first import your raw absorbance values. Then highlight and select all wells pertaining to a single curve (including all replicates) and then press the blue “S” button in the top row of icons. After that, the Auto Fill dialog will pop up. For “Starting Value,” enter “51200″ and use “2″ as your dilution factor. Then choose how many replicates you have per sample and the dilution direction of your plate.

Click “Apply and View” to make sure the dilution factors are inputted corrected. After this, simply press the “Best Fit” button and you should have your curve.

If you would like me to do the analysis for you, you can send me your raw data file (aliu@miraibio.com) and we can continue this conversation offline.

I hope this information helps.

Allen

Hi aliu:

When I import my absorbance values i don´t have any problem but when I have to generate my standard curve the values changes, for example 1.24 (DO 450nm) change to 24…..and another question how is the best formule to calculate a serum titre : endpoint titer or EC50…a lot of question aliu

Carmen

Hi Carmen,

I would definitely like to take a look at your project file to see if I can see what is happening. If you can, please email that to me.

For EC50 or IC50, I would use the 4 Parameter Logistic/4PL nonlinear regression model. Once you generate the standard curves, you will be interested in the C parameter as that will be your EC50 or IC50 values depending on the type of assay you are doing.

I hope this helps.

Allen

Hi,

I just wanted to point out that the link from your ELISA tips post to may be very confusing. The 5PL is not as easy to interpret as the 4PL. The “C” parameter in a 5PL curve is not going to be at the EC50 value.

Dan

Hi Dan,

You are absolutely right! I had actually forgotten to put my little blurb on calculating the EC50/IC50 from the 5PL. I had it in a different blog post but simply forgot to paste it over. It is taken care of now. Thanks for notifying me of this.

Allen

Great blog by the way =)

Dear Allen

Indeed, I have a drug need to study the pharmacokinetic of it, and before this I need to calculate the dose response curve of such drug, so my question is how can I do it ? , if I am going to use the experimnetal animal such as Rabbit, rats , …etc

Hi Eltayeb,

The blog post above includes a “Quick Guide” that shows you how to calculate the EC50/IC50 of your assay. If I misunderstood your question, please give me some more details about your issue and I’ll be more than happy to help out.

Allen Liu

Hi ,

What if i had only 3 data point but there is 50% reduction can I used this software to do that? If yes, how should I do that? If not, which software should I use?

Hi mint,

Although, it would be ideal to have more data points, the curve fit will depend on the quality of your data set. I can definitely give your data set a try though to see if it will work. Can you give me your raw data? If you do not feel comfortable replying in this blog post, feel free to email me your data. My email address is “aliu at miraibio dot com”.

Thanks,

Allen

Hi. I’m an undergraduate student from University of the Philippines Visayas. I’m currently doing an undergraduate thesis regarding an antioxidant assay using DPPH. I’m about to calculate its EC100 using this program which I have installed the 14-day trial just yesterday. How can I calculate for the EC100 when the range is 0<x<100? thank you

Donna,

After you input your data, indicate your plate layout and fit your curve go to the “Fit Curves” tab. Click the Statistics Toolbox icon which will show the EC/IC calculation options. By default EC50 will be displayed. In the text box type 100 and click Add. You will now see the values for EC100. You can do this for any value between 1 and 100.

Robert

Hello, I’m doing also an undergraduate thesis but confused on calculating the IC50 using DPPH assay.

I have 5 concentrations (+ control and standard), 3 replicates each. How can I calculate the IC50 then?

Thanks!

Hi Rowel,

This is Charles from support. This is a great question.

First of all let’s talk about what IC 50 is. IC50 is “half maximal inhibitory concentration”, which means that you are looking for the half point mark between the highest/lowest ranges, and then calculate for the

concentrationat that “half” response point. To calculate that, you will only need to have your standard points because that is what you’ll be using to determine the standard curve. Yourcontrolwill only validate if the curve and assay was performed within the acceptable range. If your curve was done using the 4 parameter logistic model equation, then the “c” parameter will be your IC 50.Thanks,

Charles

Senior Technical Support

On thing more:

I want to know what this “serial dilution” all about? How to determine it? If I have 5 concentrations ranging from 50 – 1000 mcg/mL, what will be my serial dilution? Correct me if I’m wrong: Is serial dilution the same with the upper- and lowermost ranges? What about dilution factor? Thanks much!

Hi Rowel,

Serial dilution is just another fancy word to say that your known concentrations for the standard curve are diluted with the same factor. For example, if your 5 concentrations are starting from 1000 with 2 fold dilution (meaning diluted by half), then your 5 concentrations would be 1000, 500, 250, 125 and 62.5. This is an example of a serial dilution.

In contrast, if your serial dilutions are 1000, 750, 500, 100, 50 then as you can see the known concentration are not diluted with any specific factor. Thus it is not a serial dilution.

With that said, you can easily work with both serial and non-serial dilution in MasterPlex ReaderFit and ReaderFit.com.

Thanks,

Charles

Senior Technical Support

Hi,

I want to determine the IC50 for a PTEN inhibitor I tested with an competitive ELISA assay. I have standards for the the product of the PTEN reaction, PIP2, in duplicate. I also have a normal reaction, with PTEN enzyme and PIP3 substrate in duplicate, as well as reactions with 3 different concentrations of inhibitor (PTEN+PIP3+Inhibitor) in duplicate. I am confused as to how to calculate the IC50 of the inhibitor. The x axis would be the concentration of inhibitor but what would the y axis be? Thanks!

Hi Alyssa,

The y-axis would usually be indicating the reading of the instrument. This is usually measured in the terms MFI (Mean Fluorescent Intensity)or RLU (Relative Light Unit).

Best regards,

Charles Ma

Senior Technical Support

I want to know how much dose to give 300g rat if my IC50 on cells is 3 mg/mL

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