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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.

Adjusting to allow for Non-Specific Background (NSB)

ELISA is a robust assay, but it is inevitable that there will be some binding of proteins to the plate that is not intended. Using NSB wells in your plate layout will give you the option to subtract out this background noise from your intensity readings before analysis.

To do this with ELISAAnalysis.com your should specify the NSB wells in your plate layout in step 2 of the work flow and select the check box “Subtract Non-Specific Binding (NSB) Control well average OD from all other ODs?” in step 3. ELISAAnalysis.com will do the rest.

Data Transformations using control wells, B/B0, Zero wells, NSB wells

ELISA Analysis now has the ability to adjust your data (transform it) using the control wells in your plate layout before proceeding to the curve fitting and calculation steps.

Adjusting to allow for Non-Specific Background (NSB)

ELISA is a robust assay, but it is inevitable that there will be some binding of proteins to the plate that is not intended. Using NSB wells in your plate layout will give you the option to subtract out this background noise from your intensity readings before analysis.

To do this with ELISAAnalysis.com your should specify the NSB wells in your plate layout in step 2 of the work flow and select the check box “Subtract Non-Specific Binding (NSB) Control well average OD from all other ODs?”  in step 3. ELISAAnalysis.com will do the rest.

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.

Invalid Results: What are they and how can I fix them?

An “Invalid Result” in the analysis report means that the OD for the mean of an unknown replicate falls outside of the range of the standard curve and as a result it is not possible to provide a predicted value.

Often the reason for this problem is that the unknown values have much lower or higher OD than then standard curve ODs. One possible solution to the problem may be to rerunning the experiment with additional standard curve points with lower or higher concentrations as is appropriate. Though it should be noted that if the lower standard curve ODs are close to the background noise then this may not improve the results and a more sensitive assay may be required.

The 4PL curve has upper and lower asymptotes that define the range of the curve on the OD axis. The lower asymptote is given by d and the upper asymptote is given by a in the 4PL equation.

 

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

What does the option “starting reference number” mean in Step 2 when creating a layout?

The “starting reference number” relates to the next sample number the investigator is going to mark on the plate layout. It can either be the standard or the unknowns.  For example, in the plate layout if you have already marked wells as Unknowns 1-3, then the “starting reference number” should be set to 4 for the next Unknown sample (this should automatically happen). The same concept is followed for the standards. Soon we will be offering more powerful labelling in our plate layouts to specify detailed descriptions rather than just number references.

What should I do when I have controls/negatives for two different conditions on the same plate?

This experiment involves comparing pairs of treatment and control replicates on one plate. At present ELISAAnalysis software does not have the option of associating specific control/negative wells with specific treatment wells. Any wells marked as negatives are used to calculate the background cut-off for all unknowns on the plate.

There are two options for working around this limitation:

  1. Split the plate into two smaller data sets and analyse these data sets separately with ELISAAnalysis.com
  2. Specify the controls and treatment wells as “Unknowns” in the plate layout. ELISAAnalysis will calculate the concentrations for both and the investigator can then manually compare the controls vs. treatment group in the results.

Which wells should be considered “blank” or “negative” while creating your layout?

ELISAAnalysis uses “Negatives” for calculating the Background Cut-off Threshold.  So if you feel that your control could be a factor for potential noise in the experiment, then you should specify your controls as “Negatives” in the plate layout. Please note that “Blanks” are completely ignored for calculations in our software.  A knowledge base article on background cut-off could be helpful for you before you proceed to calculation step and can be found here: http://elisaanalysis.com/knowledge-base/elisa-software-background-cut-off-or-threshold-calculation/