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How can I set-up a %B/B0 assay?

Document ID: DQ057
Relevant to following products: WorkOut 2.0 Manta
Document description: Application Example: Prostaglandin Assay



The Prostaglandin assay comprises of the following samples: Blank, NSB, B0 (the maximum binding), Standards (concentrations in duplicate), Unknowns in triplicate.
 
Absorbance measurements are taken of the whole plate.
 
%B/B0 is calculated as follows:
 
  • The average of the Blank wells is used to blank correct all wells.
  • The corrected maximum binding is calculated by subtracting the NSB average from the B0 average.
  • %B/B0 (% sample bound/ maximum binding) is calculated for all samples by subtracting the average NSB from the corrected absorbance measurement and divide by the blank corrected B0, then multiply by 100 to obtain %B/B0.
 
A curve fit is then performed on semi log axes.
 
To set-up this assay: create a new assay protocol with the Generic Wizard. Use this wizard to set-up the required Absorbance measurements. Then create a microplate layout containing the various sample types laid out as required. Complete the wizard and run the test.
 
After the measurements have been made you will see that the wizard has automatically added a blank correction transform and you can see the blank corrected results. Now to set-up the rest of the analysis: press Edit Transforms and Create a Matrix Expression transform. Under the Expression tab enter the expression:
 
100 * ((x-NSB)/(B0 - NSB))
 
This expression will perform the required %B/B0 calculation for each well. Note, referring to the groups such as NSB and B0 refers to the average of the replicates in the group (if any of those replicates are flagged they are not included in the average calculation)
 
Finally, create a Standard Curve Fit transform and specify the concentration values and set-up the various fit parameters as required.



Related articles:
DQ013: What is a transform?
Definition: Transform

DQ016: What is the Microplate Layout?
Definition: Microplate Layout

DQ040: What is the quickest way to take measurements now?
How to: Take measurements with an instrument now and setup some analysis afterwards…

DQ032: How do I use the changes I have made to the result analysis in future runs of the assay?
How To: Extract a version of the protocol for use in the future…





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