PhD Scientific Days 2025

Poster Session I. - I: Theoretical and Translational Medicine

Development and characterisation of a cell-based assay for measuring antibody-induced complement activating potential of human serum

Name of the presenter

Horánszky Dénes

Institute/workplace of the presenter

Semmelweis University, Department of Internal Medicine and Haematology, Research Laboratory

Authors

Dénes Horánszky¹

1: Semmelweis University, Department of Internal Medicine and Haematology, Research Laboratory

Text of the abstract

Introduction
We previously developed a flow cytometry- (FC-) based kinetic functional assay to study the complement-mediated effect of rituximab in a Raji B-cell model. We confirmed that rituximab induces lysis of a subset of cells in a complement-mediated manner and found that certain cell surface molecules significantly affect the complement-mediated effect of rituximab.

Aims
To improve our method and investigate its repeatability and capacity to distinguish human sera with different complement functional activity.

Method
Raji cells (immortalised CD20-positive Burkitt's lymphoma) were suspended in HBSS buffer containing 7.5 m/v% bovine serum albumin (BSA), treated with 1.5 μL/10^5 cells of rituximab, and labelled with 3 μL/10^5 cells of 7AAD (non-membrane-permeable fluorescent DNA dye). As a complement source, 10 or 20% human serum was added to the cells, which were incubated at 37°C and repeatedly measured for 90 minutes by FC. Tested serum samples included normal human serum (NHS), heat-inactivated NHS (ΔNHS), their mixtures, and samples of patients with complement alternative (AP) or classical pathway (CP) dysregulation.

Results
The final increment of 7AAD-positive (i.e. membrane-permeable) cell proportions increased in parallel with the percentage of NHS in the NHS:ΔNHS mixtures. The 100:0 mix had an increment of 75% and 86%, at 10% and 20% serum concentrations, respectively. Results were repeatable across measurements: the average standard deviation (SD) was 2.1% and 2.7% for 3 measurements with 10% total serum concentration and 2 with 20%, respectively. The final increment in NHS:ΔNHS mixtures with a NHS content below 60% (at 10% serum cc.) or below 40% (at 20% serum cc.) did not differ significantly from that in ΔNHS only. Importantly, however, these increments all differed from each other in samples with higher NHS contents.
In a series of patient serum samples with AP dysregulation, the final proportion of 7AAD-positive cells increased in parallel with AP activity determined by conventional methods.
In a patient sample with CP dysregulation, the ratio of 7AAD positive cells did not increase, substantially (by 4%).

Conclusion
Our results support that our improved method has an appropriate repeatability and is able to distinguish human serum samples with different complement activity.

Funding
SUPPORTED BY 2024-2.1.1-EKÖP-2024-00004 PROGRAMME.