The enzyme linked immunosorbent assay (ELISA) is a plate-based technique that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. The layout of controls, samples, and replicates across a 96-well plate is used to evaluate the meaning of data collected from those wells.

Overview

LabKey's ELISA assay type imports and interprets raw data from BioTek Microplate readers. A calibration curve is generated, tracking absorption over concentration. The coefficient of determination is also calculated automatically.

Like other built-in plate based assays, each ELISA assay design you create is associated with a specific 96-well plate template. The plate template clarifies what samples and controls are in which wells and is used by the assay design to associate result values in the run data with the correct well groups and specimens.

An example application of the ELISA assay is to analyze saliva samples for protein biomarkers that might indicate cancer or autoimmune disease. Samples are applied to the plate at various concentrations. The absorption rate at each concentration is read, plotted, and can be analyzed further to better understand biomarkers.

Topics

• Tutorial: ELISA Assay
• ELISA Run Details View
• ELISA Assay Reference: Details about plate template layouts and assay properties and fields.

Related Topics

• Specialty Plate-Based Assays

The enzyme linked immunosorbent assay (ELISA) is a plate-based technique that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. The layout of controls, samples, and replicates across a 96-well plate is used to evaluate the meaning of data collected from those wells.

LabKey's ELISA assay type imports and interprets raw data from BioTek Microplate readers.

This tutorial shows you how to:

• Set up an ELISA Plate Template and Assay Design
• Import ELISA Experiment Data
• Visualize the Data

Set up an ELISA Plate Template and Assay Design

To begin, create an assay folder to work in:

Next configure a plate template that corresponds to the plate of your ELISA experiment.

• In the Assay List web part, click Manage Assays.
• Click Configure Plate Templates.
• Select "new 96 well (8X12) ELISA default template" from the dropdown.
• Click Create to open the editor.
• Enter a Template Name, for example, "ELISA Tutorial Plate 1".
• Review the shape of the template, clicking the Control, Specimen, and Replicate tabs to see the well groupings. Notice that you could edit the template to match alternate well groupings if needed; instructions can be found in Customize Plate Templates. For the purposes of this tutorial, simply save the default.

• When finished, click Save & Close.

Now you can create the assay design using that template:

• Click the ELISA Tutorial link near the top of the page to return to the main folder page.
• Click New Assay Design.
• Click the Specialty Assays tab.
• Select ELISA (if it is not already chosen by default and choose "Current Folder (ELISA Tutorial)" from the Assay Location dropdown, then click Choose ELISA Assay.

• In the Assay Properties section, enter:
• A Name, for example, "ELISA Tutorial Assay".
• Select a Plate Template: "ELISA Tutorial Plate 1" (it may already be selected by default).
• Review the fields defined in each of the other sections by clicking each heading to page through them. You could add or edit fields, but for this tutorial, leave the default design unchanged.
• Click Save.
• You will return to the main folder page and see your new design on the assay list.

Import ELISA Experiment Data

• Download sample data:
• biotek_01.xls
• biotek_02.xls
• biotek_03.xls
• In the ELISA Tutorial folder's Assay List, click the name you gave your assay design, here: ELISA Tutorial Assay.
• Click Import Data.
• For Batch Properties, accept the default settings by clicking Next.
• In the Run Data field, click Choose File (or Browse) and select one of the sample data files you just downloaded. Make no other changes on this page, then click Next.
• On the next page, enter Concentrations for Standard Wells as shown here:

• Click Save and Import Another Run.
• Repeat the import process for the other two files, selecting the other two Run Data files in turn. The concentrations for standard wells will retain their values so you do not need to reenter them.
• Click Save and Finish after the last file.

Visualize the Data

When you are finished importing, you'll see the runs grid showing the files you just uploaded. Since you did not specify any other Assay IDs, the filenames are used. Browse the data and available visualizations:

• Hover over the row for Assay Id "biotek_01.xls", then click (details).

• You will see a visualization of the Calibration Curve.
• Use the Selection options to customize the way you view your data. Learn more about this page in the topic: ELISA Run Details View.

• Below the plot, you can click to View Results Grid, which will show you the results data for the specific run as if you had clicked the AssayId instead of the (details) link.

You have now completed the ELISA tutorial. You can continue to explore the options available on the details page in this topic:

• ELISA Run Details View

Related Topics

• ELISA Run Details View
• ELISA Assay Reference
• Specialty Plate-Based Assays
• Tutorial: Import Experimental / Assay Data
• Customize Plate Templates

• ELISA Run Details Plot
• Data Selections
• Plot Options
• Curve Fit
• Calibration Curve

ELISA Run Details Plot

When you import ELISA Assay data, you can view details for any run in an interactive visualization panel. By default, we display the standard curve plotted for concentration versus absorption (or raw signal). The calculated concentrations for the sample well groups are plotted as points around the standard curve. The curve is a linear least squares fit and we pass in the parameters of the curve fit (offset and slope).

• Data Selections
• Plot Options
• Curve Fit
• Calibration Curve

Data Selections

In the Data Selections panel, you can choose:

• Samples:
• Check the box to Show all Samples. (Default)
• Otherwise use the dropdown to select which Samples to plot.
• Controls:
• Check the box to Show all Controls. (Default)
• Otherwise use the dropdown to select which Controls to plot.

Plot Options

• X-Axis: The default is a Linear scale, check the box to switch to Log scale.
• The default is Concentration. Use the menu to select another metric to plot on this axis:
• Y-Axis: The default is a Linear scale, check the box to switch to Log scale.
• The default is Absorption. Use the menu to select another metric to plot on this axis.
• Display Options:
• Show legend: Checked by default. Uncheck to hide it.

Additional Metrics to Plot

For both axes, the set of metrics available include:

• Absorption
• Absorption CV
• Absorption Mean
• Concentration
• Concentration CV
• Concentration Mean
• Dilution
• Percent Recovery
• Percent Recovery Mean
• Std Concentration

Curve Fit

• Show curve fit line: Checked by default. Uncheck to hide it.
• When checked, the panel will display the following for this curve fit:
• R Squared
• Fit Parameters: Parameters specific to the curve fit.

• intercept
• slope

Calibration Curve

On the right, you'll see the plotted Calibration Curve based on how you've set the above selectors.

Below the plot, you have three buttons:

• View Results Grid: View the results data for this run.
• Export to PNG: Export the current plot display as a PNG file.
• Export to PDF: Export the current plot display as a PDF file.

Related Topics

• Tutorial: ELISA Assay
• ELISA Assay Reference

This topic provides a reference for plate layouts, assay properties, and fields used in the LabKey ELISA Assay implementation. When you design your own plate template and assay design, you can customize and add to these defaults as needed to suit your experiment.

• ELISA Plate Templates
• Default Template
• Undiluted Template
• ELISA Assay Properties
• ELISA Assay Fields

ELISA Plate Templates

There are two base templates you can start from to best fit your ELISA experiment design.

• In your assay folder, select (Admin) > Manage Assays.
• Click Configure Plate Templates.
• Select either the default or undiluted ELISA template to use as a starting place, depending on the structure of your experiment.

• Click Create to open the editor.
• The Template Name is required and must be unique.
• Learn about customizing plate templates in this topic: Customize Plate Templates.
• Click Save & Close when finished.

ELISA Default Template

The ELISA Default Template is suitable for an application where you have 5 specimens and a control, typically applying dilution across the wells in the plate. There are three layers, or tabs, in the plate design template: Control, Specimen, and Replicate. The defaults for well layout are as shown:

You can change the well layouts, well group names, and add well group properties before saving your new plate template for use.

ELISA Undiluted Template

The ELISA Undiluted Template is suitable for an application where you have up to 42 specimens and a control. Each specimen is present in two wells. There are three layers, or tabs, in the plate design template: Control, Specimen, and Replicate. The defaults for well layout are as shown:

You can change the well layouts, well group names, and add well group properties before saving your new plate template for use.

ELISA Assay Properties

The default ELISA assay includes the standard assay properties, except for Editable Results and Import in Background. In addition, ELISA 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.

ELISA Assay Fields

The default ELISA assay is preconfigured with batch, run, sample, and results fields. You can customize a named design to suit the specifics of your experiment, adding new fields and changing their properties. Learn more in Field Editor.

Batch Fields

• Participant/Visit: See Participant/Visit Resolver Field
• Target Study (Optional): See Link Assay Data into a Study

Run Fields

• RSquared: Coefficient of determination of the calibration curve
• CurveFitParams: Curve fit parameters

Sample Fields

• SpecimenID
• ParticipantID
• VisitID
• Date

Results Fields

• SpecimenLsid
• Well Location
• WellgroupLocation
• Absorption: Well group value measuring the absorption.
• Concentration: Well group value measuring the concentration.

Related Topics

• Specialty Plate-Based Assays
• Customize Plate Templates
• ELISA Assay
• Tutorial: ELISA Assay

Enhancements to LabKey's ELISA Assay tools are under development, including import of high-throughput data file formats which contain multiple plates and multiple analyte values per well. When completed, the tools will support:

• A 4 plex data format. Plates will be able to have up to 4 values per well location, one for each protein/analyte combination.
• Additional curve fit options for Standard curves, including 4 and 5 parameter (4pl and 5pl) alongside the existing linear curve fit.
• High throughput data support: The current ELISA assay uses a 96 well plate. With these enhancements, 384 well plates are supported.
• Input of plate data and metadata from files, where the current ELISA assay requires that sample and concentration information be added manually.
• Additional statistics calculations beyond what is supported currently.

Enable Experimental Feature

To use these enhanced ELISA assay options, a Site Administrator must enable the experimental feature.

Note that experimental features are under development and there is no guarantee that future changes will be backwards compatible with current behavior.

4-plex Data Format

The existing ELISA assay will be extended so that it can import a single file which contains the 4-plex format, in which some of the plates will have 4 values per well location. There will be a row per well/analyte combination. For every row, sample information will also be included and will be parsed out so that sample properties can be stored separately from the plate data.

Additional Curve Fit Options

When using the enhanced ELISA assay functionality, the Run Properties contain an additional field:

• CurveFitMethod

• 4 Parameter
• 5 Parameter
• Linear

High Throughput Plate Template

Before creating any ELISA assay design, create the custom plate templates you require. You can start from our default and make changes as needed to match your experiments. With the experimental features available, in addition to the low-throughput plates supported for ELISA assays, you can now use high-throughput 384 well plates:

• Selecting (Admin) > Manage Assays.
• Click Configure Plate Templates.
• From the Create New Template dropdown, select new 384 well (16x24) ELISA high-throughput (multi plate) template.

• Click Create.
• Give your new definition a unique Template Name (even if you make no changes to the defaults).
• Use the plate designer to customize the default multiplate layout to suit your needs.

• Click Save & Close.

Create ELISA Assay

With the experimental features enabled, you gain the ability to submit a Combined File Upload including metadata alongside your data, instead of requiring the user input metadata manually.

• In the Assay List web part, or by selecting (Admin) > Manage Assays, click New Assay Design.
• In the Choose Assay Type panel, click the tab for Specialty Assays.
• Under Use Instrument Specific Data Format, select ELISA (if it is not already selected by default).
• Under Assay Location, select where you want to define the assay. This selection determines where it will be available.
• Click Choose ELISA Assay.

• Under Assay Properties, give your new Assay Design a Name.
• Select the Plate Template you defined.
• Select the Metadata Input Format you will use:
• Manual
• Combined File Upload (metadata and run data)
• Enter other properties as needed.

• Review the Batch, Run, Sample, and Results Fields sections, making adjustments as needed.
• Click Save.

Related Topics

• ELISA Assay
• ELISA Run Details View