57 LC-MS/MS-method development quantifying FVIII 24 hours was only 30%. Since the signal intensity at 24 hour was significantly higher than 5 hours, an incubation period of 24 hours was chosen. An even longer incubation period than 24 hour was not deemed necessary since the difference in signal intensity between 5 and 24 hours was only 15%. Figure 3. On the x-axis binding time between camelid nanobodies and the light chain fragment of FVIII is plotted versus signature peptide signal intensity from a volunteer sample on the y-axis, error bars represent mean with SD, n = 3. Effect of sample volume on signal intensity This experiment was set-up to determine if sample matrix interferes with FVIII binding or LC-MS/MS signature peptide measurement. The signal intensity obtained after sample purification and measurement was corrected for sample volume. Figure 4 shows that volume 50 and 25 µL produced similar (p = 0.051) signal intensities when corrected for volume. However, when sample volume lower than 25 µL was purified, lower signal intensity was obtained than expected. Internal standard signal was stable for all samples indicating that no ionization differences was present due to matrix. A possible reason for the diminished signal intensity could be due to other coagulation factors being present in lower levels which might have affected FVIII activation. FVIIIa light chain is smaller and can easily be captured compared to intact FVIII. Since a low detection level were required, 50 µL sample was used. The fibrin blood clot obtained after overnight incubation was too big with higher >50 µL sample volumes. This made sample handling such as decanting and washing difficult to perform. 96 well plate brand and capacity test Streptavidin and biotin interactions are amongst strongest biological interactions known with binding strengths equivalent to covalent bonds. Furthermore, biotin and streptavidin bind selectively with each other, thus limiting cross-reactivity. 3
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