ELISpot Assay Excellence: Why PBMC Quality and Standardisation Define Success

The enzyme-linked immunospot (ELISpot) assay has established itself as an indispensable tool for monitoring immune responses at the single-cell level, underpinning advances in vaccine development, gene therapy, and immuno-oncology. Its capacity to detect rare, antigen-specific T-cell or B-cell responses with high sensitivity makes it the assay of choice for clinical trials and translational research. The reliability of ELISpot data is fundamentally tied to the quality of the starting material—namely, peripheral blood mononuclear cells (PBMCs)—and the rigour of assay standardisation.

The Central Role of PBMC Viability

It is often said that “an assay is only as good as its sample,” and nowhere is this truer than with ELISpot. PBMCs are typically isolated from whole blood, cryopreserved, and later thawed for use in the assay. Their viability and functional integrity directly influence the assay’s sensitivity and reproducibility. Even the most meticulously optimised ELISpot protocol will falter if the PBMCs are not “healthy and happy”.

At Synexa, we have observed that PBMC viability above 70% is essential for reliable ELISpot results—a threshold supported by both internal validation and external multi-site proficiency testing. Achieving this standard requires attention to several critical factors:

• Isolation Protocols: Standardisation is key. Variability in centrifugation speed, temperature, or anticoagulant choice can compromise cell health.
• Cryopreservation and Thawing: Controlled freezing and careful thawing at 37°C are crucial. Use of validated cryoprotective agents help preserve cell function.
• Time-to-Processing: Delays in processing blood samples can impact PBMC viability and function.

Standardisation—From Sample to Spot

The reproducibility of ELISpot data is not just a function of cell quality but also of rigorous standardisation at every step. This extends from sample handling to plate washing, reagent preparation, and analysis. The field has made significant strides, with guidelines now available for validation and standardisation. At Synexa, we have implemented multi-site harmonisation of protocols, ensuring that samples processed across our sites yield comparable results.

• Internal Controls: Internal QC samples are run with each batch to monitor isolation and assay consistency.
• Antigen Stimulation: Titration of cell numbers and peptide pools used for stimulation to balance maximal immune activation and minimal background.
• Reagent Stability: Longitudinal stability testing is essential for custom peptide pools. Where manufacturer data are lacking, in-house validation ensures that reagents perform consistently over time.

Troubleshooting and Best Practices

Even with best practices, challenges remain. Elevated background in negative controls or low spot counts in antigen wells can confound interpretation. To address these:

• Serum Selection: The choice of serum lot can influence background noise. Pre-screening and validating serum lots for low background and high antigen-specific responses is recommended.
• Automated Spot Counting: Employing automated readers and following a well-defined Standard Operating Procedure (SOP) reduces user bias and improves reproducibility.

The Synexa Perspective

Our experience in supporting global clinical trials has shown that investing in PBMC quality and rigorous standardisation pays dividends in data reliability and regulatory acceptance. At Synexa Life Sciences, our ELISpot (Enzyme-Linked ImmunoSpot) assay services enable precise detection and quantification of antigen-specific immune responses at the single-cell level. This highly sensitive assay measures the secretion of cytokines, antibodies, or other immune markers, providing valuable insights into immune cell activity and function.

For further reading on our approach to ELISpot and immune monitoring, visit Synexa’s ELISpot page, or check out our latest webinar by clicking below.