Table of Contents

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2022-09-24
     ELISpot Assay
       Tutorial: ELISpot Assay Tutorial
         Import ELISpot Data
         Review ELISpot Data
       ELISpot Properties and Fields

ELISpot Assay


Premium Feature — Available with all Premium Editions of LabKey Server. Learn more or contact LabKey.

The Enzyme-Linked ImmunoSpot (ELISpot) assay is a highly sensitive method for analysis of antigen-specific responses at the cellular level and is widely used to monitor immune responses in humans and other animals. A variety of instruments provide raw ELISpot data, including CTL, Zeiss, and AID. LabKey Server provides a built in ELISpot assay type to support these commonly used assay machines. You can use the built-in assay type, as shown in the tutorial, or you can customize it to your specifications.

You can see a sample of detail available for ELIspot results in our interactive example.

Topics

Reference




Tutorial: ELISpot Assay Tutorial


Premium Feature — Available with all Premium Editions of LabKey Server. Learn more or contact LabKey.

This tutorial can be completed using a free 30-day trial version of LabKey Server.

The Enzyme-Linked ImmunoSpot (ELISpot) assay is a highly sensitive method for analysis of antigen-specific responses at the cellular level and is widely used to monitor immune responses in humans and other animals. A variety of instruments provide raw ELISpot data, including CTL, Zeiss, and AID. LabKey Server provides a built in ELISpot assay type to support these commonly used assay machines. You can use the built-in assay type, as shown in this tutorial, or you can customize it to your specifications.

You can see sample ELISpot data in our interactive example.

Tutorial Steps

First Step




Import ELISpot Data


Premium Feature — Available with all Premium Editions of LabKey Server. Learn more or contact LabKey.

In this first step of the ELISpot tutorial, we create a workspace, design a plate template, create an assay design, and then import some ELISpot data. This tutorial uses the defaults built in to LabKey Server. In actual use, you can customize both template and design to suit your experimental data.

Upload Sample Data


  • Log in to your server and navigate to your "Tutorials" project. Create it if necessary.
    • If you don't already have a server to work on where you can create projects, start here.
    • If you don't know how to create projects and folders, review this topic.
  • Create a new subfolder named "ELISpot Tutorial". Choose the folder type "Assay."
  • In the new folder, add a Files web part on the left.

  • Drag and drop the unzipped LabKeyDemoFiles directory into the upload area of the Files web part.

Configure an ELISpot Plate Template

  • Select (Admin) > Manage Assays.
  • Click Configure Plate Templates.
  • Select new 96 well (8x12) ELISpot default template from the dropdown.
  • Click Create to open the Plate Template Editor, which allows you to configure the layouts of specimens and antigens on the plate.
  • Enter Template name: "Tutorial ELISpot Template"
  • Explore the template editor. On the Specimen tab, you will see the layout for specimens:
  • Click the Antigen tab. You will see the layout for antigens.
  • The Control tab can be used for defining additional well groups. For instance, if you are interested in subtracting background wells, see Background Subtraction for how to define a set of background wells.
  • For this tutorial, we will simply use the default template. For customization instructions, see Customize Plate Templates.
  • Click Save & Close.

You now have a new ELISpot plate template that you can use as a basis for creating new assay designs.

Create a New Assay Design Based on the Template

  • Select (Admin) > Manage Assays.
  • Click New Assay Design.
  • Click the Specialty Assays tab.
  • Under Use Instrument Specific Data Format, choose ELISpot.
  • Under Assay Location, choose "Current Folder (ELISpot Tutorial)".
  • Click Choose ELISpot Assay.
  • In the Assay Properties section, enter:
    • Name: "Tutorial ELISpot Design".
    • Plate Template: choose your new "Tutorial ELISpot Template" if it is not already selected.
    • Detection Method: choose "colorimetric" if it is not already selected.
  • Review (but do not change) the fields in the other sections, click the headings to open.
  • Click Save when finished.

Import ELISpot Runs

  • Click the ELISpot Tutorial link to return to the main page.
  • In the Files web part, click the icon to show the folder tree.
  • Open LabKeyDemoFiles > Assays > Elispot.
  • Select Zeiss_datafile.txt.
  • Click Import Data.
  • In the pop-up, select Use Tutorial ELISpot Design (the one you just created) and click Import.
Batch Properties:
  • For Participant/Visit, select Specimen/sample id. (Do not check the box for "I will also provide participant id and visit id".)
  • Click Next.
Run Properties: Enter the following:
  • AssayID: ES1
  • Experiment Date: 2009-03-15
  • Plate Reader: Select "Zeiss" from the pulldown list.
  • Specimen IDs:
    • Specimen id values are often barcoded on labels attached to the plates. Enter these sample barcodes (They are taken from the file "LabKeyDemoFiles\Specimens\Specimen Barcodes.pdf" you downloaded earlier. They will integrate with specimens in our demo study.):
      • 526455390.2504.346
      • 249325717.2404.493
      • 249320619.2604.640
      • 249328595.2604.530
  • Click Next.
Antigen Properties
  • Fill out the antigen properties according to the screenshot below.
    • The cells/well applies to all antigens, so you can just fill in the first box in this column with "40000" and click the "Same" checkbox above the column.
    • The antigen names shown are examples to match our tutorial screenshots; you could enter any values here appropriate to your research.
  • Click Save and Finish when you are done.

Explore Imported Data

You will see a list of runs for the assay design. See Review ELISpot Data for a walkthrough of the results and description of features for working with this data.

Link Assay Data to the Demo Study (Optional Step)

You can integrate this tutorial ELISpot data into a target study following steps described in the topic: Link Assay Data into a Study. If you have entered matching participant and specimen IDs, you may simply select all rows to link.

When the linking is complete, you will see the dataset in the target study. It will look similar to this online example in the demo study on our LabKey Support site.

Start Over | Next Step (2 of 2)




Review ELISpot Data


Premium Feature — Available with all Premium Editions of LabKey Server. Learn more or contact LabKey.

After importing ELISpot instrument data, you will see the list of currently uploaded runs. This topic guides your review of ELISpot data, using the example run uploaded during the previous tutorial step.

Explore Uploaded Data

  • Return to the ELISpot Tutorial folder if you navigated away.
  • Click the name of the assay, Tutorial ELISpot Design.
  • You will see a list of runs for the assay design. (There is only one run in the list at this point.)
  • Click Run Details to see the run you just uploaded.
  • You will see two grids: the data, and a well plate summary.
  • Note the columns in the first grid include calculated mean and median values for each antigen for each sample well group.
  • This view is filtered to show only data from the selected run. If you had multiple runs, you could clear that filter to see additional data by clicking the in the filter box above the data section.
The second web part grid represents the ELISpot well plate.
  • Hover over an individual well to see detailed information about it.
  • Use the radio buttons to highlight the location of samples and antigens on the plate. Recall the layouts in the plate template we created; the well groups here were defined and associated with certain cells in that template.
  • Click View Runs to return to the list of runs for our "Tutorial ELISpot Design".
  • Now click the run name, ES1. If you used the file name, it will read "Zeiss_datafile.txt".
  • You will see the assay results data.

A similar set of ELISpot assay results may be viewed in the interactive example

Handle TNTC (Too Numerous To Count) Values

ELISpot readers sometimes report special values indicating that a certain spot count in a given well is too numerous to count. Some instruments display the special value -1 to represent this concept, others use the code TNTC. When uploaded ELISpot data includes one of these special values instead of a spot count, the LabKey Server well grid representation will show the TNTC code, and exclude that value from calculations. By essentially ignoring these out of range values, the rest of the results can be imported and calculations done using the rest of the data.

If there are too many TNTC values for a given well group, no mean or median will be reported.

Background Subtraction

One option for ELISpot data analysis is to subtract a background value from measured results. When enabled, each specimen group will have a single background mean/median value. Then for each antigen group in the sample, the mean/median for the group is calculated, then the background mean/median is subtracted and the count is normalized by the number of cells per well.

Enable Background Subtraction

To enable background well subtraction, you first configure the plate template. The main flow of the tutorial did not need this setting, but we return there to add it now.

  • Select (Admin) > Manage Assays.
  • Click Configure Plate Templates.
  • Open the plate template editor for "Tutorial ELISpot Template" by clicking Edit. Or Edit a copy if you prefer to give it a new name and retain the original version.
  • On the Control tab, create a well group called "Background Wells".
    • Type the name into the New field, then click Create.
    • Click Create Multiple to create several groups at once.
  • Select the wells you want to use as this background group.
  • Save the plate template.

  • If you created a new copy of the plate template with subtraction wells, edit the assay design to point to the new plate template.

When an assay design uses a plate with background wells defined, the user can selectively choose background subtraction for imported data by checking the Background Subtraction checkbox during import. When selected, background calculations will be performed. When not selected, or when the plate template does not specify a set of background wells, no background calculations will be performed.

On the ELISpot assay runs grid, there is a column displaying whether background subtraction has been performed for a run. The user can select runs in this grid, and then use the Subtract Background button to start a pipeline job to convert existing runs:

Once background subtraction calculations have been performed, there is no one-step way to reverse it. Deleting and re-uploading the run without subtraction will achieve this result.

Previous Step




ELISpot Properties and Fields


Premium Feature — Available with all Premium Editions of LabKey Server. Learn more or contact LabKey.

ELISpot Assays support import of raw data files from Zeiss, CTL, and AID instruments, storing the data in sortable/filterable data grids.

The default ELISpot assay type includes some essential properties and fields beyond the defaults included in standard assay designs. You can also add additional properties when you create a new assay design. This topic describes the properties in the default ELISpot assay design and how they are used.

Assay Properties

Assay properties are set by an administrator at the time of assay design and apply to all batches and runs uploaded using that design. The default ELISpot assay includes the standard assay properties, except for Editable Results and Import in Background. In addition, ELISpot assays use:

  • Plate Template
    • Choose an existing template from the drop-down list.
    • Edit an existing template or create a new one via the "Configure Templates" button. For further details, see Customize Plate Templates.
  • Detection Method: Choose the method used for detection. Options:
    • colorimetric
    • fluorescent

Batch Fields

Batch fields receive one value during import of a given batch of runs and apply to all runs in the batch. The default ELISpot assay does not add additional fields to the standard assay type. Data importers will be prompted to enter:

Run Fields

The user is prompted to enter values for run fields which apply to the data in a single file, or run. Run-level fields are stored on a per-well basis, but used for record-keeping and not for calculations.

Included by default:

  • AssayID: The unique name of the run - if not provided, the filename will be used.
  • Comments
  • ProtocolName
  • LabID
  • PlateID
  • TemplateID
  • ExperimentDate
  • SubtractBackground: Whether to subtract background values, if a background well group is defined. See Background Subtraction for more information.
  • PlateReader (Required): Select the correct plate reader from the dropdown list. This list is populated with values from the ElispotPlateReader list.
  • Run Data (Required): Browse or Choose the file containing data for this run.

Sample Fields

For each of the sample/specimen well groups in the chosen plate template, enter the following values in the grid:

  • SpecimenID: Enter the specimenID for each group here. These values are often barcoded on labels attached to the plates.
  • SampleDescription: A sample description for each specimen group. If you click the checkbox, all groups will share the same description.

Antigen Fields

The user will be prompted to enter these values for each of the antigen well groups in their chosen plate template, or they will be read from the data file or calculated. Use the Same checkbox to apply the same value to all rows when entering antigen IDs, names, and cells per well.

  • AntigenID: The integer ID of the antigen.
  • SpecimenLsid
  • WellgroupName
  • Mean
  • Median
  • AntigenWellgroupName
  • AntigenName
  • Analyte
  • Cytokine
  • CellsWell: Cells per well

Analyte Fields

The analyte fields are by default uploaded as part of the data file.

  • CytokineName

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