Validation of an Endopep-suspension immunoassay for the diagnostics of human botulism_suppl

Supplementary methods Testing long-term reagent stabilityIn order to provide a robust in vitro method to replace the MBA, the assay reagents need to remain stable over a defined period of time to ensure reliability of the measurement results.Since most reagents used for the Endopep-SIA were produced as assay prototype in house, it was important to estimate the reagent stability.To this end, two assay reagent kits were produced independently and reagents tested on the day of production as well as 60, 90, 180 and 360 days later.Antibody-coupled beads for toxin extraction, substrate-coupled beads and streptavidin-R-phycoerythrin were stored at 4 C.All other reagents, including the cleavage buffer, were stored at -20C.BoNT/A and B standards were stored as single-use aliquots at -20C.For stability measurements standard curves were recorded with each assay kit.As an estimate of stability, the quantitative range (main manuscript, Tab. 1) was further investigated to ensure the assay was still able to detect and quantify low BoNT concentrations (Fig. S3A, gray area).The concentrations were quantified by considering every measurement of every day as a sample and relative deviation from the respective standard concentration was calculated according to main manuscript, equation (1) for every time point (Fig. S3, colors).If bias was outside of the predefined accuracy range ( 25 % bias + 25 % imprecision + inter-assay CV of the 'low spike', see main manuscript Tab. 2), the timepoint was considered quantitatively instable (Fig. S3B, red dotted lines).Qualitative instability was attributed to time points with relative deviations below the lower accuracy range (Fig. S3B, red shaded area).