How Not to Draw Standard Curves

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  • Using a linear curve fit (or regression) in Excel (y = mx + c)

    • Excel is an extremely powerful tool designed for a wide range of applications but drawing standard curves and interpolating unknown concentrations for ELISAs assays is not one of them. Bioassays or immunoassays resulting from biological systems follow a trend that is more analogous to the four paramater logistic (4PL) or five parameter parameter logistic (5PL) models rather than a linear model because there are limits to every biological system whereas linear curve fits go to infinity.
      4 Parameter Logistic Model

      Sigmoidal 4PL Curve Fit

    • Simply plugging in 0.5 for y and solving for x using a linear equation will get you the midpoint of the line but it is not an accurate IC50 or EC50 value because we are starting off with a very crude equation or approximation of the true curve fit. Simply put, you are forcing a square peg into a round hole. The more accurate method of obtaining the IC50 or EC50 value would be to use the 4PL model equation to do the curve fit and using the calculated C parameter.
      C Parameter - Inflection Point of the curve that also represents the EC50 or IC50

      C Parameter – Inflection Point of the curve that also represents the EC50 or IC50

  • Using the trendline feature with a scatter plot in Excel to get the equation and parameter values

    • Excel only offers linear, logarithmic, polynomial, power, exponential, and moving average as options for the trendline (reference). None of these are sufficient enough to describe the sigmoidal shape of immuno and bioassays.
  • Drawing standard curves without using weighting to offset heteroscedasticity

    • Heteroscedasticity is a phenomenon in which the variance of the dependent variable varies across the data. When dealing with [MFI/absorbance values/RLU] and concentration values, the concentrations usually increase as the [MFI/absorbance values/RLU] increases. When dealing with the high end of the standard curve, it is natural for the concentration values to have a greater variance when compared to the small concentration values on the low end of the standard curve. The use of weighting algorithms such as 1/y or 1/y^2 can be used to deal with the variance. More details can be found here.

The correct way of doing 4PL curve fits in Excel usually involve having to install an add-on that can do 4PL curve fitting but not all add-ons support weighting.

ReaderFit provides 4PL and 5PL curve fits with 4 different weighting algorithms.

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

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New features launched on ReaderFit.com

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You have spoken and we have listened. ReaderFit.com has been updated to include some new features:

  • Editable Projects
    Many of you have requested this and your wish has been granted. The raw data for all existing ReaderFit.com projects can now be edited. This is good for cases in which you would like to eliminate certain points after you do the initial curve fit. You can now go back and simply delete the data for that particular sample and your results will be updated to exclude that sample.
  • Remember Curve Fit Preferences
    ReaderFit.com will now remember your last curve fit settings you have used for any future curve fits (e.g. manual vs. Best Fit, model equation, weighting algorithm). This way, you do not have to constantly pull up the curve fit parameters to select that one model you always want to use.
  • “Enter” key in Raw Data table
    Pressing the “Enter” key in the Raw Data table will now navigate you down the row. Note: Pressing the “Tab” key will navigate you to the next input textbox.

That is it for this update. We’ll keep you posted on our next update soon which we are already working hard on =)

-The ReaderFit Team

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New ReaderFit.com Released

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ReaderFit.com, our online ELISA analysis web application, has undergone a facelift with a host of new features.

ReaderFit.com Home Page

Here are some of the biggest changes:

  • Saved Projects
    Projects can now be saved.

    Saved Projects in ReaderFit.com
  • Enhanced Security
    Security has improved using SSL for data encryption.

    Enhanced security for ReaderFit.com with SSL
  • New Pro Plan
    The basic application still remains free but we have implemented a Pro Plan for the power users who need higher throughput, unlimited projects, and other goodies.
  • Replicate Statistics*
    Get replicate statistics such as mean, %CV, and standard deviation for replicate groups.
  • Best Fit*
    The Best Fit feature iterates through all the possible model equations and weighting algorithms and comes up with the best combination for you automatically.

Sign up for a free account today and get started!


* Available only on the Pro Plan.

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ELISA Curve-Fitting Analysis with ReaderFit.com – 10/26/2011

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ELISA Curve-Fitting Analysis with ReaderFit.com – 10/26/2011

Join us for a Webinar on October 26
Space is limited.
Reserve your Webinar seat now at:
https://www1.gotomeeting.com/register/684921576
Join us for our upcoming webinar, “ELISA Curve-Fitting Analysis with ReaderFit.com,” on Wednesday October 26th, 11AM to 12PM PDT.

In this complimentary webinar hosted by Hitachi Solutions, presenter Charles Ma will demonstrate the streamlined workflow of ReaderFit.com.

ReaderFit.com is an online web application designed for ELISA curve fitting analysis with a slick easy-to-use interface.  This is a useful tool for plotting standard curves and interpolating your unknown values.  It can even be used for EC50/IC50 determination on dose response curves.

This webinar is designed for new users to ReaderFit.com.  Topics will include:

- Registering for new account

- Importing raw data

- Acquiring the curve fit

- Viewing results

- Exporting results
Title: How to use ReaderFit.com – 10/26/2011
Date: Wednesday, October 26, 2011
Time: 11:00 AM – 12:00 PM PDT
After registering you will receive a confirmation email containing information about joining the Webinar.
System Requirements
PC-based attendees
Required: Windows® 7, Vista, XP or 2003 Server
Macintosh®-based attendees
Required: Mac OS® X 10.5 or newer

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Hitachi Solutions America, Ltd. partners with ELISAlink.com

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The MiraiBio group of Hitachi Solutions America, Ltd. (HISAL) has partnered with ELISAlink.com to offer ReaderFit.com, a free online curve fitting tool for ELISA analysis, to ELISAlink.com users.

ELISALink.com is an online ELISA community, knowledge base and e-commerce site focused on serving the global ELISA Industry. The site will be anchored by a community-generated ELISA knowledge base and discussion platform and also feature direct access to a wide range of ELISA kits, components, equipment and services.

ReaderFit.com is a free online curve fitting application that allows users to both fit curves and optionally interpolate unknown values off the curve. Users upload response and independent values and then choose from one of 6 model equations: 4PL, 5PL, Quadratic, Log-Logit, Log-Log or Linear and one of four optional weighting algorithms: 1/Y, 1/Y2, 1/X and 1/X2. The resulting curve image and calculated results can then be easily exported from the web application.

“ELISAlink.com is an exciting concept that leverages “web 2.0” and social media features in a way that has been missing in the life science community. People in general have come to expect and rely on some of things that ELISAlink.com offers in other parts of their life. ELISA researchers now have a place to go for all of their ELISA needs. We are excited to offer ReaderFit.com as added value at ELISAlink.com,” said Robert Lynde, Deputy Director of the MiraiBio Group of HISAL.

“The team at HISAL really got it right with ReaderFit. The program is intuitive and designed from the perspective of an ELISA user, providing a streamlined and easy to use platform that is an invaluable tool to researchers and clinical users alike. The powerful Desktop Edition and the 21 CFR Part 11 compliant Security Edition were complimented earlier this year with a completely Online Edition – a natural fit for ELISAlink.com and our mission to provide the best and most powerful ELISA resources on the web,” said David Barka, Founder of ELISAlink.com

Want to be the first to know when ELISAlink.com is launched?  Sign up to receive email notifications and qualify for rewards!

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Hitachi Solutions launches free online curve fitting tool at ReaderFit.com

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The MiraiBio Group of Hitachi Solutions launched ReaderFit.com today for all users that registered at the pre-launch site. ReaderFit.com will be available to the public on May 16th, 2011 and will be featured in the Hitachi booth at the Immunology 2011 conference in San Francisco (May 14-16).

ReaderFit.com is a free online curve fitting application that allows users to both fit curves and optionally interpolate unknown values off the curve. Users upload response and independent values and then choose from one of 6 model equations: 4PL, 5PL, Quadratic, Log-Logit, Log-Log or Linear and one of four optional weighting algorithms: 1/Y, 1/Y2, 1/X and 1/X2. The resulting curve image and calculated results can then be easily exported from the web application.

Although ReaderFit.com is brand new, the algorithm behind the calculations has been in development for more than 8 years. MasterPlex ™ QT and MasterPlex ReaderFit are 2 desktop products offered by Hitachi Solutions that use the same algorithm. “We want users to understand that the calculations in ReaderFit.com are generated from the same algorithms used in the MasterPlex products. We have developed, improved and maintained these algorithms for the last 8 years and MasterPlex is used in many labs across the world. Not only are the algorithms the same, it is the same exact software code!” says Allen Liu, one of the lead developers of ReaderFit.com at Hitachi Solutions.

“We see ReaderFit.com as a way to offer the value of the MasterPlex desktop products to customers who don’t necessarily need the full desktop applications. Most importantly ReaderFit.com allows us to offer this value across platforms and devices. All you need is a web browser to use the tool,” added Robert Lynde, Deputy Director of the MiraiBio Group of Hitachi Solutions.

Robert Lynde went on to hint that more services will be added this year for users who like the convenience of a web-based, OS-independent tool but need more advanced features. Visit ReaderFit.com to create your free account and try the tool. We’d love to hear your comments below.

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View MiraiBio Webinar Series Recordings

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The following webinar recordings have been made available for viewing at your own leisure:

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