Customised labelling of antibodies: A powerful tool for multi-parameters analyses in Flow Cytometry

Flow cytometry is widely used to quantitatively identify and characterise the biological activity of cell samples, particularly in Quality Control applications for cell products. However, designing customised biomarker panels remains challenging due to the limited availability of antibodies conjugated to a broad range of fluorochromes, especially for stem cell markers. This limitation can restrict multi-parameter analyses and reduce the full exploitation of modern cytometers.

This application note presents an analytical strategy for generating customised labelled antibodies using multiple fluorochrome classes, assessing their quality, and validating their performance. It demonstrates how in-house labelling can address panel design constraints while supporting robust analytical workflows compliant with validation guidelines. The full technical application note is available for download below.

KEY TECHNICAL OUTCOMES

• Customised antibodies labelled with three fluorochromes (AF488, AF647 and PB) compatible with blue, red and violet lasers
• Comparable accuracy and coefficient of variation to commercially available antibodies across a full dilution range
• Stability demonstrated up to 6 months at 4°C and -20°C with no significant differences (p > 0.05)
• Accuracy results ranging from 80%–103% (QC Med), 70%–124% (LOQ), depending on concentration levels
• Full validation performed according to ICH Q2(R1), including specificity, linearity, precision, LOD, LOQ and range

HOW THIS SUPPORTS YOUR PROGRAMME?

This approach supports the development and validation of tailored flow cytometry panels, particularly for complex cell products such as stem cells, where flexibility and robustness are essential.

• Enables customised panel design beyond the limitations of commercially available fluorochrome-antibody combinations
• Supports multi-parameter analyses aligned with modern flow cytometry instrumentation (multi-laser systems)
• Ensures data reliability through demonstrated accuracy, precision and robustness
• Facilitates compliance with international validation guidelines (ICH Q2(R1))
• Provides strong statistical support through an experimental design suitable for comprehensive performance assessment
• Maintains analytical consistency with negligible impact from multicolour staining, fixation or light exposure

Overall, this strategy provides a validated and flexible solution to enhance flow cytometry assay development and Quality Control applications.

APPLICATIONS / USE CASES

• Characterisation of hematopoietic progenitor stem cells
• Quality Control testing of cell-based products using flow cytometry
• Development of customised multi-marker panels for complex cell populations
• Method validation of flow cytometry assays following regulatory guidelines