Validation for soluble C5b-9 detection and comparative analysis of three quantification methods

Soluble C5b-9 (sC5b-9) is a biomarker of complement activation, representing the soluble form of the Terminal Complement Complex (TCC), which is released into circulation rather than embedding in the cell membrane. Measurement of sC5b-9 is increasingly utilized for detecting complement activation, particularly in conditions like thrombotic microangiopathy and during complement inhibitor therapy. Accurate detection of sC5b-9 can be useful for guiding therapeutic decisions, especially in patients receiving anti-C5 inhibitors. This study aimed to validate the Quidel sC5b-9 ELISA kit and compare its performance with two other assays, Hycult and SVAR, focusing on assay linearity and therapeutic monitoring.

The analytical performance of the Quidel sC5b-9 ELISA kit was evaluated using 60 split samples over a wide concentration range, in collaboration with a peer laboratory. Precision, sensitivity, linearity, and reference limits were assessed, mainly using artificially activated samples with magnesium chloride and Zymosan, covering a broad range of sC5b-9 concentrations. Four patient samples with complement-mediated diseases were also tested. The correlation between the Quidel, Hycult, and SVAR assays was analyzed with 40 samples, while linear ranges were compared using 11 proportionally diluted samples. Additionally, the impact of anti-C5 inhibitors on sC5b-9 concentrations was evaluated by spiking anti-C5 biosimilar drugs into activated plasma.

The Quidel sC5b-9 assay showed accuracy, with an R² of 0.90 and a qualitative concordance of 93.3 %. Very few discrepancies were noted in samples near the reference limit. Both intra- and inter-assay precision were below 20% CV for samples above the Limit of Quantification (LOQ) of 170 ng/mL. Linearity studies defined the reportable range for Quidel's assay as 220-1800 ng/mL. Comparisons among Quidel, SVAR, and Hycult assays revealed strong correlations, with the Hycult kit demonstrating a broader linear range, maintaining linearity even at concentrations 10 times higher than its highest calibrator. All assays showed a significant reduction in sC5b-9 concentrations after the addition of 50 μg/mL of Eculizumab biosimilar, with SVAR and Quidel reaching values close to their lowest calibrators. In contrast, the Hycult assay maintained a wide dynamic range even at 100 μg/mL of anti-C5 inhibitor addition.

This study confirms that the Quidel sC5b-9 ELISA kit is a reliable tool for detecting complement activation and inhibition. However, the Hycult assay offers a broader dynamic range which can be an important consideration when assessing assay suitability for for disease monitoring and therapeutic assessments.