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Application Example: Interpolating Antibody Dilution Curves

Document ID: DQ261
Relevant to following products: WorkOut 2.0 Manta
Document description: Application Example: Interpolating Antibody Dilution Curves



A series of log titrations is made on six samples in duplicate with 8 dilutions. The absorbance OD is plotted on the Y axis and the dilution value along the X. The dilution value for each sample at 0.6 OD is to be calculated from its dilution curve.

To set this up:

  1. From the Organiser select Create a new protocol
  2. Select the IC50 Wizard
  3. Setup the Data Acquisition as required
  4. Setup the Microplate Layout containing 6 Unknowns in duplicate
  5. Setup the Concentration Replicates to be 2 and enter the dilutions
  6. Continue and complete the wizard specifying additional options as required.
  7. Run the protocol for the first time to get your raw data in.
  8. Inspect your dilution curves.
  9. To add the interpolation of the curves, add two new transforms: The first transform makes it easier if you want to change the OD value from 0.6. The second transform calculates the dilution for each group at the specified OD:
        i)  Create an Expression transform. Under the Expression tab, enter OD for the Result Name and under the Expression enter 0.6 
        ii) Create a Matrix Expression transform. Under Expression tab, paste the following expression:

             #1,g,c*pow((#1,g,d-#2.OD)/(#2.OD-#1,g,a), 1/#1,g,b)

            This expression calculates the dilution at the specified OD for each group. (See below for a more detailed explanation)

            Under the Output Matrix enter a meaningful name for this data, such as Dilution @ OD
  10. Recalculate the results and you will see the new column in the main table of the report “Dilution @ OD”

    To change the OD to calculate the dilution of (i.e. a value other than 0.6), press Edit Transforms, double click on the Expression transform and change the 0.6 as required. Press Recalculate.

An example results file can be downloaded from here.

The expression: #1,g,c*pow((#1,g,d-#2.OD)/(#2.OD-#1,g,a), 1/#1,g,b) is used because there are a number of groups on the plate, each group has a curve fit calculating the coefficients of the four parameter fit: a,b,c,d – these are arranged to calculate the dilution for a given OD.   Writing this expression in the Matrix Expression transform means that the expression is calculated on each well to calculate the dilution at the specified OD for its group. The #1,g part of the expression refers to the calculated coefficients of the curve fit of each group (caclulated in the first transform). #2.OD refers to the OD to calculate the dilution (specified in the second transform).

The underlying expression is: c*pow((d-OD)/(OD-a), 1/b) evaluated on each well on the plate. 






Related articles:
DQ267: How To: Show the calculated coefficients of each fit with the Competitive Bindings transform
How To: Show the calculated coefficients of each fit with the Competitive Bindings transform

DQ153: How can I calculate the concentrations of my unknowns using standards data measured in a previous assay?
How To: Use archived standards data?

DQ159: IC50 Example
Application Example: Inhibition Concentration of Malaria Parasites in Red Blood Cells





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