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

Tracie Profaizer¹, Abdulrahman Saadalla², Vijayalakshmi Nandakumar³

¹ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States of America.

Journal of Immunological Methods

|May 24, 2025

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The Quidel sC5b-9 ELISA kit accurately detects complement activation, but the Hycult assay provides a wider dynamic range for monitoring complement inhibitor therapy. Both assays show effectiveness in therapeutic assessments.

Area of Science:

Immunology
Biochemistry
Clinical Diagnostics

Background:

Soluble C5b-9 (sC5b-9) is a key biomarker for complement system activation.
sC5b-9 measurement is crucial for diagnosing conditions like thrombotic microangiopathy and monitoring complement inhibitor therapies.
Accurate sC5b-9 detection aids therapeutic decisions, especially for patients on anti-C5 inhibitors.

Purpose of the Study:

To validate the Quidel sC5b-9 ELISA kit for detecting complement activation.
To compare the performance of the Quidel sC5b-9 ELISA kit with Hycult and SVAR assays.
To assess assay linearity and suitability for therapeutic monitoring.

Main Methods:

Analytical performance evaluation of the Quidel sC5b-9 ELISA kit using split samples.

Keywords:

Complement-mediated activation ELISA Eculizumab Terminal complement Cascade (TCC)Thrombotic microangiopathy (TMA)sC5b-9

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Assessment of precision, sensitivity, linearity, and reference limits with artificially activated samples and patient samples.

Correlation analysis between Quidel, Hycult, and SVAR assays, and comparison of linear ranges.

Evaluation of the impact of anti-C5 inhibitors on sC5b-9 concentrations.

Main Results:

The Quidel sC5b-9 assay demonstrated accuracy (R²=0.90) and high qualitative concordance (93.3%).
The reportable range for the Quidel assay was determined as 220-1800 ng/mL, with LOQ at 170 ng/mL.
The Hycult kit exhibited a broader linear range compared to Quidel and SVAR assays, maintaining linearity at higher concentrations.
All tested assays showed reduced sC5b-9 levels after anti-C5 inhibitor addition, with Hycult maintaining a wide dynamic range.

Conclusions:

The Quidel sC5b-9 ELISA kit is a reliable tool for detecting complement activation and inhibition.
The Hycult assay's broader dynamic range is advantageous for disease monitoring and therapeutic assessments.
Assay selection should consider the specific requirements for sensitivity and dynamic range in clinical applications.