The range of technologies available for protein biomarker analysis has expanded considerably. Researchers can now measure proteins that were previously below the detection limits of conventional immunoassays, profile hundreds of targets from a small sample and generate detailed biomarker datasets across large or longitudinal studies.

However, greater choice can make platform selection more complicated.

NULISA, Simoa and MSD are all sensitive immunoassay technologies, but they are not interchangeable. Each platform has different strengths in sensitivity, multiplexing, assay availability, sample requirements, quantification and customisation. The right choice therefore depends on more than selecting the platform with the highest stated sensitivity or largest panel. It depends on the biological question, the expected abundance of the targets, the available sample matrix and volume, the stage of the biomarker programme and how the resulting data will be used.

Start with the study objective

A discovery study may aim to identify previously unknown biomarkers or biological pathways. This usually benefits from broad multiplexing.

A validation study focuses on a smaller number of candidate biomarkers and may prioritise sensitivity, reproducibility and quantitative performance.

A monitoring study repeatedly measures established markers over time, requiring a robust and scalable targeted assay.

In many cases, different platforms can be used at different stages of the same biomarker programme.

When is NULISA suitable?

NULISA combines immunoassay specificity with highly sensitive nucleic-acid-based detection. In the NULISAseq format, target-specific DNA barcodes are measured using next-generation sequencing, allowing hundreds of proteins to be analysed simultaneously.

This technology achieves exceptional sensitivity through a proprietary dual-capture and wash process coupled with proximity ligation chemistry. This approach reduces non-specific background while amplifying true target signals, enabling reliable measurement of proteins present at ultra-low abundance.

NULISA may be particularly useful when:

• The study is exploratory or discovery-focused.

• A broad protein signature is required.

• Low-abundance cytokines or chemokines need to be measured.

• Sample volume is limited.

• Pathway-level changes are of interest.

• The most relevant biomarkers are not yet known.

Its combination of high sensitivity and multiplexing makes NULISA particularly well suited to inflammatory and neurological biomarker research.

Importantly, NULISA is not limited to large discovery panels. Lower-plex panels, including focused options such as the Alzheimer’s disease 5-plex, are available alongside singleplex assays and custom assay development. This enables the technology to support both broad profiling and targeted measurement.

High-multiplex studies do, however, generate more complex datasets. Statistical analysis, multiple-testing correction, inter-individual variability and biological interpretation should therefore be considered during study design.

When is Simoa suitable?

Simoa, or single-molecule array technology, is a digital immunoassay designed to measure proteins at extremely low concentrations.

Target molecules are captured on antibody-coated beads and isolated in very small wells, allowing individual immunocomplexes to be detected. This makes Simoa particularly useful for proteins that may be below the detection range of conventional immunoassays.

Simoa may be appropriate when:

• The study focuses on a small number of defined biomarkers.

• The targets are expected to be present at extremely low concentrations.

• Candidate biomarkers have already been identified.

• Highly sensitive quantitative measurement is required.

• Longitudinal changes in an established biomarker need to be monitored.

Simoa is widely used for low-abundance neurological and inflammatory biomarkers. Its main trade-off is multiplexing, as it is generally better suited to focused target measurement than broad protein discovery.

When is MSD suitable?

MSD uses electrochemiluminescence detection. Capture antibodies are placed on electrodes within the assay plate, and light is produced when an electrical signal activates labels attached to the detection antibodies.

The platform offers sensitive detection, low background and a broad dynamic range. Assays can be run in singleplex or multiplex formats, with established panels available for many cytokines and other biomarkers. Up to 10 analytes can also be combined within a custom or self-defined multiplex panel, allowing researchers to build a focused assay around their target list.

MSD may be suitable when:

• The target list is already defined.

• Moderate multiplexing is sufficient.

• Proteins occur across a broad concentration range.

• A scalable workflow is needed for a larger cohort.

• A suitable commercial panel is available.

• Custom multiplex assay development is required.

MSD may not provide sufficient sensitivity for every extremely low-abundance target. However, when biomarkers fall within its quantitative range, it can provide a robust and practical option for targeted validation studies.

These categories are not absolute. Performance also depends on the target, assay reagents, sample matrix and study design.

Example study scenarios

A researcher investigating broad inflammatory responses without a predefined target list may benefit from NULISA multiplex panels because hundreds of proteins can be profiled simultaneously.

A study validating one low-abundance neurological biomarker in plasma may be better suited to Simoa, where focused sensitivity is more important than multiplexing.

A project measuring an established panel of eight cytokines across several hundred samples may benefit from MSD, particularly if a suitable panel already exists.

Can more than one platform be used?

Yes. A staged strategy may provide the strongest approach.

A high-plex technology such as NULISA can first be used to discover candidate biomarkers. The most promising proteins can then be prioritised according to biological relevance, effect size, detectability and reproducibility.

Simoa or MSD may subsequently be used to validate selected targets in a larger or independent cohort. Measuring a candidate using a second technology can also provide confidence that the result is reproducible and not specific to one platform.

Choosing around the biological question

There is no single best platform for every biomarker study.

NULISA is well suited to broad and sensitive protein discovery. Simoa offers highly sensitive measurement of selected low-abundance biomarkers. MSD provides a flexible and scalable approach for defined target panels.

Platform selection should consider:

• The purpose of the study

• The number and abundance of targets

• Available sample volume

• Sample matrix

• Cohort size

• Quantification requirements

• Future validation plans

At Altheome, we work across NULISA, Simoa and MSD, alongside custom assay development, protocol optimisation and biomarker data interpretation. This allows the analytical strategy to be designed around the scientific question rather than restricted to a single platform.

Researchers planning a protein biomarker study can speak with the Altheome scientific team to assess target feasibility, sample requirements and the most appropriate platform.