Category Archives: General ELISA Analysis Topics

How to calculated B/B0, % bound, for your competitive ELISA

Competitive ELISA is one of the many types of ELISA protocol. Competitive ELISA differs from sandwich ELISA in that the higher the concentration of the antigen in the sample, the lower the intensity reading. The maximum intensity is achieved when no antigen is present in the sample. To assess this maximum level in a competitive ELISA there is usually a control well in the plate layout that specifically contains no antigen and this is known as a Zero well or B0. Then the sample intensities can be compared to the B0 well to obtain a percentage bound. This is symbolized as B/B0 where B is the intensity of a sample well and B0 is the maximum intensity.

To calculate B/B0 with ELISAAnalysis.com your should specify the B0 wells in your plate layout in step 2 of the work flow and select the check box “Calculate % bound (B/B0) for all samples using Maximum Binding (B0) controls?”  in step 3. ELISAAnalysis.com will do the rest.

What a user should do if they have two standard curves on one plate?

We understand that most experiments may use only half or few wells of the plate.  Hence two or sometime three readouts with their respective standard curves can be designed into one single ELISA plate. Currently, ELISAAnalysis.com allows users to insert only one standard curve per plate. In order to analyze layouts with two or more standard curves (Figure A) with ELISAAnalysis, the user must split the raw data manually into partial data sets and analyze the OD readings separately for each partial data set (Figure B).

Figure A:  Raw data in Excel

Figure A: Raw data in Excel

Figure B: Split data into two plate layouts in Excel

Figure B: Split data into two plate layouts in Excel

How to Tighten ELISA Standard Curve Confidence Intervals

Now that ELISAAnalysis.com provides 95% confidence intervals, many clients have asked how they can tighten the confidence intervals for their standard curves.  Here are some comments and suggestions:

  • You will usually see the confidence intervals widening for higher concentrations.  This is normal when the percentage variability is roughly constant for all concentrations.  For example, if the confidence interval is plus or minus 10% then for higher concentrations this will translate into a larger confidence interval.
  • For a given level of variability, to tighten the confidence interval you should increase the number of replicates for your standard curve points.  While duplicates for standard curve points are common, from a statistical point of view this is a very low level of replication and as a result this leads to low confidence.
  • For a given number of replicates, to tighten the confidence interval you should explore strategies for reducing the variability between replicates.  ELISA involves a large number of steps and the challenge is to implement experimental procedures to reduce variability at each step.
  • The 4PL curve has asymptotes at both the positive and negative extremes.  As the curve moves closer to the asymptote, the confidence intervals will widen.  For a given concentration, the tightest confidence intervals for a 4PL curve will be near the inflection point of the curve, where it looks roughly like a linear curve at 45%.  If your unknown values are near the asymptotes then you could consider diluting the samples to move them towards the centre of the curve or consider a more sensitive kit.

ELISA Standard Curve Confidence Intervals Log Scale

Linear ELISA Curve, Linear Curve Fitting

A linear curve can be used for fitting an ELISA standard curve and can provide a reasonable fit for readings in the middle of the assay range.  If you want to get a more detailed understanding of linear curve fitting then we suggest that you refer to page 327 of the The Immunoassay Handbook, Forth Edition.  A preview of the relivant pages can be found on Google Books via the following link: http://books.google.com.au/books?id=xuYf6tcVdqYC&lpg=PA329&dq=4pl%20elisa%20curve%20fitting&pg=PA327#v=onepage&q&f=false

5PL ELISA Curve, 5 Parameter Logistic Curve Fitting

The 5 Parameter Logistic (5PL) curve is recommended by some ELISA kit manufactures for fitting a standard curve.  For those without a statistics degree (most of us in the life sciences) it can sound pretty intimidating.  The good news is that ELISA Software like elisaanalysis.com will do the hard work for you by fitting the curve and predicting your unknown values without you needing to know the details.  But if you want to get a more detailed understanding of the 5PL curve then we suggest that you refer to page 327 of the The Immunoassay Handbook, Forth Edition.  A preview of the relivant pages can be found on Google Books via the following link: http://books.google.com.au/books?id=xuYf6tcVdqYC&lpg=PA329&dq=4pl%20elisa%20curve%20fitting&pg=PA327#v=onepage&q&f=false

4PL ELISA Curve, 4 Parameter Logistic Curve Fitting

The 4 Parameter Logistic (4PL) curve is the most common curve recommended by ELISA kit manufactures for fitting a standard curve.  For those without a statistics degree (most of us in the life sciences) it can sound pretty intimidating.  The good news is that ELISA Software like elisaanalysis.com will do the hard work for you by fitting the curve and predicting your unknown values without you needing to know the details.  But if you want to get a more detailed understanding of the 4PL curve then we suggest that you refer to page 327 of the The Immunoassay Handbook, Forth Edition.  A preview of the relivant pages can be found on Google Books via the following link: http://books.google.com.au/books?id=xuYf6tcVdqYC&lpg=PA329&dq=4pl%20elisa%20curve%20fitting&pg=PA327#v=onepage&q&f=false

ELISA Analysis Most Popular Layouts

At elisaanalysis.com we have two primary objectives: 1. To make ELISA analysis straightforward and 2. To provide ELISA analysis software functionality that is not available elsewhere.  We have a number of exciting long term plans that will become available later in 2013, however we are excited to be able to offer our users some great functionality now.

Our Most Popular Layouts functionality allows users to select from a list of ELISA plate layouts based on there popularity.  One of the powerful advantages of having an internet based services is that popularity is based on the plate layout usage of all of our users.

ELISA Analysis Most Popular LayoutsYou can choose to see the community favorites or just your most popular layouts.  We also show the last used layouts, which is particularly useful for users who are repeating a similar experiment.  We hope that this feature will come in handy for new users by allowing them to select from existing plate layouts rather than needing to create new ones.  And don’t worry, if you want to keep your layouts private you can.

We have plans to improve this feature by allow access to all popular layouts and not just the top ten.  If you have some other ideas please send them through!

ELISA Kit Reader Software – What is it?

ELISA kit reader software is a broad term that describes software products that are used to control ELISA kit reader equipment and analyze the results.  This software is often provided along with the microplate reader equipment by the supplier, however there are also many software products that are designed to work with equipment from many different suppliers.  Reader specific software can have the advantage of  being more tightly integrated and allowing access to more advanced features of the reader equipment.  The ability to accept data from many different types of equipment offer by equipment interdependent software is useful for researchers who operate in a lab with many different types of readers and where the software is installed on a computer separated from the equipment.  It also allows researchers to use one software product over their career and to store their data in a standard format.

ELISA Software Background Cut-off or Threshold Calculation

ELISA kits offer a reliable and repeatable method for detecting any given target in may types of sample.  However ELISA kits involve several steps and reagents and in some cases significant background noise can result from the interaction of these reagents.  For this reason it is important to ensure that your results are statistically significant when compared to the natural background noise of the ELISA system.

The ELISA Analysis platform calculates the background cut-off automatically whenever you include negative controls in your experiment.  Here is how it works:

1. Specify the wells that are negative controls in your plate layout.  The more negatives you include the better as this will increase the statistical accuracy of the cut-off calculation.

This is an example of an ELISA kit plate plate layout that can be used for calculating an accurate background cut-off value.

2. When you click “Analyze & Create Report” we will take the negative OD values, calculate the mean OD for the negatives and calculate the standard deviation (SD) for the negatives.  The background cut-off is then calculated as the mean plus three times the standard deviation:

Background Cut-off = Mean of Negatives + (3 x SD of Negatives)

3. In your ELISA Analysis report you will see that the Cut-off Threshold is displayed at the top along with the Baseline value which is equal to the mean of the negative values.  Where a mean OD reading is below the Background Cut-off value then the calculated Mean Concentration is not provided and there is a cross marked in the “Excluded: Optical Density below cutoff” column.

ELISA Analysis Report Showing Background Cut-offs

 

Note: If you do not include any negative wells in your plate layout then the Background cut-off value will default to zero.

We hope that you will find this article useful.  Please feel free to contact us with any questions and feedback regarding this feature and how we could improve it.

ELISA Analysis, ELISA Data Analysis – What is it?

ELISA Analysis is a broad term that describes the analysis of the data obtained from a ELISA based experiment.  A basic ELISA experiment involves a 96 well microplate with a series of wells used to produce a standard curve from known concentrations, a small number of blank wells and the remainder of the plate used for samples with unknown concentrations.  ELISA Analysis involves the following steps:

  1. Fitting of a model curve to the standard data to produce a Standard Curve
  2. Use the blank wells to identify the level of background noise and exclude data that is undifferentiatable form the background noise
  3. Use the Standard Curve to predict the concentrations of the unknown samples

There are many advanced ELISA Analysis techniques and these will be discussed in specific knowledge base posts.