Antibody titration is the process of determining the optimal concentration of an antibody that provides the best resolution between the positive and negative signal. Titration is important to help reduce non-specific binding, reduce spectral spillover (for fluorescent antibodies), and minimize background noise. Titration is normally done with a serial dilution series and for each new lot of antibodies.
OMIQ allows you to calculate the Stain Index, Separation Index, and Signal-To-Noise Ratio in a single task. These are measures that help determine the relative intensity of an antibody by comparing the positive and negative signals.
The Stain/Separation Index task in OMIQ allows you to analyze a Titration Series or Compare the Relative Brightness of dyes.
This allows you to examine a single dye at various concentrations, identifying the concentration with the highest level of stain / separation index or signal-to-noise ratio. This is the concentration yielding the best separation between positive and negative populations.
1.1 Add Metadata to your Dataset
Antibody concentration metadata is required to analyze a titration series. This metadata can represent the dilutions or concentrations in your titration assay. The values can be numbers (e.g. 5, 2.5) or fractions (e.g. 1/20, 1/160). You can optionally add units to these values. The example above shows a metadata for volume of antibody added.
To learn more about how to add metadata to your dataset, please view our article on How to Set Metadata for Files in OMIQ.
1.2 Add a Stain/Separation Index Task to your Workflow
Click Add new child task and select Stain/Separation Index from the task selector.
Your workflow may look different from above, depending on whether a Compensation task is present. The important thing is to add the Stain/Separation Index task after a Scaling task.
1.3 Choose the Titration Method
Choose Titration as the Method.
1.4 Choose the Metadata for your Titration Series
Choose the appropriate Titration Series Metadata that denotes the antibody concentration/dilution as entered in Step 1.1.
1.5 Choose the Channel (Feature) for Titration
Click on Choose Channel (Feature).
Select the channel (Feature) to use for the calculation.
Click Submit.
1.6 Select the Files for Titration
OMIQ attempts to select the files for titration automatically. However, it is best to review that the selected files are correct.
To review the file selections, click on Configure Files.
Review the File selection for the Channel (Feature).
Click on Submit.
1.7 Generate Gates and Set Position
In the gating plot, select the File you would want to draw clean up gates on. Here, we have drawn a Scatter gate and a Singlets gate.
In this example, we would want to generate our gates for titration on our Singlets gate. We would need to activate our Singlets gate. Right Click on the Singlet node in the Gating Tree and select Activate. An active node is denoted by the Green Arrow on the gating tree.
Click on Generate Gates to generate the positive and negative gates that will be used for titration.
Adjust the positive and negative gates for each file in your titration. Turn on Per-File Positioning to adjust the gates on a per file basis by right clicking on the gate and selecting Enable per file positioning.
1.8 Calculate the Titration Statistics
Click Calculate Statistics.
The task will create a table that contains the different statistics calculated and the values used for the calculations. Click on Copy Table to copy the data into a spreadsheet editor.
1.9 View the Results in a Figure Task
Click on Save Task and return to your workflow.
Click on Add new child task and select Figure in the task selector.
Click on Actions then go to Figure Actions then select View Stain Index Results.
Select the Stain Index Task that you would want to view the results for and click Submit.
OMIQ will generate a special x axis plot, the gating plots, and regression plots for the Stain Index, Separation Index, and Signal to Noise Ratio of your titration series.
You can compare the stain index, separation index, and signal-to-noise ratio of different dyes for a particular antibody clone. Often, the best concentration candidate, as determined by the titration series, is chosen for its respective channel (feature).
2.1 Add a Stain/Separation Index Task
Click Add new child task and select Stain/Separation Index from the task selector.
Your workflow may look different from above, depending on whether a Compensation task is present. The important thing is to add the Stain/Separation Index task after a Scaling task.
2.2 Choose the Relative Brightness Comparison Method
Choose Relative Brightness Comparison as the Method.
2.3 Choose the Channels (Features)
Click on Choose Channels (Features). This will bring up a dialog box that allows you to Choose the Channels (Features) you would want to compare the relative brightness for. Click Submit.
2.4 Configure the Files for the Selected Channels (Features)
Click on Configure Files. This will open up a dialog box that allows you to select the file of the best candidate concentration for the corresponding channel (feature). Click Submit.
It is best to review and confirm the file to channel (feature) mappings generated by OMIQ.
2.5 Generate the Gates and Set the Position
In the gating plot, select the File you would want to draw clean up gates on. Here, we have drawn a Scatter gate and a Singlets gate.
In this example, we would want to generate our gates for titration on our Singlets gate. We would need to activate our Singlets gate. Right Click on the Singlet node in the Gating Tree and select Activate. An active node is denoted by the Green Arrow on the gating tree.
Click on Generate Gates to generate the positive and negative gates that will be used for the relative brightness comparison.
Adjust the positive and negative gates for each file that you want to perform the relative brightness comparison on.
Clicking on the nodes in the gating tree should load the file you have chosen for the feature in Step 2.4.
2.6 Calculate the Statistics
Click on Calculate Statistics.
The task will create a table that contains the different statistics calculated and the values used for the calculations. Click on Copy Table to copy the data into a spreadsheet editor.
2.7 View the Results in a Figure Task
Click on Save Task and return to your workflow.
Click on Add new child task and select Figure in the task selector.
Click on Actions then go to Figure Actions then select View Stain Index Results.
Select the Stain Index Task that you would want to view the results for and click Submit.
OMIQ will generate the gating plots and bar charts that compare the Stain Index, Separation Index, and Signal to Noise Ratio for the chosen channels (features).