Supplementary Material for: Development and characterization of novel ELISAs for the specific quantification of the factor H-related proteins 2, 3, 4 and 5

Background: The complement system's alternative pathway relies on factor H (FH) for immune homeostasis. Next to FH, a group of highly similar proteins was described known as FH-related (FHR) proteins. The FH protein family includes FH, factor H-like protein 1, and five FHR proteins (FHR-1 to -5). The exact function of the FHRs is still unknown, necessitating further research. However, the lack of highly specific assays has hindered studying their role in health and disease. This study aimed to develop novel ELISAs for reliably and specifically quantifying levels of the FHRs in human blood. Methods: Novel FHR specific antibodies were generated. Positive hybridoma clones were taken to monoclonality, verified for target specificity via ELISA and western blot, and antibody pairs were selected for further ELISA development. During development, ELISAs were characterized and validated for specificity, stability, accuracy and reproducibility among others. Results: Monoclonal antibodies specific for FHR-2, -3, -4, or FHR-5 were generated. Using these antibodies, four ELISAs were developed capable of quantifying FHR levels in an accurate and robust manner. Each assay showed high target specificity, good analyte recovery and strong reproducibility between replicates, test-runs and test laboratories. Conclusions: These assays enable specific and accurate quantification of FHR-2, -3, -4, and -5 in human blood. They facilitate large-scale screening of patient cohorts in a standardized manner and contribute to understanding the role of the FHRs in health and disease.