LabKey Server's tool for Luminex® assays help you to manage, quality control, analyze, share, integrate and export results from BioPlex instruments. Luminex immunoassays are plate-based assays that can measure multiple analytes independently in each well.
Additional information can be found in this paper:
There are two tutorials which introduce the Luminex features using some example data. They are independent of each other, but we recommend completing them in order to learn your way around the tools. The tutorial scenario is that we want to evaluate binding between a panel of HIV envelope proteins (antigens) and plasma immunoglobulin A (antibodies) from participants in a study.
LabKey Server supports multiplexed immunoassays based on Luminex xMAP® technology
. A Luminex assay multiplexes analysis of up to 500 analytes in each plate well. In contrast, an ELISA requires a separate well for analysis of each individual analyte.
Luminex immunoassays often aim to do one or both of the following:
- Measure the strength of binding between a set of analytes (e.g., virus envelope antigens) and unknowns (e.g., blood serum samples with unknown antibody activity).
- Find concentrations of unknowns using dose-response curves calculated for titrated standards.
LabKey Server supports using an R transform script to customize Luminex analyses and using Levey-Jennings plots for performing cross-run quality control.
Binding and Concentrations
Each analyte is bound to a different type of bead. Each bead contains a mixture of red and infrared dyes in a ratio whose spectral signature can identify the bead (and thus the analyte).
In each plate well, analyte-bound beads of many types are combined with a sample. The sample added to each plate well is typically a replicate for one of the following:
- A titrated standard whose concentrations are known and used to calculate a reference curve
- A titrated quality control used for verification
- An unknown, such as serum from a study participant
- A background well, which contains no active compound and is used for subtracting background fluorescence
Bead-analyte-sample complexes are rinsed, then allowed to react with a phycoerythrin-bound detection reagent, then rinsed again. The fluorochrome attached to the detection reagent serves as the reporter of binding.
Next, each bead is illuminated by two lasers to detect the bead/analyte type (from the red/infrared ratio) and sample binding (from the fluorochrome's fluorescence). The instrument reports the median fluorescence intensity (FI) for all beads attached to a given analyte type for each well, among other measures.
The LabKey Luminex transform script
The LabKey Luminex tool can be configured to run a custom R transform script that applies custom curve fits to titrated standards. The script also uses these curve fits to find estimated concentrations of samples and other desired parameters.
The data used by the script for such analyses can be customized based on the latest lab techniques; for example, the default script allows users to make choices in the user interface that enable subtraction of negative bead FI to account for nonspecific binding to beads.
The methods and parameters used for the curve fit can be fully customized, including the weighting used for squared errors to account for trends in variance, the equation used to seed the fitting process, and the optimization technique.
Developers can customize the R transform script to use the latest R packages to provide results that reflect the most advanced algorithms and statistical techniques. Additional calculations can be provided automatically by the script, such as calculations of a result’s “positivity” (increase relative to a baseline measurement input by the user).
Levey-Jennings plots can help labs execute cross-run quality control by visualizing trends and identifying outliers.
LabKey Server automatically generates Levey-Jennings plots for a variety of metrics for the curve fits determined from titrations of standards. See Step 5: Track Analyte Quality Over Time
for more information.