Design, Optimisation, Validation, and Analysis Framework for Multiplex Bead‑Based Biomarker Assays
Tegwen Marlais, Chris Drakeley & Chrissy h. Roberts
Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, UK
This book can be found here > https://chrissyhroberts.github.io/Mos-Def/
Files and Data needed to reproduce the results and visualisations are provided in this repo > https://github.com/chrissyhroberts/Mos-Def
1 Overview
This resource provides a generalised, platform‑agnostic wet‑lab and in silico framework for the design, optimisation, validation, use and analysis of data from multiplex bead‑based biomarker assays (e.g., Luminex xMAP) .
The method is presented through the lens of a novel multimarker immunoassay for evaluating febrile disease, but by using the concepts, methods and code included in this repo, you should be able to create and validate your own multiplexes.
These protocols also provide information on how to perform quality assurance experiments and to evaluate performance parameters such as the coefficient of variation. We also provide some analysis protocols which will support you to import, process and perform a quality controlled analysis of data from this or any multiplexed immuno-bead assay created on the MagPix platform.
1.1 Citation
To cite this work, please use:
Marlais, T., Drakeley, C., & Roberts, C.H. (2025). Design, Optimisation, Validation, and Analysis Framework for Multiplex Bead‑Based Biomarker Assays. London School of Hygiene & Tropical Medicine. Retrieved [YYYY-MM-DD] from https://chrissyhroberts.github.io/Mos-Def/ DOI pending.
1.2 About the method
The assay is a multiplexed sandwich assay designed for use with the Luminex MagPix and related devices. During the assay, analytes are bound to addressable microbeads using bead-bound, analyte-specific antibodies. The analytes bound to the beads then form complexes with analyte-specific biotin-conjugated detection antibodies followed by streptavidin-phycoerythrin (SA-PE) fluorescent reporter. This sandwich assay offers high specificity for the analytes, ensuring that each bead becomes fluorescent only when conjugated with the intended analyte. The level of SA-PE fluorescence serves as a proxy for the quantity of analyte bound to each bead. The addressable signals of the microbeads then allow for the identification of which analyte corresponds to a given SA-PE fluorescence signal. When performed with a plurality of capture-antibody conjugated beads and a corresponding set of biotin conjugated detection antibodies, the platform allows a highly specific estimate of analyte concentration across the 12 targets. To allow full reproduction of our panel of markers for febrile diseases, we provide exhaustive detail in the supplement.
1.3 Why This Method?
- Platform‑agnostic – applicable to most bead‑based multiplex immunoassay systems. Tested to work with Luminex MagPix
- Generalised principles – not tied to a specific disease or biomarker panel.
- Reproducible – includes full rationale for each design step.
- Citable – stable DOI for use in publications.
- Updatable – versioned for traceability and improvements over time.
1.4 Method Summary
- This framework guides you through the complete lifecycle of a multiplex bead‑based biomarker assay — from concept and reagent preparation to clinical testing, data analysis, and prognostic evaluation.
Project Overview & Planning
Define intended use, panel scope, and platform requirements.
Compile initial marker lists, shopping lists, and resource planning tools.
Reagent Preparation
Preparation of buffers and reagents.
Capture antibody preparation.
Detection antibody preparation.
Antigen preparation and storage.
Bead Coupling & Verification
Coupling capture antibodies to bead regions.
Confirmation of coupling efficiency.
Assay Design & Cross‑Reactivity Testing
Selection and pairing of capture/detection antibodies.
Systematic cross‑reactivity screening.
Mitigation strategies for problematic analytes.
Standard Curve & Calibration
Preparation of assay standards.
Dilution series design and use of calculators.
Standard curve modelling and performance checks.
Instrument Protocols
Luminex MagPix operating procedures.
Recommended plate layouts and run settings.
Assay Optimisation & Performance Characterisation
Coefficient of variation (intra‑ and inter‑assay) determination.
Spike‑and‑recovery testing.
Dilution linearity evaluation.
Specimen Testing
Volume requirements and handling guidance.
Integration of QC samples into every run.
Data Analysis
Importing and preprocessing raw MagPix output.
Standard curve fitting (e.g., 5‑PL) and concentration back‑calculation.
Replicate handling and quality control flagging.
Batch effect assessment and correction.
Prognostic and Diagnostic Modelling
Statistical evaluation of biomarker associations with clinical outcomes.
ROC curve generation, AUC, sensitivity, specificity, PPV, NPV.
Development of predictive models and multi‑marker signatures.
Documentation, Transparency & Reproducibility
Full reagent and protocol traceability.
Version‑controlled code and protocol sharing.
Contributor roles (CRediT taxonomy).
References and further reading.
1.5 Citing This Method
If you use or adapt this protocol in your own work, please cite:
Marlais, T., Drakeley, C. & Roberts, Ch. Design, Optimisation, Validation, and Analysis Framework for Multiplex Bead‑Based Biomarker Assays. London School of Hygiene & Tropical Medicine; 2025. Available from: https://github.com/chrissyhroberts/multiplex-assay-method doi:[DOI to be assigned]
1.6 Versioning & DOI
This method will be versioned using GitHub Releases.
Each public release will be archived in Zenodo, which will assign a permanent DOI.
The DOI for this version will be added once minted.
1.7 License
This work is released under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.
You are free to share and adapt the method, provided you give appropriate credit.
1.8 Contact
For questions, feedback, or collaboration requests:
Dr Chrissy H Roberts
Email: Chrissy.Roberts@lshtm.ac.uk
GitHub: @chrissyhroberts